Development of propidium monoazide-recombinase polymerase amplification (PMA-RPA) assay for rapid detection of Streptococcus pyogenes and Streptococcus agalactiae

Author(s):

Jing Chen,Yuanyang Wang,Xiaoqing Liu,Guopei Chen,Xuejian Chen,Jiaping Chen,Zhongdong Liu,Jingwen Gong,Guowu Yang,Quanxue Lan

Journal:

Molecular and cellular probes

Year:

2018

Abstract:

Streptococcus pyogenes (Group A Streptococcus, GAS) and Streptococcus agalactiae (Group B Streptococcus, GBS) are common pathogens that threaten public health. In this study, a double recombinase polymerase (RPA) amplification assay was developed to rapidly detect these pathogens. Specificity tests revealed that the GAS and GBS strains were positive for speB and SIP genes, respectively. In clinical samples, the double assay performed similarly to the traditional biochemical method. The limits of detection were both ≤100 copies per reaction. In tests for simulant-contaminated samples, bacterial-culture media containing 103 CFU/mL original concentrations of S. pyogenes and S. agalactiae were positive in RPA assays after incubating for 4 h. Results can be obtained at 37 °C in 20 min. To determine whether propidium monoazide (PMA) can eliminate the influence of DNA extracted from dead cells, a bacterial suspension was treated with PMA before DNA extraction. Findings of RPA assay showed that DNA extracted from dead cells had no fluorescence signal. Therefore, the PMA-RPA assay is a promising technology for field tests and rapid point-of-care diagnosis.

PrimerBankID	Target Pathogen	Target Gene
RPB0166	Streptococcus pyogenes	speB
RPB0167	Streptococcus pyogenes	SIP
RPB0512	Group A Streptococcus	speB
RPB0513	Group B Streptococcus	SIP

Rapid Detection of Avian Influenza A Virus (H7N9) by Lateral Flow Dipstick Recombinase Polymerase Amplification

Author(s):

Shiwei Ma,Xue Li,Bo Peng,Weihua Wu,Xin Wang,Hui Liu,Lihong Yuan,Shisong Fang,Jiahai Lu

Journal:

Biological & pharmaceutical bulletin

Year:

2018

Abstract:

Avian influenza A (H7N9) virus has caused several epidemics and infection in both human and poultry. With mutation, the H7N9 virus gained its fifth endemic in China. Early diagnosis is crucial for the control of viral spread in poultry and prognosis of infected patients. In this study, we developed and evaluated a lateral flow dipstick recombinase polymerase amplification (LFD-RPA) assay for rapid detection of both hemagglutinin and neuraminidase gene of H7N9. Our H7-LFD-RPA and N9-LFD-RPA assay were able to detect 32 fg H7N9 nucleic acid which is more convenient and rapid than previous methods. Through detecting 50 influenza positive samples, cross-reaction was not found with other subtypes of influenza virus. The 100% analytical specificity and sufficient analytical sensitivity results agreed the real time RT-PCR assay. The results data demonstrated that our method performed well and could be applied to the detection of H7N9 virus. This LFD-RPA assay provides a candidate method for rapid point-of-care diagnosis of H7N9.

PrimerBankID	Target Pathogen	Target Gene
RPB0101	Influenza A virus (H7N9)	HA gene
RPB0102	Influenza A virus (H7N9)	NA gene

Reverse transcription recombinase polymerase amplification with lateral flow dipsticks for detection of influenza A virus and subtyping of H1 and H3

Author(s):

Ning Sun,Weiping Wang,Jie Wang,Xinyue Yao,Fangfang Chen,Xiaojun Li,Yi Yinglei,Bo Chen

Journal:

Molecular and cellular probes

Year:

2018

Abstract:

Three reverse transcription recombinase polymerase amplification assays with lateral flow dipsticks (RT-RPA-LFD) were developed for identification of the matrix and hemagglutinin (HA) genes to detect influenza A virus and distinguish subtypes H1 and H3. Assessment of the assays' specificity showed that there was no cross-reactivity with other targets. Their limits of detection were 123.6 copies per reaction for the matrix gene, 677.1 copies per reaction for the H1 HA gene, and 112.2 copies/reaction for the H3 HA gene. Of 111 samples tested by RT-RPA-LFD assays, 27 were positive for influenza A virus, 14 were positive for H1, and 10 were positive for H3. Compared to the results obtained from real-time RT-PCR assays, the sensitivity of RT-RPA-LFD assays was 75%, 93.33% and 71.43% for the matrix, H1, and H3, with 100% specificity. The sensitivity of RT-RPA-LFD assays is lower than that of real-time RT-PCR, comparable or better than that of conventional RT-PCR, and much better than that of RIDTs. In conclusion, these assays offer an efficient and reliable tool for identification and subtyping of influenza A virus (subtype H1 and H3) in the resource-limited setting.

PrimerBankID	Target Pathogen	Target Gene
RPB0098	Influenza A virus	matrix gene
RPB0099	Influenza A virus	H1
RPB0100	Influenza A virus	H3

Rapid detection of Mycobacterium ulcerans with isothermal recombinase polymerase amplification assay

Author(s):

Michael Frimpong,Hubert Senanu Ahor,Ahmed Abd El Wahed,Bernadette Agbavor,Francisca Naana Sarpong,Kenneth Laing,Mark Wansbrough-Jones,Richard Odame Phillips

Journal:

PLoS neglected tropical diseases

Year:

2019

Abstract:

Background: Access to an accurate diagnostic test for Buruli ulcer (BU) is a research priority according to the World Health Organization. Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect Mycobacterium ulcerans (M. ulcerans), the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Isothermal DNA amplification systems such as the recombinase polymerase amplification (RPA) have emerged as a molecular diagnostic tool with similar accuracy to PCR but having the advantage of amplifying a template DNA at a constant lower temperature in a shorter time. The aim of this study was to develop RPA for the detection of M. ulcerans and evaluate its use in Buruli ulcer disease. Methodology and principal findings: A specific fragment of IS2404 of M. ulcerans was amplified within 15 minutes at a constant 42°C using RPA method. The detection limit was 45 copies of IS2404 molecular DNA standard per reaction. The assay was highly specific as all 7 strains of M. ulcerans tested were detected, and no cross reactivity was observed to other mycobacteria or clinically relevant bacteria species. The clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. All results were compared to a highly sensitive real-time PCR. The clinical specificity of the Mu-RPA assay was 100% (95% CI, 84-100), whiles the sensitivity was 88% (95% CI, 77-95). Conclusion: The Mu-RPA assay represents an alternative to PCR, especially in areas with limited infrastructure.

PrimerBankID	Target Pathogen	Target Gene
RPB0118	Mycobacterium ulcerans	IS2404

Development of a reverse transcription recombinase polymerase amplification assay for rapid detection of human respiratory syncytial virus.

Author(s):

Xi, Yun; Xu, Chang-Zhi; Xie, Zhi-Zhi; Zhu, Dong-Lin; Dong, Jie-Ming; Xiao, Gang;

Journal:

MOL CELL PROBE

Year:

2019

Abstract:

Respiratory syncytial virus (RSV) is one of the most important causative agents that causing respiratory tract infection in children and associated with high morbidity and mortality. A diagnostic method would be a robust tool for identification of RSV infection, especially in the resource-limited settings. Recombinase polymerase amplification (RPA) is a novel isothermal amplification technique which has been widely employed to detect human/animal pathogens. In present study, a probe-based reverse transcription RPA (RT-RPA) assay was established for the detection of RSV. The primers and probe were designed based on the sequences of the conserved nucleocapsid (N) gene. The minimal detection limit of the RT-RPA assay for the detection of RSV B was 19 copies of RNA molecules at 95% probability, whereas the detection limit for RSV A was 104 copies molecule. The assay was RSV-specific since it had no non-specific reactions with other common human pathogens. The clinical performance of the RT-RPA assay was validated using 188 nasopharyngeal aspirates (NPAs). The nucleic acid extraction of the samples was performed by use of the magnetic bead-based kit which didn't require the heavy and expensive centrifuge. The coincidence rates between RT-RPA and qRT-PCR for the clinical samples was 96%, indicating the RT-RPA assay had good diagnostic performance on clinical samples. The real-time RT-RPA assay combined with the manual genome extraction method make it potential to detect clinical samples in field, providing a possible solution for RSV diagnosis in remote rural areas in developing countries.

PrimerBankID	Target Pathogen	Target Gene
RPB0050	RSV A/B	N gene
RPB0129	RSV	(N) gene

Utilization of recombinase polymerase amplification method combined with lateral flow dipstick for visual detection of respiratory syncytial virus.

Author(s):

Xu, Yu-Zhong; Fang, Du-Zhi; Chen, Fang-Fang; Zhao, Qin-Fei; Cai, Chao-Ming; Cheng, Ming-Gang;

Journal:

MOL CELL PROBE

Year:

2020

Abstract:

Respiratory syncytial virus (RSV) is a major causative agent of respiratory tract infection necessitating hospitalization in children. A rapid diagnostic method would facilitate early detection of RSV infection and timely implementation of special treatment. Here, a reverse transcription recombinase polymerase amplification (RT-RPA) assay combined with lateral flow dipstick (LFD) was evaluated for rapid visual detection of RSV. The primers were designed to target the conserved L gene. The RT-RPA-LFD assay could simultaneously detect RSV subtype A and B with the same detection limit of 10 copies of a given RNA molecule. Moreover, the assay showed no cross-reactivity with other common human pathogens. The performance of the RT-RPA-LFD assay was evaluated by testing 136 nasopharyngeal aspirates (NPAs). The agreement of the detection results between RT-RPA-LFD and qRT-PCR was 100% (34 positive and 102 negative cases). In summary, the developed RT-RPA-LFD assay had good performance in detecting RSV in clinical specimens, thus providing a novel alternative solution for the detection of RSV under field conditions.

PrimerBankID	Target Pathogen	Target Gene
RPB0049	RSV A/B	UTR gene
RPB0128	RSV	conserved L gene

Rapid Detection of SARS-CoV-2 by Low Volume Real-Time Single Tube Reverse Transcription Recombinase Polymerase Amplification Using an Exo Probe with an Internally Linked Quencher (Exo-IQ).

Author(s):

Behrmann, Ole; Bachmann, Iris; Spiegel, Martin; Schramm, Marina; Abd El Wahed, Ahmed; Dobler, Gerhard; Dame, Gregory; Hufert, Frank T;

Journal:

CLIN CHEM

Year:

2020

Abstract:

BACKGROUND:The current outbreak of SARS-CoV-2 has spread to almost every country with more than 5 million confirmed cases and over 300,000 deaths as of May 26, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance. METHODS:We used computational and manual designs to generate a suitable set of reverse transcription recombinase polymerase amplification (RT-RPA) primer and exonuclease probe, internally quenched (exo-IQ), sequences targeting the SARS-CoV-2 N gene. RT-RPA sensitivity was determined by amplification of in vitro transcribed RNA standards. Assay selectivity was demonstrated with a selectivity panel of 32 nucleic acid samples derived from common respiratory viruses. To validate the assay against full-length SARS-CoV-2 RNA, total viral RNA derived from cell culture supernatant and 19 nasopharyngeal swab samples (8 positive and 11 negative for SARS-CoV-2) were screened. All results were compared to established RT-qPCR assays. RESULTS:The 95% detection probability of the RT-RPA assay was determined to be 7.74 (95% CI: 2.87-27.39) RNA copies per reaction. The assay showed no cross-reactivity to any other screened coronaviruses or respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n = 20). CONCLUSIONS:With a run time of 15 to 20 minutes and first results being available in under 7 minutes for high RNA concentrations, the reported assay constitutes one of the fastest nucleic acid based detection methods for SARS-CoV-2 to date and may provide a simple-to-use alternative to RT-qPCR for first-line screening at the point of need.

PrimerBankID	Target Pathogen	Target Gene
RPB0079	SARS-CoV-2	N gene

An Isothermal Method for Sensitive Detection of Mycobacterium tuberculosis Complex Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a Cis and Trans Cleavage

Author(s):

Haipo Xu,Xiaolong Zhang,Zhixiong Cai,Xiuqing Dong,Geng Chen,Zhenli Li,Liman Qiu,Lei He,Bin Liang,Xiaolong Liu,Jingfeng Liu

Journal:

The Journal of molecular diagnostics : JMD

Year:

2020

Abstract:

Tuberculosis is one of the most serious infectious diseases, resulting in death worldwide. Traditional detection methods are not enough to meet the clinical requirements of rapid diagnosis, high specificity, and high sensitivity. Fast, sensitive, and accurate detection of Mycobacterium tuberculosis (MTB) is urgently needed to treat and control tuberculosis disease. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas12a) exhibit strong nonspecific degradation ability of exogenous single-strand nucleic acids (trans cleavage) after specific recognition of target sequence. We purified Cas12a protein and selected a proper guide RNA based on conserved sequences of MTB from designed guide RNA library. Then, we proposed a novel detection method based on recombinase polymerase amplification and CRISPR/Cas12a nuclease system for specific and sensitive detection of MTB DNA. The assay, based on fluorescence detection, showed 4.48 fmol/L of limit of detection and good linear correlation of concentration with fluorescence value (R2 = 0.9775). It also showed good performance in distinguishing other bacteria. Furthermore, its clinical performance was evaluated by 193 samples and showed sensitivity of 99.29% (139/140) and specificity of 100% (53/53) at 99% CI, compared with culture method. Taken together, the CRISPR/Cas12a system showed good specificity, excellent sensitivity, and excellent accuracy for MTB detection, and it meets requirements of MTB detection in clinical samples and has great potential for clinical translation.

PrimerBankID	Target Pathogen	Target Gene
RPB0117	Mycobacterium	IS1081

Recombinase polymerase amplification with polymer flocculation sedimentation for rapid detection of Staphylococcus aureus in food samples

Author(s):

Jinqiang Hu,Yi Wang,Huimin Ding,Chunpeng Jiang,Yao Geng,Xincheng Sun,Jianzhou Jing,Hui Gao,Zhangcun Wang,Caiwen Dong

Journal:

International journal of food microbiology

Year:

2020

Abstract:

Currently, rapid, sensitive, and convenient visual detection methods for Staphylococcus aureus (S. aureus) are scarce. In this study, a novel detection method based on recombinase polymerase amplification (RPA) and polymer flocculation sedimentation (PFS) was developed. Twelve effective primer combinations derived from four forward primers F1, F2, F3, F4, and three reverse primers R1, R2, R3 targeting the nuc gene of S. aureus were designed and screened by a polymerase chain reaction and RPA methods. RPA reaction conditions, including temperature, time, and volume as well as PEG8000 and NaCl concentrations range, were optimized. Moreover, the specificity and sensitivity of the RPA-PFS assay were further analyzed. Finally, the potential use of the RPA-PFS assay was evaluated using artificially S. aureus contaminated food samples, including pork, beef, shrimp, fish, cheese, cabbage, leftover rice, egg, milk, and orange juice. Results showed that the SA5 (F2/R2) combination was the optimal primer candidate. The optimal temperature range, the shortest time and the minimal volume of RPA reaction were 40-42 °C, 10 min and 10 μL, respectively and the optimal PEG8000/NaCl concentrations were 0.2 g/mL and 2.5 M, respectively, for the adsorption between magnetic beads and RPA products. The RPA-PFS method could detect as little as 13 fg genomic DNA of S. aureus and was also specific for five target S. aureus as well as twenty-seven non-target foodborne bacteria. The limit of detection of RPA-PFS for S. aureus in artificially contaminated food samples was 38 CFU/mL (g). Besides, RPA-PFS has directly been judged by the naked eye and has totally taken less than 20 min. In short, the assay RPA-PFS developed in this study is a rapid, sensitive, and specific visual detection method for S. aureus.

PrimerBankID	Target Pathogen	Target Gene
RPB0159	Staphylococcus aureus	nuc gene

Ultra-sensitive and high-throughput CRISPR-p owered COVID-19 diagnosis.

Author(s):

Huang, Zhen; Tian, Di; Liu, Yang; Lin, Zhen; Lyon, Christopher J; Lai, Weihua; Fusco, Dahlene; Drouin, Arnaud; Yin, Xiaoming; Hu, Tony; Ning, Bo;

Journal:

BIOSENS BIOELECTRON

Year:

2020

Abstract:

Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.

PrimerBankID	Target Pathogen	Target Gene
RPB0077	SARS-CoV-2	ORF1ab and N gene
RPB0078	SARS-CoV-2	N gene