Development of RPA-Cas12a-fluorescence assay for rapid and reliable detection of human bocavirus 1

Author(s):

Weidong Qian,Xuefei Wang,Ting Wang,Jie Huang,Qian Zhang,Yongdong Li,Si Chen

Journal:

Animal Models and Experimental Medicine

Year:

2024

Abstract:

Human bocavirus (HBoV) 1 is considered an important pathogen that mainly affects infants aged 6-24 months, but preventing viral transmission in resource-limited regions through rapid and affordable on-site diagnosis of individuals with early infection of HBoV1 remains somewhat challenging. Herein, we present a novel faster, lower cost, reliable method for the detection of HBoV1, which integrates a recombinase polymerase amplification (RPA) assay with the CRISPR/Cas12a system, designated the RPA-Cas12a-fluorescence assay. The RPA-Cas12a-fluorescence system can specifically detect target gene levels as low as 0.5 copies of HBoV1 plasmid DNA per microliter within 40 min at 37°C without the need for sophisticated instruments. The method also demonstrates excellent specificity without cross-reactivity to non-target pathogens. Furthermore, the method was appraised using 28 clinical samples, and displayed high accuracy with positive and negative predictive agreement of 90.9% and 100%, respectively. Therefore, our proposed rapid and sensitive HBoV1 detection method, the RPA-Cas12a-fluorescence assay, shows promising potential for early on-site diagnosis of HBoV1 infection in the fields of public health and health care. The established RPA-Cas12a-fluorescence assay is rapid and reliable method for human bocavirus 1 detection. The RPA-Cas12a-fluorescence assay can be completed within 40 min with robust specificity and sensitivity of 0.5 copies/μl.

PrimerBankID	Target Pathogen	Target Gene
RPB0401	Human bocavirus 1	NP1

Portable Paper-Based Nucleic Acid Enrichment for Field Testing

Author(s):

Junyang Mei, Dandan Wang, Yiheng Zhang, Dan Wu, Jinhui Cui, Mingzhe Gan, Peifeng Liu

Journal:

Advanced Science

Year:

2023

Abstract:

Point-of-care testing (POCT) can be the method of choice for detecting infectious pathogens; these pathogens are responsible for not only infectious diseases such as COVID-19, but also for certain types of cancers. For example, infections by human papillomavirus (HPV) or Helicobacter pylori (H. pylori) are the main cause of cervical and stomach cancers, respectively. COVID-19 and many cancers are treatable with early diagnoses using POCT. A variety of nucleic acid testing have been developed for use in resource-limited environments. However, questions like unintegrated nucleic acid extraction, open detection systems increase the risk of cross-contamination, and dependence on expensive equipment and alternating current (AC) power supply, significantly limit the application of POCT, especially for on-site testing. In this paper, a simple portable platform is reported capable of rapid sample-to-answer testing within 30 min based on recombinase polymerase amplification (RPA) at a lower temperature, to detect SARS-CoV-2 virus and H. pylori bacteria with a limit of detection as low as 4 × 102 copies mL-1 . The platform used a battery-powered portable reader for on-chip one-pot amplification and fluorescence detection, and can test for multiple (up to four) infectious pathogens simultaneously. This platform can provide an alternative method for fast and reliable on-site diagnostic testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0013	SARS-CoV-2	ORF1ab
RPB0396	SARS-CoV-2	ORF1ab gene

Fast and visual detection of nucleic acids using a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM

Author(s):

Kangfeng Lin,Jianguang Guo,Xiangju Guo,Qinghan Li,Xiao Li,Zhen Sun,Zehang Zhao,Jiao Weng,Jinzhun Wu,Rui Zhang,Boan Li

Journal:

Analytica Chimica Acta

Year:

2023

Abstract:

CRISPR-Cas12a (Cpf1) is widely used for pathogen detection. However, most Cas12a nucleic acid detection methods are limited by a PAM sequence requirement. Moreover, preamplification and Cas12a cleavage are separate. Here, we developed a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM sequence with high sensitivity and specificity that offers one-tube, rapid, and visually observable detection of nucleic acids. In this system, Cas12a detection and RPA amplification are performed simultaneously, without separate preamplification and product transfer steps, and 0.2 copies/μL of DNA and 0.4 copies/μL of RNA can be detected. In the ORCD system, the activity of Cas12a is the key to the nucleic acid detection; specifically, reducing Cas12a activity increases the sensitivity of ORCD assay detection of the PAM target. Furthermore, by combining this detection technique with a nucleic acid extraction-free method, our ORCD system can be used to extract, amplify and detect samples within 30 min, as verified with tests of 82 Bordetella pertussis clinical samples with a sensitivity and specificity of 97.30% and 100% compared with PCR. We also tested 13 SARS-CoV-2 samples with RT-ORCD, and the results were consistent with RT-PCR.

PrimerBankID	Target Pathogen	Target Gene
RPB0399	SARS-CoV-2	N gene
RPB0400	Bordetella pertussis CS	IS1663 gene

Centrifugal microfluidic-based multiplex recombinase polymerase amplification assay for rapid detection of SARS-CoV-2

Author(s):

Ruoxu Li,Ning Su,Xiaodong Ren,Xiange Sun,Wenman Li,Yuwei Li,Jin Li,Chen Chen,Hong Wang,Weiping Lu,Shaoli Deng,Qing Huang

Journal:

iScience

Year:

2023

Abstract:

The COVID-19 pandemic has spread worldwide, and rapid detection of the SARS-CoV-2 virus is crucial for infection surveillance and epidemic control. This study developed a centrifugal microfluidics-based multiplex reverse transcription recombinase polymerase amplification (RT-RPA) assay for endpoint fluorescence detection of the E, N, and ORF1ab genes of SARS-CoV-2. The microscope slide-shaped microfluidic chip could simultaneously accomplish three target genes and one reference human gene (i.e., ACTB) RT-RPA reactions in 30 min, and the sensitivity was 40 RNA copies/reaction for the E gene, 20 RNA copies/reaction for the N gene, and 10 RNA copies/reaction for the ORF1ab gene. The chip demonstrated high specificity, reproducibility, and repeatability. Chip performance was also evaluated using real clinical samples. Thus, this rapid, accurate, on-site, and multiplexed nucleic acid test microfluidic chip would significantly contribute to detecting patients with COVID-19 in low-resource settings and point-of-care testing (POCT) and, in the future, could be used to detect emerging new variants of SARS-CoV-2.

PrimerBankID	Target Pathogen	Target Gene
RPB0014	SARS-CoV-2	N
RPB0015	SARS-CoV-2	ORF1ab
RPB0016	SARS-CoV-2	E

Evaluation of three rapid low-resource molecular tests for Nipah virus

Author(s):

Nina M Pollak,Malin Olsson,Glenn A Marsh,Joanne Macdonald,David McMillan

Journal:

Frontiers in Microbiology

Year:

2023

Abstract:

Accurate and timely diagnosis of Nipah virus (NiV) requires rapid, inexpensive, and robust diagnostic tests to control spread of disease. Current state of the art technologies are slow and require laboratory infrastructure that may not be available in all endemic settings. Here we report the development and comparison of three rapid NiV molecular diagnostic tests based on reverse transcription recombinase-based isothermal amplification coupled with lateral flow detection. These tests include a simple and fast one-step sample processing step that inactivates the BSL-4 pathogen, enabling safe testing without the need for multi-step RNA purification. The rapid NiV tests targeted the Nucleocapsid protein (N) gene with analytical sensitivity down to 1,000 copies/μL for synthetic NiV RNA and did not cross-react with RNA of other flaviviruses or Chikungunya virus, which can clinically present with similar febrile symptoms. Two tests detected 50,000-100,000 TCID50/mL (100-200 RNA copies/reaction) of the two distinct strains of NiV, Bangladesh (NiVB) and Malaysia (NiVM), and took 30 min from sample to result, suggesting these tests are well suited for rapid diagnosis under resource-limited conditions due to rapidity, simplicity, and low equipment requirements. These Nipah tests represent a first step toward development of near-patient NiV diagnostics that are appropriately sensitive for first-line screening, sufficiently robust for a range of peripheral settings, with potential to be safely performed outside of biohazard containment facilities.

PrimerBankID	Target Pathogen	Target Gene
RPB0443	Nipah virus	Nucleocapsid protein (N) gene

Rapid Detection of Staphylococcus aureus in Milk and Pork via Immunomagnetic Separation and Recombinase Polymerase Amplification

Author(s):

Runan Cheng,Lei Li,Sihui Zhen,Honglei Liu,Zhouhui Wu,Yu Wang,Zhen Wang

Journal:

Microbiology spectrum

Year:

2023

Abstract:

Separation processes using immunomagnetic beads (IMBs) are advantageous for the rapid detection of Staphylococcus aureus (S. aureus). Herein, a novel method, based on immunomagnetic separation using IMBs and recombinase polymerase amplification (RPA), was employed to detect S. aureus strains in milk and pork. IMBs were formed by the carbon diimide method using rabbit anti-S. aureus polyclonal antibodies and superparamagnetic carboxyl-Fe3O4 MBs. The average capture efficiency for 2.5 to 2.5 × 105 (CFU)/mL gradient dilution of S. aureus with 6 mg of IMBs within 60 min were a range of 62.74 to 92.75%. The detection sensitivity of the IMBs-RPA method in artificially contaminated samples was 2.5 × 101 CFU/mL. The entire detection process was completed within 2.5 h, including bacteria capture, DNA extraction, amplification, and electrophoresis. Among 20 actual samples, one case of raw milk sample and two cases of pork samples were tested positive using the established IMBs-RPA method, which were verified by the standard S. aureus inspection procedure. Therefore, the novel method shows potential for food safety supervision owing to its short detection time, higher sensitivity, and high specificity. IMPORTANCE Our study established IMBs-RPA method, which simplified the steps of bacteria separation, shortened the detection time, and realized the convenient detection of S. aureus in milk and pork samples. IMBs-RPA method was also suitable for the detection of other pathogens, providing a new method for food safety monitoring and a favorable basis for rapid and early diagnosis of diseases.

PrimerBankID	Target Pathogen	Target Gene
RPB0155	Staphylococcus aureus	nuc

Recombinase-Aided Amplification Assay for Rapid Detection of Hypervirulent Klebsiella pneumoniae (hvKp) and Characterization of the hvKp Pathotype

Author(s):

Chao Yan,Yao Zhou,Shuheng Du,Bing Du,Hanqing Zhao,Yanling Feng,Guanhua Xue,Jinghua Cui,Lin Gan,Junxia Feng,Zheng Fan,Tongtong Fu,Ziying Xu,Qun Zhang,Rui Zhang,Xiaohu Cui,Ziyan Tian,Yujie Chen,Ting Zhang,Lei Huang,Jing Yuan

Journal:

Microbiology Spectrum

Year:

2023

Abstract:

Hypervirulent Klebsiella pneumoniae (hvKp) is a major human pathogen associated with liver abscess, pneumonia, meningitis, and endophthalmitis. It is challenging to differentiate hvKp from classical Klebsiella pneumoniae (cKp) using conventional methods, necessitating the development of a rapid, sensitive, and convenient assay for hvKp detection. In this study, we constructed a recombinase-aided amplification (RAA) method targeting hvKp genes peg344 and rmpA, and also analyzed the pathogenic characteristics of hvKp. We optimized the reaction temperature and system, and evaluated its sensitivity, specificity, and clinical application. The primer and probe sets peg344-set1 and rmpA-set2 delivered significant fluorescent signals at 39°C with the shortest gene amplification times (sensitivity: 20 copies/reaction). This RAA assay showed no cross-reactivity with 15 other common pathogenic bacteria. Its applicability was confirmed by the evaluation of 208 clinical specimens, of which 45 were confirmed to be hvKp. The sensitivity and specificity of the RAA assay were both 100% compared with real-time PCR as the reference standard. To verify the assay, we also assessed the diversity of molecular characteristics among the hvKp isolates and identified serotype K1 and sequence type ST23 as the dominant clone. Virulence factors iroN and iutA were highly associated with virulence level. In conclusion, our novel RAA assay is a powerful tool for early diagnosis and epidemiological surveillance of hvKp. IMPORTANCEKlebsiella pneumoniae is the most common opportunistic bacterial species and a major threat to public health. Since the 1990s, hvKp has received increasing attention from public health officials and infectious disease specialists. Hypervirulent strains differ from classical strains in terms of phenotypic features and clinical outcomes. It is hard to identify hvKp from cKp using the conventional methods including colony morphology analysis, serum killing assays, mouse lethality assays, string tests, and real-time PCR. In this study, we established a rapid, sensitive and convenient recombinase-aided amplification assay for hvKp detection targeting virulence genes peg344 and rmpA. Our RAA assay provides an important tool for the rapid diagnosis of infectious diseases caused by hvKp, particularly in primary laboratories.

PrimerBankID	Target Pathogen	Target Gene
RPB0374	Klebsiella pneumoniae	peg344 gene
RPB0375	Klebsiella pneumoniae	rmpA gene

Cas12a/Guide RNA-Based Platforms for Rapidly and Accurately Identifying Staphylococcus aureus and Methicillin-Resistant S. aureus

Author(s):

Xiaoying Cao,Yanbin Chang,Chunqing Tao,Sen Chen,Qiuxia Lin,Chao Ling,Shifeng Huang,Hengshu Zhang

Journal:

Microbiology spectrum

Year:

2023

Abstract:

In order to ensure the prevention and control of methicillin-resistant Staphylococcus aureus (MRSA) infection, rapid and accurate detection of pathogens and their resistance phenotypes is a must. Therefore, this study aimed to develop a fast and precise nucleic acid detection platform for identifying S. aureus and MRSA. We initially constructed a CRISPR-Cas12a detection system by designing single guide RNAs (sgRNAs) specifically targeting the thermonuclease (nuc) and mecA genes. To increase the sensitivity of the CRISPR-Cas12a system, we incorporated PCR, loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA). Subsequently, we compared the sensitivity and specificity of the three amplification methods paired with the CRISPR-Cas12a system. Finally, the clinical performance of the methods was tested by analyzing the fluorescence readout of 111 clinical isolates. In order to visualize the results, lateral-flow test strip technology, which enables point-of-care testing, was also utilized. After comparing the sensitivity and specificity of three different methods, we determined that the nuc-LAMP-Cas12a and mecA-LAMP-Cas12a methods were the optimal detection methods. The nuc-LAMP-Cas12a platform showed a limit of detection (LOD) of 10 aM (~6 copies μL-1), while the mecA-LAMP-Cas12a platform demonstrated a LOD of 1 aM (~1 copy μL-1). The LOD of both platforms reached 4 × 103 fg/μL of genomic DNA. Critical evaluation of their efficiencies on 111 clinical bacterial isolates showed that they were 100% specific and 100% sensitive with both the fluorescence readout and the lateral-flow readout. Total detection time for the present assay was approximately 80 min (based on fluorescence readout) or 85 min (based on strip readout). These results indicated that the nuc-LAMP-Cas12a and mecA-LAMP-Cas12a platforms are promising tools for the rapid and accurate identification of S. aureus and MRSA. IMPORTANCE The spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to global health. Isothermal amplification combined with the trans-cleavage activity of Cas12a has been exploited to generate diagnostic platforms for pathogen detection. Here, we describe the design and clinical evaluation of two highly sensitive and specific platforms, nuc-LAMP-Cas12a and mecA-LAMP-Cas12a, for the detection of S. aureus and MRSA in 111 clinical bacterial isolates. With a limit of detection (LOD) of 4 × 103 fg/μL of genomic DNA and a turnaround time of 80 to 85 min, the present assay was 100% specific and 100% sensitive using either fluorescence or the lateral-flow readout. The present assay promises clinical application for rapid and accurate identification of S. aureus and MRSA in limited-resource settings or at the point of care. Beyond S. aureus and MRSA, similar CRISPR diagnostic platforms will find widespread use in the detection of various infectious diseases, malignancies, pharmacogenetics, food contamination, and gene mutations.

PrimerBankID	Target Pathogen	Target Gene
RPB0153	Staphylococcus aureus	nuc
RPB0154	Staphylococcus aureus	mecA

CESSAT: A chemical additive-enhanced single-step accurate CRISPR\Cas13 testing system for field-deployable ultrasensitive detection and genotyping of SARS-CoV-2 variants of concern

Author(s):

Yunxiang Wang,Hong Chen,Huixia Gao,Hongjuan Wei,Yuling Wang,Kai Mu,Liyan Liu,Erhei Dai,Zhen Rong,Shengqi Wang

Journal:

Biosensors and Bioelectronics

Year:

2023

Abstract:

The continued emergence of SARS-CoV-2 variants of concern (VOCs) has raised great challenges for epidemic prevention and control. A rapid, sensitive, and on-site SARS-CoV-2 genotyping technique is urgently needed for individual diagnosis and routine surveillance. Here, a field-deployable ultrasensitive CRISPR-based diagnostics system, called Chemical additive-Enhanced Single-Step Accurate CRISPR/Cas13 Testing system (CESSAT), for simultaneous screening of SARS-CoV-2 and its five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) within 40 min was reported. In this system, a single-step reverse transcription recombinase polymerase amplification-CRISPR/Cas13a assay was incorporated with optimized extraction-free viral lysis and reagent lyophilization, which could eliminate complicated sample processing steps and rigorous reagent storage conditions. Remarkably, 10% glycine as a chemical additive could improve the assay sensitivity by 10 times, making the limit of detection as low as 1 copy/μL (5 copies/reaction). A compact optic fiber-integrated smartphone-based device was developed for sample lysis, assay incubation, fluorescence imaging, and result interpretation. CESSAT could specifically differentiate the synthetic pseudovirus of SARS-CoV-2 and its five VOCs. The genotyping results for 40 clinical samples were in 100% concordance with standard method. We believe this simple but efficient enhancement strategy can be widely incorporated with existing Cas13a-based assays, thus leading a substantial progress in the development and application of rapid, ultrasensitive, and accurate nucleic acid analysis technology.

PrimerBankID	Target Pathogen	Target Gene
RPB0444	SARS-CoV-2	N gene
RPB0445	SARS-CoV-2	S gene
RPB0446	SARS-CoV-2 (Alpha)	H69V70Del
RPB0447	SARS-CoV-2 (Beta)	K417N
RPB0448	SARS-CoV-2 (Gamma)	H655Y
RPB0449	SARS-CoV-2 (Delta)	T478K
RPB0450	SARS-CoV-2 (Omicron)	49X

A Rapid Antimicrobial Resistance Diagnostic Platform for Staphylococcus aureus Using Recombinase Polymerase Amplification

Author(s):

Chuangxin Lin,Yongmei Zeng,Zhihong Zhu,Jiayu Liao,Tiandan Yang,Yaqun Liu,Huagui Wei,Jiamin Li,Jibin Ma,Xiaoqing Wu,Guangyu Lin,Liyun Lin,Liying Chen,Huiying Huang,Weizhong Chen,Junli Wang,Feiqiu Wen,Min Lin

Journal:

Microbiology spectrum

Year:

2023

Abstract:

Antimicrobial resistance (AMR) has posed a global threat to public health. The Staphylococcus aureus strains have especially developed AMR to practically all antimicrobial medications. There is an unmet need for rapid and accurate detection of the S. aureus AMR. In this study, we developed two versions of recombinase polymerase amplification (RPA), the fluorescent signal monitoring and lateral flow dipstick, for detecting the clinically relevant AMR genes retained by S. aureus isolates and simultaneously identifying such isolates at the species level. The sensitivity and specificity were validated with clinical samples. Our results showed that this RPA tool was able to detect antibiotic resistance for all the 54 collected S. aureus isolates with high sensitivity, specificity, and accuracy (all higher than 92%). Moreover, results of the RPA tool are 100% consistent with that of PCR. In sum, we successfully developed a rapid and accurate AMR diagnostic platform for S. aureus. The RPA might be used as an effective diagnostic test in clinical microbiology laboratories to improve the design and application of antibiotic therapy. IMPORTANCE Staphylococcus aureus is a species of Staphylococcus and belongs to Gram-positive. Meanwhile, S. aureus remains one of the most common nosocomial and community-acquired infections, causing blood flow, skin, soft tissue, and lower respiratory tract infections. The identification of the particular nuc gene and the other eight genes of drug-resistant S. aureus can reliably and quickly diagnose the illness, allowing doctors to prescribe treatment regimens sooner. The detection target in this work is a particular gene of S. aureus, and a POCT is built to simultaneously recognize S. aureus and analyze genes representing four common antibiotic families. We developed and assessed a rapid and on-site diagnostic platform for the specific and sensitive detection of S. aureus. This method allows the determination of S. aureus infection and 10 different AMR genes representing four different families of antibiotics within 40 min. It was easily adaptable in low-resource circumstances and professional-lacking circumstances. It should be supported in overcoming the continuous difficulty of drug-resistant S. aureus infections, which is a shortage of diagnostic tools that can swiftly detect infectious bacteria and numerous antibiotic resistance indicators.

PrimerBankID	Target Pathogen	Target Gene
RPB0132	Staphylococcus aureus	nuc gene
RPB0133	Staphylococcus aureus	blaZ
RPB0134	Staphylococcus aureus	mecA
RPB0135	Staphylococcus aureus	ermA
RPB0136	Staphylococcus aureus	ermB
RPB0137	Staphylococcus aureus	ermC
RPB0138	Staphylococcus aureus	msrA
RPB0139	Staphylococcus aureus	tetK
RPB0140	Staphylococcus aureus	tetM
RPB0141	Staphylococcus aureus	aadD
RPB0142	Staphylococcus aureus	nuc
RPB0143	Staphylococcus aureus	blaZ
RPB0144	Staphylococcus aureus	mecA
RPB0145	Staphylococcus aureus	ermA
RPB0146	Staphylococcus aureus	ermB
RPB0147	Staphylococcus aureus	ermC
RPB0148	Staphylococcus aureus	msrA
RPB0149	Staphylococcus aureus	tetK
RPB0150	Staphylococcus aureus	tetM
RPB0151	Staphylococcus aureus	aadD

A multi-country phase 2 study to evaluate the suitcase lab for rapid detection of SARS-CoV-2 in seven Sub-Saharan African countries: Lessons from the field

Author(s):

Arianna Ceruti,Ndongo Dia,Adeleye Solomon Bakarey,Judah Ssekitoleko,Soa Fy Andriamandimby,Padra Malwengo-Kasongo,Rasheeda H A Ahmed,Rea Maja Kobialka,Jean Michel Heraud,Moussa Moise Diagne,Marie Henriette Dior Dione,Idrissa Dieng,Martin Faye,Ousmane Faye,Jean Théophile Rafisandratantsoa,Jean-Pierre Ravalohery,Claudio Raharinandrasana,Tsiry H Randriambolamanantsoa,Norosoa Razanajatovo,Iony Razanatovo,Joelinotahina H Rabarison,Phillipe Dussart,Louis Kyei-Tuffuor,Abigail Agbanyo,Olubusuyi Moses Adewumi,Adeola Fowotade,Muideen Kolawole Raifu,Patient Okitale-Talunda,Gracia Kashitu-Mujinga,Christelle Mbelu-Kabongo,Steve Ahuka-Mundeke,Anguy Makaka-Mutondo,Enas M Abdalla,Sanaa M Idris,Wisal A Elmagzoub,Rahma H Ali,Eman O M Nour,Rasha S M Ebraheem,Huda H H Ahmed,Hamadelniel E Abdalla,Musab Elnegoumi,Izdihar Mukhtar,Muatsim A M Adam,Nuha Y I Mohamed,Shahinaz A Bedri,Hamdan Mustafa Hamdan,Magid Kisekka,Monica Mpumwiire,Sharley Melissa Aloyo,Joanita Nabwire Wandera,Andrew Agaba,Rogers Kamulegeya,Hosea Kiprotich,David Patrick Kateete,Paul Kadetz,Uwe Truyen,Kamal H Eltom,Anavaj Sakuntabhai,Julius Boniface Okuni,Sheila Makiala-Mandanda,Vincent Lacoste,George Olusegun Ademowo,Michael Frimpong,Amadou Alpha Sall,Manfred Weidmann,Ahmed Abd El Wahed

Journal:

Journal of Clinical Virology

Year:

2023

Abstract:

Background: The COVID-19 pandemic led to severe health systems collapse, as well as logistics and supply delivery shortages across sectors. Delivery of PCR related healthcare supplies continue to be hindered. There is the need for a rapid and accessible SARS-CoV-2 molecular detection method in low resource settings. Objectives: To validate a novel isothermal amplification method for rapid detection of SARS-CoV-2 across seven sub-Sharan African countries. Study design: In this multi-country phase 2 diagnostic study, 3,231 clinical samples in seven African sites were tested with two reverse transcription Recombinase-Aided Amplification (RT-RAA) assays (based on SARS-CoV-2 Nucleocapsid (N) gene and RNA-dependent RNA polymerase (RdRP) gene). The test was performed in a mobile suitcase laboratory within 15 min. All results were compared to a real-time RT-PCR assay. Extraction kits based on silica gel or magnetic beads were applied. Results: Four sites demonstrated good to excellent agreement, while three sites showed fair to moderate results. The RdRP gene assay exhibited an overall PPV of 0.92 and a NPV of 0.88. The N gene assay exhibited an overall PPV of 0.93 and a NPV 0.88. The sensitivity of both RT-RAA assays varied depending on the sample Ct values. When comparing sensitivity between sites, values differed considerably. For high viral load samples, the RT-RAA assay sensitivity ranges were between 60.5 and 100% (RdRP assay) and 25 and 98.6 (N assay). Conclusion: Overall, the RdRP based RT-RAA test showed the best assay accuracy. This study highlights the challenges of implementing rapid molecular assays in field conditions. Factors that are important for successful deployment across countries include the implementation of standardized operation procedures, in-person continuous training for staff, and enhanced quality control measures.

PrimerBankID	Target Pathogen	Target Gene
RPB0437	SARS-CoV-2	N
RPB0438	SARS-CoV-2	RdRP

A Novel Sample-to-Answer Visual Nucleic Acid Detection System for Adenovirus Detection

Author(s):

Kui Sun,Xiaodong Yang,Yanan Wang,Qun Guan,Wenliang Fu,Chao Zhang,Qin Liu,Wenzheng An,Yongqi Zhao,Weiwei Xing,Donggang Xu

Journal:

Microbiology Spectrum

Year:

2023

Abstract:

Human adenoviruses (HAdVs) are common viruses that can cause local outbreaks in schools, communities and military camps, posing a huge threat to public health. An ideal POCT device for adenovirus detection in resource-limited settings is critical to control the spread of the virus. In this study, we developed an integrated and electricity-independent sample-to-answer system that can complete nucleic acid extraction, amplification, and detection at room temperature. This system is suitable for field and on-site detection because of its rapidity, sensitivity, lack of contamination, and lack of requirements of high-precision instruments and skilled technicians. It consists of two separate modules, ALP FINA (alkaline lysis with the paper-based filtration isolation of nucleic acid) and SV RPA (sealed and visual recombinase polymerase amplification). The extraction efficiency of ALP FINA can reach 48 to 84%, which is close to that of the conventional centrifuge column. The detection sensitivity of SV RPA is close to 10 copies/μL of AdvB and AdvE without aerosol contamination after repeated operations. When SV RPA was applied to the detection of nasopharyngeal swab samples of 19 patients who were infected with AdvB or AdvE as well as 10 healthy volunteers, its sensitivity and specificity reached 100%, respectively. IMPORTANCE HAdV infections are readily transmittable and, in some instances, highly contagious. Early and rapid diagnosis is essential for disease control. In this work, we developed a portable, disposable, and modularized sample-to-answer detection system for AdvB and AdvE, which rendered the entire test to be completely independent of electricity and other laboratory infrastructure. Thus, this detection system can be applied in resource-limited settings, and it has the potential to be further developed as an early diagnosis method in the field.

PrimerBankID	Target Pathogen	Target Gene
RPB0419	HAdV	AdvB AdvE

CDetection.v2: One-pot assay for the detection of SARS-CoV-2

Author(s):

Xinge Wang,Yangcan Chen,Xuejia Cheng,Si-Qi Wang,Yanping Hu,Yingmei Feng,Ronghua Jin,Kangping Zhou,Ti Liu,Jianxing Wang,Kai Pan,Bing Liu,Jie Xiang,Yanping Wang,Qi Zhou,Ying Zhang,Weiye Pan,Wei Li

Journal:

Frontiers in Microbiology

Year:

2023

Abstract:

Introduction: The ongoing 2019 coronavirus disease pandemic (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, is a global public health threat. Early diagnosis and identification of SARS-CoV-2 and its variants plays a critical role in COVID-19 prevention and control. Currently, the most widely used technique to detect SARS-CoV-2 is quantitative reverse transcription real-time quantitative PCR (RT-qPCR), which takes nearly 1 hour and should be performed by experienced personnel to ensure the accuracy of results. Therefore, the development of a nucleic acid detection kit with higher sensitivity, faster detection and greater accuracy is important. Methods: Here, we optimized the system components and reaction conditions of our previous detection approach by using RT-RAA and Cas12b. Results: We developed a Cas12b-assisted one-pot detection platform (CDetection.v2) that allows rapid detection of SARS-CoV-2 in 30 minutes. This platform was able to detect up to 5,000 copies/ml of SARS-CoV-2 without cross-reactivity with other viruses. Moreover, the sensitivity of this CRISPR system was comparable to that of RT-qPCR when tested on 120 clinical samples. Discussion: The CDetection.v2 provides a novel one-pot detection approach based on the integration of RT-RAA and CRISPR/Cas12b for detecting SARS-CoV-2 and screening of large-scale clinical samples, offering a more efficient strategy for detecting various types of viruses.

PrimerBankID	Target Pathogen	Target Gene
RPB0405	SARS-CoV-2	RdRp

CRISPR\Cas9-based point-of-care lateral flow biosensor with improved performance for rapid and robust detection of Mycoplasma pneumonia

Author(s):

Rong Zhu,Han Jiang,Chaoyou Li,Yan Li,Min Peng,Junfeng Wang,Qian Wu,Chao Yan,Qingli Bo,Jie Wang,Chenlin Shen,Panzhu Qin

Journal:

Analytica Chimica Acta

Year:

2023

Abstract:

Screening of acute respiratory infections causes serious challenges in urgent point-of-care scenarios where conventional methods are impractical and alternative techniques suffer from low accuracy, poor robustness, and reliance on sophisticated instruments. As an improvement to this paradigm, we report a point-of-care lateral flow biosensor (LFB) based on the recognition property of clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (Cas9) and apply it to the detection of Mycoplasma pneumoniae (M. pneumoniae). The designed biosensor employs CRISPR/Cas9 for secondary recognition after preamplification of target gene using specific primer set, avoiding false positives caused by nontarget factors. The high amplification efficiency and low applicable temperatures of recombinase polymerase amplification brings the detection limit of the biosensor to 3 copies even at a preamplification temperature of 25 °C. Its practical application is further demonstrated with 100% accuracy by testing with 43 M. pneumoniae-infected specimens and 80 uninfected specimens. Additionally, the entire detection, including pretreatment, preamplification, CRISPR/Cas9 recognition, and visual analysis, can be completed in 30 min. Featured with the combination of CRISPR/Cas9 and LFB, the biosensor we developed herein ensures excellent convenience, accuracy, and robustness, which endows promising point-of-care screening potential for infectious pathogens.

PrimerBankID	Target Pathogen	Target Gene
RPB0432	Mycoplasma pneumoniae	\

Droplet digital recombinase polymerase amplification for multiplexed detection of human coronavirus

Author(s):

Ji Wook Choi, Won Ho Seo, Taejoon Kang, Taewook Kang, Bong Geun Chung

Journal:

Lab On A Chip

Year:

2023

Abstract:

Since the outbreak of coronavirus 2019 (COVID-19), detection technologies have been attracting a great deal of attention in molecular diagnosis applications. In particular, the droplet digital PCR (ddPCR) has become a promising tool as it offers absolute quantification of target nucleic acids with high specificity and sensitivity. In recent years, the combination of the isothermal amplification strategies has made ddPCR a popular method for on-site testing by enabling amplification at a constant temperature. However, the current isothermal ddPCR assays are still challenging due to inherent non-specific amplification. In this paper, we present a multiplexed droplet digital recombinase polymerase amplification (MddRPA) with precise initiation of the reaction. First, the reaction temperature and dynamic range of reverse transcription (RT) and RPA were characterized by real-time monitoring of fluorescence intensities. Using a droplet-based microfluidic chip, the master mix and the initiator were fractionated and rapidly mixed within well-confined droplets. Due to the high heat transfer and mass transfer of the droplets, the precise initiation of the amplification was enabled and the entire assay could be conducted within 30 min. The concentrations of target RNA in the range from 5 copies per μL to 2500 copies per μL could be detected with high linearity (R2 > 0.999). Furthermore, the multiplexed detection of three types of human coronaviruses was successfully demonstrated with high specificity (>96%). Finally, we compared the performance of the assay with a commercial RT-qPCR system using COVID-19 clinical samples. The MddRPA assay showed a 100% concordance with the RT-qPCR results, indicating its reliability and accuracy in detecting SARS-CoV-2 nucleic acids in clinical samples. Therefore, our MddRPA assay with rapid detection, precise quantification, and multiplexing capability would be an interesting method for molecular diagnosis of viral infections.

PrimerBankID	Target Pathogen	Target Gene
RPB0006	HCoV-229E	\
RPB0007	HCoV-OC43	\
RPB0008	HCoV-NL63	\

Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation

Author(s):

Harmen J van der Veer, Eva A van Aalen , Claire M S Michielsen, Eva T L Hanckmann, Jeroen Deckers, Marcel M G J van Borren, Jacky Flipse, Anne J M Loonen, Joost P H Schoeber, Maarten Merkx

Journal:

ACS Central Science

Year:

2023

Abstract:

Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/μL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing. Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/μL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0009	SARS-CoV-2	ORF1a_L
RPB0010	SARS-CoV-2	ORF1a_S
RPB0011	SARS-CoV-2	N1_L
RPB0012	SARS-CoV-2	N1_S

A rapid and highly sensitive multiple detection of human adenovirus type 3, type 7 and respiratory syncytial virus by recombinase-aided reverse transcription PCR

Author(s):

Guohao Fan,Xiaozhou He,Ruiqing Zhang,Fengyu Tian,Xiuli Sun,Mengyi Zhang,Fengyu Li,Xinxin Shen,Xuejun Ma

Journal:

Journal of Clinical Laboratory Analysis

Year:

2023

Abstract:

Background: Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase-aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology. Methods: In this study, we developed and validated the multiplex reverse transcription recombinase-aided PCR (multiplex RT-RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process. Results: Using recombinant plasmids, the sensitivity of multiplex RT-RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT-RAP showed no cross-reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT-RAP and the results were found to be consistent with those of corresponding RT-qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT-RAP was two to eightfold higher than that of corresponding RT-qPCR. Conclusion: We conclude the multiplex RT-RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load.

PrimerBankID	Target Pathogen	Target Gene
RPB0422	HAdV3	\
RPB0423	HAdV7	\
RPB0424	HRSV A	\

RNA extraction-free workflow integrated with a single-tube CRISPR-Cas-based colorimetric assay for rapid SARS-CoV-2 detection in different environmental matrices

Author(s):

Yuliang Kang, Jiali Wang, Wensi Zhang, Yuhang Xu, Bohui Xu, Guangbo Qu, Yanyan Yu, Bing Yan, Gaoxing Su

Journal:

Journal Of Hazardous Materials

Year:

2023

Abstract:

On-site environmental surveillance of viruses is increasingly important for infection prevention and pandemic control. Herein, we report a facile single-tube colorimetric assay for detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from environmental compartments. Using glycerol as the phase separation additive, reverse transcription recombinase polymerase amplification (RT-RPA), CRISPR-Cas system activation, G-quadruplex (G4) cleavage, and G4-based colorimetric reaction were performed in a single tube. To further simplify the test, viral RNA genomes used for the one-tube assay were obtained via acid/base treatment without further purification. The whole assay from sampling to visual readout was completed within 30 min at a constant temperature without the need for sophisticated instruments. Coupling the RT-RPA to CRISPR-Cas improved the reliability by avoiding false positive results. Non-labeled cost-effective G4-based colorimetric systems are highly sensitive to CRISPR-Cas cleavage events, and the proposed assay reached the limit of detection of 0.84 copies/µL. Moreover, environmental samples from contaminated surfaces and wastewater were analyzed using this facile colorimetric assay. Given its simplicity, sensitivity, specificity, and cost-effectiveness, our proposed colorimetric assay is highly promising for applications in on-site environmental surveillance of viruses.

PrimerBankID	Target Pathogen	Target Gene
RPB0003	SARS-CoV-2	N

CRISPR Cas12a-enabled biosensors coupled with commercial pregnancy test strips for the visible point-of-care testing of SARS-CoV-2 Mycoplasma pneumonia

Author(s):

Peijie Shen,Zhenjun Si,Di Huang,Zhipeng Xu,Ziyi Wang,Mengjun Fang,Zhinan Xu

Journal:

Analyst

Year:

2023

Abstract:

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has promoted the development of nucleic acid diagnosis technology. Several platforms with isothermal amplification methods have achieved sensitive and specific detection of SARS-CoV-2. However, they still suffer from complicated operations, delicate instruments, and unintuitive signal output modes. Here, a system consisting of CRISPR Cas12a-based biosensors and commercial pregnancy test strips (CRISPR-PTS) was established for the point-of-care testing of SARS-CoV-2. The target viral nucleic acids were finally reflected on the test strips through four steps, namely sample pretreatment, RT-RAA amplification, CRISPR Cas12a reaction, and separation-free hCG detection. This CRISPR-PTS assay possessed an outstanding sensitivity of as low as 1 copy per μL for SARS-CoV-2 detection and showed an excellent specificity in distinguishing the SARS-CoV-2 pseudovirus as well as other SARS-like viral clinical samples. In addition, the CRISPR-PTS assay performed well in practical applications, with 96.3% agreement versus RT-qPCR in spiked samples. With the advantages of low reagent cost, simple operation procedure, and visible signal output, CRISPR-PTS assay was expected to provide a strong supplement in the prevention and early diagnosis of infectious diseases in resource-limited situations.

PrimerBankID	Target Pathogen	Target Gene
RPB0433	SARS-CoV-2	E gene
RPB0434	MERS-CoV	ORF1a
RPB0435	MERS-CoV	upE
RPB0436	MERS-CoV	ORF1b

Rapid detection of Nipah virus using the one-pot RPA-CRISPR\Cas13a assay

Author(s):

Jing Miao,Lulu Zuo,Dongmei He,Zhixin Fang,Nicolas Berthet,Chao Yu,Gary Wong

Journal:

Virus Research

Year:

2023

Abstract:

Nipah virus (NiV) is a zoonotic pathogen with airborne transmission and high case fatality rates in humans. There is currently no treatment or vaccine against NiV infection approved for humans or animals, therefore early diagnosis is the key to control any potential outbreaks. In this study, we developed an optimized one-pot assay using recombinase polymerase amplification (RPA) coupled to CRISPR/Cas13a for the molecular detection of NiV. The one-pot RPA-CRISPR/Cas13a assay for NiV detection was specific and did not cross-react against other selected (re)-emerging pathogens. The sensitivity of the one-pot RPA-CRISPR/Cas13a assay for NiV detection can detect as little as 103 cp/μL of total synthetic NiV cDNA. The assay was then validated with simulated clinical samples. The results for the one-pot RPA-CRISPR/Cas13a assay could be visualized with either fluorescence or lateral flow strips for convenient clinical or field diagnostics, providing a useful supplement to the gold-standard qRT-PCR assay for detecting NiV detections.

PrimerBankID	Target Pathogen	Target Gene
RPB0384	Nipah virus	N gene

Real-time recombinase-aided amplification assay for rapid amplification of the IS1081 gene of Mycobacterium tuberculosis

Author(s):

Yuanyuan Liu,Weicong Ren,Zhongtan Xue,Yuedong Miao,Wei Wang,Xuxia Zhang,Cong Yao,Yuanyuan Shang,Shanshan Li,Fengling Mi,Yu Pang

Journal:

European Journal of Clinical Microbiology & Infectious Diseases

Year:

2023

Abstract:

Mycobacterium tuberculosis (MTB), the etiological agent of tuberculosis (TB), is the leading cause of death due to a single infectious agent worldwide. Rapid and accurate diagnosis of MTB is critical for controlling TB especially in resource-limited countries, since any diagnosis delay increases the chances of transmission. Here, a real-time recombinase-aided amplification (RAA) assay targeting conserved positions in IS1081 gene of MTB, is successfully established to detect MTB. The intact workflow was completed within 30 min at 42 °C with no cross-reactivity observed for non-tuberculous mycobacteria and other clinical bacteria, and the detection limit for recombinant plasmid of MTB IS1081 was 163 copies/reaction at 95% probability, which was approximately 1.5-fold increase in analytical sensitivity for the detection of MTB, compared to conventional quantitative real-time PCR (qPCR; 244 copies/reaction). Furthermore, the result of clinical performance evaluation revealed an increased sensitivity of RAA assay relative to qPCR was majorly noted in the specimens with low bacteria loads. Our results demonstrate that the developed real-time RAA assay is a convenient, sensitive, and low-cost diagnostic tool for the rapid detection of MTB.

PrimerBankID	Target Pathogen	Target Gene
RPB0365	Mycobacterium tuberculosis H37Rv	IS1081 gene

Fully integrated sample-in-answer-out platform for viral detection using digital reverse transcription recombinase polymerase amplification (dRT-RPA)

Author(s):

Seder, Islam; Coronel-Tellez, Rodrigo; Helalat, Seyed Hossein; Sun, Yi;

Journal:

BIOSENS BIOELECTRON

Year:

2023

Abstract:

Recombinase polymerase amplification (RPA) is one of the most promising diagnostic methods for pathogen detection, owing to the simplified isothermal amplification technique. Using one-step digital reverse transcription RPA (dRT-RPA) to detect viral RNA provides a fast diagnosis and absolute quantification. Here, we present a chip that purifies, digitalizes, and detects viral RNA of SARS-CoV-2 in a fully automated and sensitive manner. The chip purifies the RNA using the surface charge concept of magnet bead-RNA binding, then mixes the RNA with the amplification reagents, digitalizes the amplification mixture, and performs dRT-RPA. RNA-bead complex is transported among purification buffers that are separated by an oil phase. For reagent manipulation and mixing, a magnetic valve system is integrated on the chip, where an external magnet controls the reagent direction and time of addition. Besides, a novel vacuum system is suggested to drive and regulate the reagents into two fluid systems simultaneously in ∼2 min. We also developed a cost-effective way to perform fluorescent detection for dRT-RPA on chip by using EvaGreen® dye. With integrated heating and optical detection system, the on-chip dRT-RPA presents a sample-to-answer detection platform for absolute viral RNA quantitation in 37 min and a sensitivity as low as 10 RNA copies/μL. Hence, this platform is expected to be a useful tool for accurate and automated diagnosis of infectious diseases.

PrimerBankID	Target Pathogen	Target Gene
RPB0056	SARS-CoV-2	\

FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity

Author(s):

Yi Ma,Haiping Wu,Shan Chen,Chunmei Xie,Jingjing Hu,Xiemin Qi,Xueping Ma,Yanan Chu,Jingwen Shan,Yan Lu,Lunbiao Cui,Bingjie Zou,Guohua Zhou

Journal:

Biosensors and Bioelectronics

Year:

2023

Abstract:

Recombinase polymerase amplification (RPA) running at 37-42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics.

PrimerBankID	Target Pathogen	Target Gene
RPB0357	SARS-CoV-2	RNase P gene
RPB0358	SARS-CoV-2 (Alpha)	S gene
RPB0359	SARS-CoV-2 (Beta)	S gene
RPB0360	SARS-CoV-2 (Delta)	S gene
RPB0361	SARS-CoV-2 (Omicron)	S gene
RPB0362	SARS-CoV-2	ORF1ab gene
RPB0363	SARS-CoV-2	N gene

Rapid Detection of SARS-CoV-2 RNA Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA) with Lateral Flow for N-Protein Gene and Variant-Specific Deletion-Insertion Mutation in S-Protein Gene

Author(s):

Jose L Malaga, Monica J Pajuelo, Michiko Okamoto, Emmanuel Kagning Tsinda, Kanako Otani, Pablo Tsukayama, Lucero Mascaro, Diego Cuicapuza, Masamichi Katsumi, Kazuhisa Kawamura, Hidekazu Nishimura, Akie Sakagami, Yo Ueki, Suguru Omiya, Satoshi Okamoto, Asami Nakayama, Shin-Ichi Fujimaki, Chuyao Yu, Sikandar Azam, Eiichi Kodama, Clyde Dapat, Hitoshi Oshitani, Mayuko Saito

Journal:

Viruses

Year:

2023

Abstract:

Rapid molecular testing for severe acute respiratory coronavirus 2 (SARS-CoV-2) variants may contribute to the development of public health measures, particularly in resource-limited areas. Reverse transcription recombinase polymerase amplification using a lateral flow assay (RT-RPA-LF) allows rapid RNA detection without thermal cyclers. In this study, we developed two assays to detect SARS-CoV-2 nucleocapsid (N) gene and Omicron BA.1 spike (S) gene-specific deletion-insertion mutations (del211/ins214). Both tests had a detection limit of 10 copies/µL in vitro and the detection time was approximately 35 min from incubation to detection. The sensitivities of SARS-CoV-2 (N) RT-RPA-LF by viral load categories were 100% for clinical samples with high (>9015.7 copies/µL, cycle quantification (Cq): < 25) and moderate (385.5-9015.7 copies/µL, Cq: 25-29.9) viral load, 83.3% for low (16.5-385.5 copies/µL, Cq: 30-34.9), and 14.3% for very low (<16.5 copies/µL, Cq: 35-40). The sensitivities of the Omicron BA.1 (S) RT-RPA-LF were 94.9%, 78%, 23.8%, and 0%, respectively, and the specificity against non-BA.1 SARS-CoV-2-positive samples was 96%. The assays seemed more sensitive than rapid antigen detection in moderate viral load samples. Although implementation in resource-limited settings requires additional improvements, deletion-insertion mutations were successfully detected by the RT-RPA-LF technique.

PrimerBankID	Target Pathogen	Target Gene
RPB0004	SARS-CoV-2	N
RPB0005	SARS-CoV-2	S

A CRISPR-based approach using dead Cas9-sgRNA to detect SARS-CoV-2

Author(s):

Mustapha Aouida,Maryam Saifaldeen,Dana E Al-Ansari,Sara Taleb,Ali Ait Hssain,Dindial Ramotar

Journal:

Frontiers in Molecular Biosciences

Year:

2023

Abstract:

Rapid, highly specific, and robust diagnostic kits to detect viruses and pathogens are needed to control disease spread and transmission globally. Of the many different methods proposed to diagnose COVID-19 infection, CRISPR-based detection of nucleic acids tests are among the most prominent. Here, we describe a new way of using CRISPR/Cas systems as a rapid and highly specific tool to detect the SARS-CoV-2 virus using the in vitro dCas9-sgRNA-based technique. As a proof of concept, we used a synthetic DNA of the M gene, one of the SARS-CoV-2 virus genes, and demonstrated that we can specifically inactivate unique restriction enzyme sites on this gene using CRISPR/Cas multiplexing of dCas9-sgRNA-BbsI and dCas9-sgRNA-XbaI. These complexes recognize and bind to the target sequence spanning the BbsI and XbaI restriction enzyme sites, respectively, and protect the M gene from digestion by BbsI and/or XbaI. We further demonstrated that this approach can be used to detect the M gene when expressed in human cells and from individuals infected with SARS-CoV-2. We refer to this approach as dead Cas9 Protects Restriction Enzyme Sites, and believe that it has the potential to be applied as a diagnostic tool for many DNA/RNA pathogens.

PrimerBankID	Target Pathogen	Target Gene
RPB0391	SARS-CoV-2	BbsI
RPB0392	SARS-CoV-2	XbaI
RPB0393	SARS-CoV-2	M gene

Strategies to Improve Multi-enzyme Compatibility and Coordination in One-Pot SHERLOCK

Author(s):

Hongzhao Li,Dominic M S Kielich,Guodong Liu,Greg Smith,Alexander Bello,James E Strong,Bradley S Pickering

Journal:

Analytical Chemistry

Year:

2023

Abstract:

While molecular diagnostics generally require heating elements that supply high temperatures such as 95 °C in polymerase chain reaction and 60-69 °C in loop-mediated isothermal amplification, the recently developed CRISPR-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can operate at 37 °C or a similar ambient temperature. This unique advantage may be translated into highly energy-efficient or equipment-free molecular diagnostic systems with unrestricted deployability. SHERLOCK is characterized by ultra-high sensitivity when performed in a traditional two-step format. For RNA sensing, the first step combines reverse transcription with recombinase polymerase amplification, while the second step consists of T7 transcription and CRISPR-Cas13a detection. The sensitivity drops dramatically, however, when all these components are combined into a single reaction mixture, and it largely remains an unmet need in the field to establish a high-performance one-pot SHERLOCK assay. An underlying challenge, conceivably, is the extremely complex nature of a one-pot formulation, crowding a large number of reaction types using at least eight enzymes/proteins. Although previous work has made substantial improvements by serving individual enzymes/reactions with accommodating conditions, we reason that the interactions among different enzymatic reactions could be another layer of complicating factors. In this study, we seek optimization strategies by which inter-enzymatic interference may be eliminated or reduced and cooperation created or enhanced. Several such strategies are identified for SARS-CoV-2 detection, each leading to a significantly improved reaction profile with faster and stronger signal amplification. Designed based on common molecular biology principles, these strategies are expected to be customizable and generalizable with various buffer conditions or pathogen types, thus holding broad applicability for integration into future development of one-pot diagnostics in the form of a highly coordinated multi-enzyme reaction system.

PrimerBankID	Target Pathogen	Target Gene
RPB0407	SARS-CoV-2	ORF1ab
RPB0408	SARS-CoV-2	E
RPB0409	SARS-CoV-2	S

Detecting SARS-CoV-2 BA.2, BA.4, and BA.5 Variants Utilizing a Robust RT-RPA-CRISPR/Cas12a-Based Method - China, 2023.

Author(s):

Luo, Meihui; Pan, Yang; He, Yaqing; A, Ruhan; Wu, Changcheng; Huang, Baoying; Lu, Roujian; Zhao, Li; Peng, Bo; Ye, Fei; Wang, Huijuan; Chen, Yuda; Li, Zhen; Zhang, Daitao; Wang, Wenling; Tan, Wenjie; 

Journal:

China CDC Wkly

Year:

2023

Abstract:

Introduction:Since 2019, numerous variants of concern for severe acute respiratory syndrome virus 2 (SARS-CoV-2) have emerged, leading to significant outbreaks. The development of novel, highly accurate, and rapid detection techniques for these new SARS-CoV-2 variants remains a primary focus in the ongoing efforts to control and prevent the coronavirus disease 2019 (COVID-19) pandemic.Methods:Reverse transcription-recombinase polymerase amplification combined with the clustered regularly interspaced short palindromic repeats-associated protein 12a (CRISPR/Cas12a) system was used to validate the detection of the Omicron BA.2, BA.4, and BA.5 variants of SARS-CoV-2.Results:Our results demonstrate that the CRISPR/Cas12a assay is capable of effectively detecting the SARS-CoV-2 BA.2, BA.4, and BA.5 variants with a limit of detection of 10, 1, and 10 copies/渭L, respectively. Importantly, our assay successfully differentiated the three SARS-CoV-2 Omicron strains from one another. Additionally, we evaluated 46 SARS-CoV-2 positive clinical samples consisting of BA.2 (n=20), BA.4 (n=6), and BA.5 (n=20) variants, and the sensitivity of our assay ranged from 90% to 100%, while the specificity was 100%.Discussion:This research presents a swift and reliable CRISPR-based method that may be employed to track the emergence of novel SARS-CoV-2 variants.

PrimerBankID	Target Pathogen	Target Gene
RPB0051	SARS-CoV-2 (Omicron)	ORF1ab gene C9866T
RPB0052	SARS-CoV-2 (Omicron)	S gene A23040G
RPB0053	SARS-CoV-2 (Omicron)	ORF7b gene G27788T
RPB0054	SARS-CoV-2 (Omicron)	M gene G26529A
RPB0055	SARS-CoV-2 (Omicron)	S gene C27889T

Field-deployable assay based on CRISPR-Cas13a coupled with RT-RPA in one tube for the detection of SARS-CoV-2 in wastewater

Author(s):

Yihan Yang,Fan Wang,Boyuan Xue,Xiaohong Zhou

Journal:

Journal Of Hazardous Materials

Year:

2023

Abstract:

CRISPR-based nucleic acid detection is easy to implement, field deployable, and always coupled with isothermal amplification to improve the sensitivity. However, the conventional detection requires two separate steps, which can cause long-lasting amplicon aerosol contaminants, hence leading to false-positive results. To address this problem, we proposed a one-tube assay based on CRISPR-Cas13a coupled with reverse transcription-recombinase polymerase amplification to avoid aerosol pollution. The one-tube assay could be completed within 40 min with a sensitivity of up to 180 copies of RNA per reaction, and exhibited no cross reactivity with two related coronaviruses. Our technology showed reproducibility with relative standard deviation of 4.6% responding to 1 fM nucleic acid for three times. It could be used to detect SARS-CoV-2 nucleic acids in raw wastewater with a limit of detection of 103 copies/mL. We also validated the practicability of this technique for viral detection in environmental water samples by detecting SARS-CoV-2 in wastewater, which were not detectable by RT-qPCR technology, showing resistance of this technology to wastewater matrix. It is anticipated that the robustness and high sensitivity will significantly promote the development of a point-of-care method in environmental virus monitoring.

PrimerBankID	Target Pathogen	Target Gene
RPB0439	SARS-CoV-2	WPRE
RPB0440	SARS-CoV-2	S gene

Rapid isothermal point-of-care test for screening of SARS-CoV-2 (COVID-19)

Author(s):

Jean-Marc Zingg, Yu-Ping Yang, Spencer Seely, Pratibha Joshi, Md Harun Or Roshid, Fabiola Iribarren Latasa, Gregory O'Connor, Jennifer Alfaro, Eduardo Riquelme, Sebastian Bernales, Emre Dikici, Sapna Deo, Sylvia Daunert

Journal:

Aspects of Molecular Medicine

Year:

2023

Abstract:

Rapid on-site diagnosis of emerging pathogens is key for early identification of infected individuals and for prevention of further spreading in a population. Currently available molecular diagnostic tests are instrument-based whereas rapid antibody and antigen tests are often not sufficiently sensitive for detection in pre-symptomatic subjects. There is a need for rapid point of care molecular screening tests that can be easily adapted to emerging pathogens and are selective, sensitive, reliable in different settings around the world. We have developed a simple, rapid (<30 ​min), and inexpensive test for SARS-CoV-2 that is based on combination of isothermal reverse transcription recombinase polymerase amplification (RT-RPA) using modified primers and visual detection with paper-based microfluidics. Our test (CoRapID) is specific for SARS-CoV-2 (alpha to omicron variants) and does not detect other coronaviruses and pathogens by in silico and in vitro analysis. A two-step test protocol was developed with stable lyophilized reagents that reduces handling by using portable and disposable components (droppers, microapplicators/swabs, paper-strips). After optimization of assay components and conditions, we have achieved a limit of detection (LoD) of 1 copy/reaction by adding a blocking primer to the lateral flow assay. Using a set of 138 clinical samples, a sensitivity of 88.1% (P ​< ​0.05, CI: 78.2-93.8%) and specificity of 93.9% (P ​< ​0.05, CI: 85.4-97.6%) was determined. The lack of need for instrumentation for our CoRapID makes it an ideal on-site primary screening tool for local hospitals, doctors' offices, senior homes, workplaces, and in remote settings around the world that often do not have access to clinical laboratories.

PrimerBankID	Target Pathogen	Target Gene
RPB0020	SARS-CoV-2	N

Rapid, ultrasensitive and highly specific diagnosis of Mycoplasma pneumoniae by a CRISPR-based detection platform

Author(s):

Juan Zhou,Fei Xiao,Jin Fu,Nan Jia,Xiaolan Huang,Chunrong Sun,Zheng Xu,Yu Zhang,Dong Qu,Yi Wang

Journal:

Frontiers in Cellular and Infection Microbiology

Year:

2023

Abstract:

Mycoplasma pneumoniae (MP) is an important causative agent of morbidity and mortality among all age groups, especially among patients of extreme ages. Improved and readily available tests for accurate, sensitive and rapid diagnosis of MP infection is sorely needed. Here, we developed a CRISPR-Cas12b-based detection platform on the basis of recombinase polymerase amplification (RPA) for rapid, simple, and accurate diagnosis of MP infection, named MP-RPA-CRISPR. The RPA was employed for amplifying the community-acquired respiratory distress syndrome (CARDS) toxin gene of MP strains at the optimal reaction temperature 37°C. The resulting amplicons were decoded by the CRISPR-Cas12b-based detection platform, which was interpreted by real-time PCR system and by naked eye under blue light. The MP-RPA-CRISPR can detected down to 5 fg of genomic DNA templates of MP strains and accurately distinguish MP strains from non-MP strains without any cross-reactivity. A total of 96 bronchoalveolar lavage fluid (BALF)samples collected from patients suspected of respiratory infection were used to evaluate the clinical performance of the MP-RPA-CRISPR assay. As a result, our assay accurately diagnosed 45 MP-infected samples and 51 non-MP infected sample, and the results obtained from MP-RPA-CRISPR were consistent with microfluidic chip technology. In conclusion, our MP-RPA-CRISPR assay is a simple, rapid, portable and highly sensitive method to diagnose MP infection, which can be used as a promising tool in a variety of settings including clinical, field, and resource-limited aeras.

PrimerBankID	Target Pathogen	Target Gene
RPB0390	Mycobacterium	CARDS

A universal all-in-one RPA-Cas12a strategy with de novo autodesigner and its application in on-site ultrasensitive detection of DNA and RNA viruses

Author(s):

Cailing Lin,Feng Chen,Dongchao Huang,Wenyan Li,Changsheng He,Yingjun Tang,Xueping Li,Can Liu,Liya Han,Yunpeng Yang,Yongchong Zhu,Ruikang Chen,Yuanju Shi,Chenglai Xia,Zhibin Yan,Hongli Du,Lizhen Huang

Journal:

Biosensors and Bioelectronics

Year:

2023

Abstract:

Revolutionary all-in-one RPA-CRISPR assays are rapidly becoming the most sought-after tools for point-of-care testing (POCT) due to their high sensitivity and ease of use. Despite the availability of one-pot methods for specific targets, the development of more efficient methods for new targets remains a significant challenge. In this study, we present a rapid and universal approach to establishing an all-in-one RPA-Cas12a method CORDSv2 based on rational balancing amplification and Cas12a cleavage, which achieves ultrasensitive detection of several targets, including SARS-CoV-2, ASFV, HPV16, and HPV18. CORDSv2 demonstrates a limit of detection (LOD) of 0.6 cp/μL and 100% sensitivity for SARS-CoV-2, comparable to qPCR. Combining with our portable device(hippo-CORDS), it has a visual detection LOD of 6 cp/μL and a sensitivity up to 100% for SARS-CoV-2 and 97% for Ct<35 ASFV samples, surpassing most one-pot visual methods. To simplify and accelerate the process for new targets, we also develop a de novo autodesigner by which the optimal couples of primers and crRNA can be selected rapidly. As a universal all-in-one RPA-CRISPR method for on-site testing, CORDSv2 becomes an attractive choice for rapid and accurate diagnosis in resource-limited settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0406	SARS-CoV-2	N gene

Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system

Author(s):

Shuang Liu,Siyuan Huang,Fang Li,Yuanyuan Sun,Jin Fu,Fei Xiao,Nan Jia,Xiaolan Huang,Chunrong Sun,Juan Zhou,Yi Wang,Dong Qu

Journal:

Frontiers in cellular and infection microbiology

Year:

2023

Abstract:

Pseudomonas aeruginosa (P. aeruginosa) is an important bacterial pathogen involved in a wide range of infections and antimicrobial resistance. Rapid and reliable diagnostic methods are of vital important for early identification, treatment, and stop of P. aeruginosa infections. In this study, we developed a simple, rapid, sensitive, and specific detection platform for P. aeruginosa infection diagnosis. The method integrated recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 12a (Cas12a) biosensing system and was termed P. aeruginosa-CRISPR-RPA assay. The P. aeruginosa-CRISPR-RPA assay was subject to optimization of reaction conditions and evaluation of sensitivity, specificity, and clinical feasibility with the serial dilutions of P. aeruginosa genomic DNA, the non-P. aeruginosa strains, and the clinical samples. As a result, the P. aeruginosa-CRISPR-RPA assay was able to complete P. aeruginosa detection within half an hour, including RPA reaction at 42°C for 20 min and CRISPR-Cas12a detection at 37°C for 10 min. The diagnostic method exhibited high sensitivity (60 fg per reaction, ~8 copies) and specificity (100%). The results of the clinical samples by P. aeruginosa-CRISPR-RPA assay were consistent to that of the initial result by microfluidic chip method. These data demonstrated that the newly developed P. aeruginosa-CRISPR-RPA assay was reliable for P. aeruginosa detection. In summary, the P. aeruginosa-CRISPR-RPA assay is a promising tool to early and rapid diagnose P. aeruginosa infection and stop its wide spread especially in the hospital settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0124	Pseudomonas aeruginosa	oprL gene

Genetic identification of Staphylococcus aureus isolates from cultured milk samples of bovine mastitis using isothermal amplification with CRISPR/Cas12a-based molecular assay

Author(s):

Meruyert Amanzholova,Aisha Shaizadinova,Aitbay Bulashev,Sailau Abeldenov

Journal:

Veterinary research communications

Year:

2023

Abstract:

Bovine mastitis, a common and costly disease in dairy cattle, is primarily caused by Staphylococcus aureus. Timely and accurate detection of this pathogen is crucial for effective disease management. In this study, we developed and validated a novel molecular diagnostic assay based on the CRISPR/Cas12a system coupled with Recombinase Polymerase Amplification (RPA) and Loop-Mediated Isothermal Amplification (LAMP). We utilized specific primers targeting the nucleotide sequences of the S.aureus genes of interest, such as nuc and sea. RPA/LAMP reactions were performed under optimized conditions, and the resulting products were subsequently subjected to CRISPR/Cas12a detection. The CRISPR/Cas12a assay successfully detected the target nuc and sea genes, with a limit of detection of 104 and 102 gene copies per reaction, respectively. All 13 S.aureus clinical isolates were identified by RPA-CRISPR/Cas12a assay. The total reaction time is approximately 1 h. The assay demonstrated high sensitivity for the detection of S.aureus in both laboratory and clinical samples.

PrimerBankID	Target Pathogen	Target Gene
RPB0152	Staphylococcus aureus	nuc
RPB0330	Staphylococcus aureus	nuc gene

CRISPR dual enzyme cleavage triggers DNA and RNA substrate cleavage for SARS-CoV-2 dual gene detection

Author(s):

Tong Jiang, Runde Liu, Jilu Shen

Journal:

Journal Of Medical Virology

Year:

2023

Abstract:

The widespread dissemination of coronavirus 2019 imposes a significant burden on society. Therefore, rapid detection facilitates the reduction of transmission risk. In this study, we proposed a multiplex diagnostic platform for the rapid, ultrasensitive, visual, and simultaneous detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 1ab (ORF1ab) and N genes. A visual diagnostic method was developed using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a/Cas13a dual-enzyme digestion system integrated with multiplex reverse transcriptase-recombinase polymerase amplification (RT-RPA). Two CRISPR-Cas proteins (Cas12a and Cas13a) were introduced into the system to recognize and cleave the N gene and ORF1ab gene, respectively. We used fluorescent or CRISPR double digestion test strips to detect the digested products, with the N gene corresponding to the FAM channel in the PCR instrument or the T1 line on the test strip, and the ORF1ab gene corresponding to the ROX channel in the PCR instrument or the T2 line on the test strip. The analysis can be completed in less than 20 min. Meanwhile, we assessed the application of the platform and determined a sensitivity of up to 200 copies/mL. Additionally, dual gene validation in 105 clinical nasopharyngeal swab samples showed a 100% positive predictive value agreement and a 95.7% negative predictive value agreement between our method and quantitative reverse transcription-polymerase chain reaction. Overall, our method offered a novel insight into the rapid diagnosis of SARS-CoV-2.

PrimerBankID	Target Pathogen	Target Gene
RPB0001	SARS-CoV-2	N
RPB0002	SARS-CoV-2	ORF1ab

One-tube RPA-CRISPR Cas12a/Cas13a rapid detection of methicillin-resistant Staphylococcus aureus

Author(s):

Yujie Liu,Hui Liu,Guanliu Yu,Wenbo Sun,Muhammad Aizaz,Guiwen Yang,Lei Chen

Journal:

Analytica chimica acta

Year:

2023

Abstract:

At present, methicillin-resistant Staphylococcus aureus (MRSA) has caused a serious impact on a global scale. The infection and carrier rate of MRSA in the community is increasing year by year, but there is still no convenient detection system for on-site rapid detection. It is very important to select a rapid detection system to accurately and quickly detect patients infected with MRSA. We have developed a high-efficient single-tube detection platform based on RPA and CRISPR reaction system to detect the genes of mecA and clfA of MRSA. Using this detection platform, visual MRSA detection could be achieved in 30 min. It was observed that this detection platform was capable to successfully detect the target genomic as low as 5 copies μL-1, and the reaction was completed in one step without opening the lid. This detection platform could only detect MRSA, but not other common clinical pathogenic bacteria, such as Salmonella, Pseudomonas aeruginosa, Staphylococcus xylosus, Aeromonas hydrophila, Escherichia coli and Staphylococcus warneri, indicated its satisfactory selectivity for MRSA without interference from other bacteria. The results of clinical samples show that the platform has outstanding advantages in sensitivity, specificity and identification of methicillin resistance. The entire reaction can be completed in one step in the handheld instrument without opening the cover, avoiding aerosol pollution during the reaction. The detection platform combined with handheld instruments will have great application potential in point-of-care testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0130	Staphylococcus aureus	clfA
RPB0131	Staphylococcus aureus	mecA

Development and preliminary assessment of a CRISPR-Cas12a-based multiplex detection of Mycobacterium tuberculosis complex

Author(s):

Jing Xiao,Jieqiong Li,Shuting Quan,Yacui Wang,Guanglu Jiang,Yi Wang,Hairong Huang,Weiwei Jiao,Adong Shen

Journal:

Frontiers in Bioengineering and Biotechnology

Year:

2023

Abstract:

Since the onset of the COVID-19 pandemic in 2020, global efforts towards tuberculosis (TB) control have encountered unprecedented challenges. There is an urgent demand for efficient and cost-effective diagnostic technologies for TB. Recent advancements in CRISPR-Cas technologies have improved our capacity to detect pathogens. The present study established a CRISPR-Cas12a-based multiplex detection (designated as MCMD) that simultaneously targets two conserved insertion sequences (IS6110 and IS1081) to detect Mycobacterium tuberculosis complex (MTBC). The MCMD integrated a graphene oxide-assisted multiplex recombinase polymerase amplification (RPA) assay with a Cas12a-based trans-cleavage assay identified with fluorescent or lateral flow biosensor (LFB). The process can be performed at a constant temperature of around 37°C and completed within 1 h. The limit of detection (LoD) was 4 copies μL-1, and no cross-reaction was observed with non-MTBC bacteria strains. This MCMD showed 74.8% sensitivity and 100% specificity in clinical samples from 107 patients with pulmonary TB and 40 non-TB patients compared to Xpert MTB/RIF assay (63.6%, 100%). In this study, we have developed a straightforward, rapid, highly sensitive, specific, and cost-effective assay for the multiplex detection of MTBC. Our assay showed superior diagnostic performance when compared to the widely used Xpert assay. The novel approach employed in this study makes a substantial contribution to the detection of strains with low or no copies of IS6110 and facilitates point-of-care (POC) testing for MTBC in resource-limited countries.

PrimerBankID	Target Pathogen	Target Gene
RPB0376	Mycobacterium tuberculosis	IS6110
RPB0377	Mycobacterium tuberculosis	IS1081

Rapid Detection of blaKPC in Carbapenem-Resistant Enterobacterales Based on CRISPR\Cas13a

Author(s):

Mingjun Liang,Bin Xiao,Lidan Chen,Xiaoyan Huang,Jinchao Li,Zhenzhan Kuang,Xinping Chen,Xiuna Huang,Zhaohui Sun,Linhai Li

Journal:

Current Microbiology

Year:

2023

Abstract:

Klebsiella pneumoniae carbapenemase (KPC) is a crucial enzyme that causes carbapenem resistance in Enterobacterales, and infections by these "superbugs" are extremely challenging to treat. Therefore, there is a pressing need for a rapid and accurate KPC detection test to control the prevalence of carbapenem-resistant Enterobacterales (CREs). In this study, we established a novel method for detection of blaKPC, the gene responsible for encoding KPC, based on a recombinase polymerase amplification (RPA) and a CRISPR/Cas13a reaction coupled to fluorophore activation (termed RPA-Cas13a assay). We carefully selected a pair of optimal amplification primers for blaKPC and achieved a lower limit of detection of approximately 2.5 copies/μL by repeatedly amplifying a recombinant plasmid containing blaKPC. The RPA-Cas13a assay demonstrated a sensitivity of 96.5% and specificity of 100% when tested on 57 blaKPC-positive CRE strains, which were confirmed by DNA sequencing. Moreover, in 311 sputum samples, the theoretical antibiotic resistance characteristics of blaKPC-positive strains obtained by the RPA-Cas13a assay were highly consistent with the results of antibiotic susceptibility test (Kappa = 0.978 > 0.81, P < 0.01). In conclusion, the RPA-Cas13a system is a simple and one-hour efficient technology for the detection of a potentially fatal antibiotic resistance gene.

PrimerBankID	Target Pathogen	Target Gene
RPB0402	Klebsiella pneumoniae	bla KPC

Infectious Disease Diagnosis and Pathogen Identification Platform Based on Multiplex Recombinase Polymerase Amplification-Assisted CRISPR-Cas12a System

Author(s):

Ziqin Lin,Baochang Sun,Xi Yang,Yayun Jiang,Sihong Wu,Binbin Lv,Yajing Pan,Qingxun Zhang,Xiaoqiong Wang,Guangxin Xiang,Yongliang Lou,Xingxing Xiao

Journal:

ACS Infectious Diseases

Year:

2023

Abstract:

Controlling and mitigating infectious diseases caused by multiple pathogens or pathogens with several subtypes require multiplex nucleic acid detection platforms that can detect several target genes rapidly, specifically, sensitively, and simultaneously. Here, we develop a detection platform, termed Multiplex Assay of RPA and Collateral Effect of Cas12a-based System (MARPLES), based on multiplex nucleic acid amplification and Cas12a ssDNase activation to diagnose these diseases and identify their pathogens. We use the clinical specimens of hand, foot, and mouth disease (HFMD) and influenza A to evaluate the feasibility of MARPLES in diagnosing the disease and identifying the pathogen, respectively, and find that MARPLES can accurately diagnose the HFMD associated with enterovirus 71, coxsackievirus A16 (CVA16), CVA6, or CVA10 and identify the exact types of H1N1 and H3N2 in an hour, showing high sensitivity and specificity and 100% predictive agreement with qRT-PCR. Collectively, our findings demonstrate that MARPLES is a promising multiplex nucleic acid detection platform for disease diagnosis and pathogen identification.

PrimerBankID	Target Pathogen	Target Gene
RPB0378	EV-A71	VP1 gene
RPB0379	CVA16	VP4 gene
RPB0380	Influenza A virus (H1N1)	HA
RPB0381	Influenza A virus (H1N1)	NA
RPB0382	Influenza A virus (H3N2)	HA
RPB0383	Influenza A virus (H3N2)	NA
RPB0529	Vibrio vulnificus	vvhA gene
RPB0530	Aeromonas hydrophila	clone Ah563 aerolysin (aerA) gene
RPB0531	Vibrio vulnificus	vvhA gene
RPB0532	Aeromonas hydrophila strain Til1	16S ribosomal RNA gene
RPB0533	eromonas hydrophila	clone F108AH1 hemolysin gene
RPB0534	enterovirus 71	EV71 VP1
RPB0535	coxsackievirus A16	CVA16 VP4
RPB0536	coxsackievirus A10	CVA10 VP1
RPB0537	coxsackievirus A6	CVA6 VP1

Establishment of two assays based on reverse transcription recombinase-aided amplification technology for rapid detection of H5 subtype avian influenza virus

Author(s):

Yang Li,Jiajing Shang,Yixin Wang,Juan Luo,Wenming Jiang,Xin Yin,Fuyou Zhang,Chunran Deng,Xiaohui Yu,HuaLei Liu

Journal:

Microbiology Spectrum

Year:

2023

Abstract:

Avian influenza virus (AIV) subtype H5 is a highly contagious zoonotic disease and a serious threat to the farming industry and public health. Traditional detection methods, including virus isolation and real-time PCR, require tertiary biological laboratories and are time-consuming and complex to perform, making it difficult to rapidly diagnose H5 subtype avian influenza viruses. In this study, we successfully developed two methods, namely, RF-RT-RAA and RT-RAA-LFD, for rapid detection of H5-AIV. The assays are characterized by their high specificity, sensitivity, and user-friendliness. Moreover, the results of the reaction can be visually assessed, which are suitable for both laboratory testing and grassroots farm screening for H5-AIV.

PrimerBankID	Target Pathogen	Target Gene
RPB0421	Influenza A virus (H5)	HA gene

A new method for the detection of Mycobacterium tuberculosis based on the CRISPR\Cas system

Author(s):

Xiaoyu Zhang,Xiaoying He,Yubo Zhang,Lei Chen,Zhaobao Pan,Yueying Huang,Heng Li

Journal:

BMC Infectious Diseases

Year:

2023

Abstract:

Object: Mycobacterium tuberculosis (MTB) is a bacterium that can cause zoonoses by aerosol transmission. Tuberculosis (TB) caused by MTB heavily burdens world public health security. Developing efficient, specific, convenient, and inexpensive MTB assays are essential for preventing and controlling TB. Methods: In this study, we established a specific detection method for MTB using the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) system, combined with recombinase mediated isothermal nucleic acid amplification (RAA) to improve the sensitivity of the detection system and achieve "two-level" amplification of the detection signal. The sensitivity and specificity of RAA combined with the CRISPR/Cas system were analyzed. Using BACTEC 960 culture as the gold standard for detecting MTB, we established the TB-CRISPR technique by testing 504 samples from patients with suspected TB. Results: MTB H37Ra could be seen as low as 3.13 CFU/mL by the CRISPR-Cas12a system targeting IS6110. With BACTEC960 culture (120 positives and 384 negatives) as the gold standard, the sensitivity of the TB-CRISPR technique was 0.883 (0.809-0.932), and the specificity was 0.940 (0.910-0.961). According to the receiver operating characteristic (ROC) curve analysis, the area under the curve (AUC) reached 0.944 (0.914-0.975) within 95% CI. The positive likelihood ratio (PLR) was 14.747 (9.870-22.035), and the negative likelihood ratio (NLR) was 0.124 (0.076-0.203). The positive predictive value (PPV) was 0.822 (0.742-0.881), and the negative predictive value (NPV) was 0.963 (0.937-0.979). Conclusion: TB-CRISPR plays an essential role in the molecular diagnosis of TB. The whole detection time is less than 1.5 h. It is easy to operate and does not need complex instruments. It is of great significance for the rapid detection of MTB and the clinical diagnosis of TB.

PrimerBankID	Target Pathogen	Target Gene
RPB0397	Mycobacterium tuberculosis	IS6110
RPB0398	Mycobacterium tuberculosis	IS1081

Multiplex bacteria detection using one-pot CRISPR\Cas13a-based droplet microfluidics

Author(s):

Yuting Shang,Gaowa Xing,Jiaxu Lin,Yuxuan Li,Yongning Lin,Shulang Chen,Jin-Ming Lin

Journal:

Biosensors and Bioelectronics

Year:

2024

Abstract:

High-throughput detection of bacteria at low levels is critical in public health, food safety, and first response. Herein, for the first time, we present a platform based on droplet microfluidics coupling with the recombinase aided amplification (RAA)-assisted one-pot clustered regularly interspaced short palindromic repeats together with CRISPR-associated proteins 13a (CRISPR/Cas13a) assay, and droplet encoding strategy for accurate and sensitive determination of nucleic acids from various foodborne pathogens. The workflow takes full advantage of CRISPR/Cas13a signal amplification and droplet confinement effects, which enhances the detection sensitivity and enables end-point quantitation. Meanwhile, by varying the color of droplets, the number of bacteria detected at the same time is greatly improved. It possesses the capability to simultaneously detect seven different types of foodborne pathogens. Notably, the system is also applied to real food samples with satisfactory results. Overall, in view of superiorities in high sensitivity, outstanding selectivity, and large-scale multiplexing, the one-pot CRISPR/Cas13a-based droplet microfluidic system could be expanded and universalized for identifying other bacteria.

PrimerBankID	Target Pathogen	Target Gene
RPB0277	Pseudomonas aeruginosa	\
RPB0278	Staphylococcus aureus	\

CRISPR-Cas13a-based detection method for avian influenza virus

Author(s):

Yuhan Wu,Jiaxing Zhan,Zhaomeng Shan,Yanbing Li,Yining Liu,Yan Li,Yixin Wang,Zhe Liu,Xuexia Wen,Xiurong Wang

Journal:

Frontiers in Microbiology

Year:

2023

Abstract:

Avian influenza virus (AIV) causes huge losses to the global poultry industry and poses a threat to humans and other mammals. Fast, sensitive, and portable diagnostic methods are essential for efficient avian influenza control. Here, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a based platform was developed to detect AIV. This novel method was developed to specifically detect H1-H16 subtypes of AIV with fluorescence and lateral flow-based readouts and exhibited no cross-reactivity with Newcastle disease virus, avian infectious bronchitis virus, or infectious bursal disease virus. The limit of detection was determined to be 69 and 690 copies/μL using fluorescence and lateral flow as readouts, respectively. The developed assay exhibited 100% consistency with quantitative real-time polymerase chain reaction in detecting clinical samples. The heating of unextracted diagnostic samples to obliterate nuclease treatment was introduced to detect viral RNA without nucleic acid extraction. Single-step optimization was used to perform reverse transcription, recombinase polymerase amplification, and CRISPR-Cas13a detection in a tube. These advances resulted in an optimized assay that could specifically detect AIV with simplified procedures and reduced contamination risk, highlighting the potential to be used in point-of-care testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0385	Influenza A virus (H1)	M gene

A Blood Drying Process for DNA Amplification

Author(s):

Jongwon Lim,Shuaizhen Zhou,Janice Baek,Alicia Yeaeun Kim,Enrique Valera,Jonathan Sweedler,Rashid Bashir

Journal:

small

Year:

2023

Abstract:

The presence of numerous inhibitors in blood makes their use in nucleic acid amplification techniques difficult. Current methods for extracting and purifying pathogenic DNA from blood involve removal of inhibitors, resulting in low and inconsistent DNA recovery rates. To address this issue, a biphasic method is developed that simultaneously achieves inhibitor inactivation and DNA amplification without the need for a purification step. Inhibitors are physically trapped in the solid-phase dried blood matrix by blood drying, while amplification reagents can move into the solid nano-porous dried blood and initiate the amplification. It is demonstrated that the biphasic method has significant improvement in detection limits for bacteria such as Escherichia coli, Methicillin-resistant Staphylococcus aureus, Methicillin-Sensitive Staphylococcus aureus using loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). Several factors, such as drying time, sample volume, and material properties are characterized to increase sensitivity and expand the application of the biphasic assay to blood diagnostics. With further automation, this biphasic technique has the potential to be used as a diagnostic platform for the detection of pathogens eliminating lengthy culture steps.

PrimerBankID	Target Pathogen	Target Gene
RPB0412	Staphylococcus aureus	vicK gene

Rapid and sensitive detection of Pseudomonas aeruginosa by isothermal amplification combined with Cas12a-mediated detection

Author(s):

Siyi Huang,Xianfeng Wang,Xinchong Chen,Xiaoyu Liu,Qiuqing Xu,Lijun Zhang,Guangtao Huang,Jun Wu

Journal:

Scientific Reports

Year:

2023

Abstract:

CRISPR based technologies have been used for fast and sensitive detection of pathogens. To test the possibility of CRISPR based detection strategy in Pseudomonas aeruginosa infections, a combined method of recombinase polymerase amplification followed by Cas12a-mediated detection via fluorescence reader or lateral flow biosensor (named Cas12a-RCFL) has been established in this study. The Cas12a-RCFL can detect as low as 50 CFU/mL Pseudomonas aeruginosa. The whole detection process can be finished within one hour with satisfied detection specificity. Cas12a-RCFL also shows good sensitivity of detecting Pseudomonas aeruginosa inStaphylococcus aureus and Acinetobacter baumannii contaminated samples. For the detection of 22 clinical samples, Cas12a-RCFL matches with PCR sequencing result exactly without DNA purification. This Cas12a-RCFL is rapid and sensitive with low cost, which shows good quality to be adopted as a point-of-care testing method.

PrimerBankID	Target Pathogen	Target Gene
RPB0366	Pseudomonas aeruginosa	\

Development of a rapid, sensitive detection method for SARS-CoV-2 and influenza virus based on recombinase polymerase amplification combined with CRISPR-Cas12a assay

Author(s):

Yuning Wang,Liqiang Wu,Xiaomei Yu,Gang Wang,Ting Pan,Zhao Huang,Ting Cui,Tianxun Huang,Zhentao Huang,Libo Nie,Chungen Qian

Journal:

Journal of Medical Virology

Year:

2023

Abstract:

Respiratory tract infections are associated with the most common diseases transmitted among people and remain a huge threat to global public health. Rapid and sensitive diagnosis of causative agents is critical for timely treatment and disease control. Here, we developed a novel method based on recombinase polymerase amplification (RPA) combined with CRISPR-Cas12a to detect three viral pathogens, including SARS-CoV-2, influenza A, and influenza B, which cause similar symptom complexes of flu cold in the respiratory tract. The detection method can be completed within 1 h, which is faster than other standard detection methods, and the limit of detection is approximately 102 copies/μL. Additionally, this detection system is highly specific and there is no cross-reactivity with other common respiratory tract pathogens. Based on this assay, we further developed a more simplified RPA/CRISPR-Cas12a system combined with lateral flow assay on a manual microfluidic chip, which can simultaneously detect these three viruses. This low-cost detection system is rapid and sensitive, which could be applied in the field and resource-limited areas without bulky and expensive instruments, providing powerful tools for the point-of-care diagnostic.

PrimerBankID	Target Pathogen	Target Gene
RPB0367	SARS-CoV-2	N gene
RPB0368	Influenza A virus	Matrix gene
RPB0369	Influenza B virus	NS gene

Establishment of RT-RPA-Cas12a assay for rapid and sensitive detection of human rhinovirus B

Author(s):

Yongdong Li,Xuefei Wang,Rong Xu,Ting Wang,Dandan Zhang,Weidong Qian

Journal:

BMC Microbiology

Year:

2023

Abstract:

Human rhinovirus B (HRV-B) is a major human viral pathogen that can be responsible for various kinds of infections. Due to the health risks associated with HRV-B, it is therefore crucial to explore a rapid, specific, and sensitive method for surveillance. Herein, we exploited a novel detection method for HRV-B by combining reverse-transcription recombinase polymerase amplification (RT-RPA) of nucleic acids isothermal amplification and the trans-cleavage activity of Cas12a. Our RT-RPA-Cas12a-based fluorescent assay can be completed within 35-45 min and obtain a lower detection threshold to 0.5 copies/µL of target RNA. Meanwhile, crRNA sequences without a specific protospacer adjacent motif can effectively activate the trans-cleavage activity of Cas12a. Moreover, our RT-RPA-Cas12a-based fluorescent method was examined using 30 clinical samples, and exhibited high accuracy with positive and negative predictive agreement of 90% and 100%, respectively. Taken together, a novel promising, rapid and effective RT-RPA-Cas12a-based detection method was explored and shows promising potential for on-site HRV-B infection in resource-limited settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0364	HRV	VP4 gene

Rapid detection of avian influenza virus based on CRISPR-Cas12a

Author(s):

Xu Zhou,Siwen Wang,Yue Ma,Yanbing Li,Guohua Deng,Jianzhong Shi,Xiurong Wang

Journal:

Virology Journal

Year:

2023

Abstract:

Background: Avian influenza (AI) is a disease caused by the avian influenza virus (AIV). These viruses spread naturally among wild aquatic birds worldwide and infect domestic poultry, other birds, and other animal species. Currently, real-time reverse transcription polymerase chain reaction (rRT-PCR) is mainly used to detect the presence of pathogens and has good sensitivity and specificity. However, the diagnosis requires sophisticated instruments under laboratory conditions, which significantly limits point-of-care testing (POCT). Rapid, reliable, non-lab-equipment-reliant, sensitive, and specific diagnostic tests are urgently needed for rapid clinical detection and diagnosis. Our study aimed to develop a reverse transcription recombinase polymerase amplification (RT-RPA)/CRISPR method which improves on these limitations. Methods: The Cas12a protein was purified by affinity chromatography with Ni-agarose resin and observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Specific CRISPR RNA (crRNA) and primers targeting the M and NP genes of the AIV were designed and screened. By combining RT-RPA with the Cas12a/crRNA trans-cleavage system, a detection system that uses fluorescence readouts under blue light or lateral flow strips was established. Sensitivity assays were performed using a tenfold dilution series of plasmids and RNA of the M and NP genes as templates. The specificity of this method was determined using H1-H16 subtype AIVs and other avian pathogens, such as newcastle disease virus (NDV), infectious bursal disease virus (IBDV), and infectious bronchitis virus (IBV). Results: The results showed that the method was able to detect AIV and that the detection limit can reach 6.7 copies/μL and 12 copies/μL for the M and NP gene, respectively. In addition, this assay showed no cross-reactivity with other avian-derived RNA viruses such as NDV, IBDV, and IBV. Moreover, the detection system presented 97.5% consistency and agreement with rRT-PCR and virus isolation for detecting samples from poultry. This portable and accurate method has great potential for AIV detection in the field. Conclusion: An RT-RPA/CRISPR method was developed for rapid, sensitive detection of AIV. The new system presents a good potential as an accurate, user-friendly, and inexpensive platform for point-of-care testing applications.

PrimerBankID	Target Pathogen	Target Gene
RPB0386	Influenza A virus	M gene
RPB0387	Influenza A virus	NP gene

CRISPR\Cas13-assisted carbapenem-resistant Klebsiella pneumoniae detection

Author(s):

Yaling Cao,Yuan Tian,Jing Huang,Ling Xu,Zihao Fan,Zhenzhen Pan,Sisi Chen,Yao Gao,Linlin Wei,Sujun Zheng,Xiangying Zhang,Yanhua Yu,Feng Ren

Journal:

Journal of Microbiology,Immunology and Infection

Year:

2024

Abstract:

Background/purpose: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is capable of causing serious community and hospital-acquired infections. However, currently, the identification of CRKP is complex and inefficient. Hence, this study aimed to develop methods for the early and effective identification of CRKP to allow reasonable antimicrobial therapy in a timely manner. Methods: K. pneumoniae (KP)-, K. pneumoniae carbapenemase (KPC)- and New Delhi metallo-β-lactamase (NDM)- specific CRISPR RNAs (crRNAs), polymerase chain reaction (PCR) primers and recombinase-aided amplification (RAA) primers were designed and screened in conserved sequence regions. We established fluorescence and lateral flow strip assays based on CRISPR/Cas13a combined with PCR and RAA, respectively, to assist in the detection of CRKP. Sixty-one clinical strains (including 51 CRKP strains and 10 carbapenem-sensitive strains) were collected for clinical validation. Results: Using the PCR-CRISPR assay, the limit of detection (LOD) for KP and the blaKPC and blaNDM genes reached 1 copy/μL with the fluorescence signal readout. Using the RAA-CRISPR assay, the LOD could reach 101 copies/μL with both the fluorescence signal readout and the lateral flow strip readout. Additionally, the positivity rates of CRKP-positive samples detected by the PCR/RAA-CRISPR fluorescence and RAA-CRISPR lateral flow strip methods was 92.16% (47/51). The sensitivity and specificity reached 100% for KP and blaKPC and blaNDM gene detection. For detection in a simulated environmental sample, 1 CFU/cm2 KP could be detected. Conclusion: We established PCR/RAA-CRISPR assays for the detection of blaKPC and blaNDM carbapenemase genes, as well as KP, to facilitate the detection of CRKP.

PrimerBankID	Target Pathogen	Target Gene
RPB0238	Klebsiella pneumoniae	blaKPC gene
RPB0239	Klebsiella pneumoniae	blaNDM gene

Development of a novel integrated isothermal amplification system for detection of bacteria-spiked blood samples

Author(s):

Jin Li,Mei-Yun Shang,Shao-Li Deng,Min Li,Ning Su,Xiao-Dong Ren,Xian-Ge Sun,Wen-Man Li,Yu-Wei Li,Ruo-Xu Li,Qing Huang,Wei-Ping Lu

Journal:

AMB Express

Year:

2023

Abstract:

Bloodstream infection (BSI) caused by bacteria is highly pathogenic and lethal, and easily develops whole-body inflammatory state. Immediate identification of disease-causing bacteria can improve patient prognosis. Traditional testing methods are not only time-consuming, but such tests are limited to laboratories. Recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) holds great promise for rapid nucleic acid detection, but the uncapping operation after amplification easily contaminates laboratories. Therefore, the establishment of a more effective integrated isothermal amplification system has become an urgent problem to be solved. In this study, we designed and fabricated a hermetically sealed integrated isothermal amplification system. Combining with this system, a set of RPA-LFD assays for detecting S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI were established and evaluated. The whole process could be completed in less than 15 min and the results can be visualized by the naked eye. The developed RPA-LFD assays displayed a good sensitivity, and no cross-reactivity was observed in seven similar bacterial genera. The results obtained with 60 clinical samples indicated that the developed RPA-LFD assays had high specifcity and sensitivity for identifying S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI. In conclusion, our results showed that the developed RPA-LFD assay is an alternative to existing PCR-based methods for detection of S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI in primary hospitals.

PrimerBankID	Target Pathogen	Target Gene
RPB0427	Staphylococcus aureus	Nuc
RPB0428	Klebsiella pneumoniae	CelB
RPB0429	Pseudomonas aeruginosa	Eta
RPB0430	Haemophilus influenzae	Fuck

Development of a recombinase-aided amplification combined with a lateral flow dipstick assay for rapid detection of H7 subtype avian influenza virus

Author(s):

Fuyou Zhang,Jiajing Shang,Juan Luo,Xin Yin,Xiaohui Yu,Wenming Jiang,Jinping Li,Liping Yuan,Guangyu Hou,Hualei Liu,Yang Li

Journal:

Frontiers in Microbiology

Year:

2023

Abstract:

Avian influenza viruses (AIV) pose a significant persistent threat to the public health and safety. It is estimated that there have been over 100 outbreaks caused by various H7 subtypes of avian influenza viruses (AIV-H7) worldwide, resulting in over 33 million deaths of poultry. In this study, we developed a recombinase-aided amplification combined with a lateral flow dipstick assay for the detection of hemagglutinin (HA) genes to provide technical support for rapid clinical detection of AIV-H7. The results showed that the assay can complete the reaction within 30 min at a temperature of 39°C. Specificity tests demonstrated that there was no cross-reactivity with other common poultry pathogens, including Newcastle disease virus (NDV) and infections bronchitis virus (IBV). The detection limit of this assay was 1 × 101 copies/μL, while RT-qPCR method was 1 × 101 copies/μL, and RT-PCR was 1 × 102 copies/μL. The κ value of the RT-RAA-LFD and RT-PCR assay in 132 avian clinical samples was 0.9169 (p < 0.001). These results indicated that the developed RT-RAA-LFD assay had good specificity, sensitivity, stability and repeatability and may be used for rapid detection of AIV-H7 in clinical diagnosis.

PrimerBankID	Target Pathogen	Target Gene
RPB0420	Influenza A virus (H7)	HA