Rapid detection of Mycobacterium tuberculosis using recombinase polymerase amplification: A pilot study

Author(s):

Michael Sciaudone,Renzo Carpena,Maritza Calderón,Patricia Sheen,Mirko Zimic,Jorge Coronel,Robert H Gilman,Natalie M Bowman

Journal:

PLOS ONE

Year:

2023

Abstract:

Tuberculosis remains one of the leading causes of death worldwide, especially in low- and middle-income countries. Tuberculosis treatment and control efforts are hindered by the difficulty in making the diagnosis, as currently available diagnostic tests are too slow, too expensive, or not sufficiently sensitive. Recombinase polymerase amplification (RPA) is a novel technique that allows for the amplification of DNA rapidly, at constant temperature, and with minimal expense. We calculated and compared the limit of detection, sensitivity, and specificity of two RPA-based assays for the diagnosis of pulmonary tuberculosis, using two sets of published primers. We also calculated and compared the assays' limits of detection and compared their performance using two different DNA extraction methods prior to amplification (a commercially available DNA extraction kit vs. the chelex method). The RPA-lateral flow assay had a limit of detection of 5 fg/μL of DNA, a sensitivity of 53.2%, and a specificity of 93.3%, while the real time-RPA assay had a limit of detection of 25 fg/μL of DNA, a sensitivity of 85.1%, and a specificity of 93.3%. There was no difference in assay performance when DNA extraction was carried out using the commercial kit vs. the chelex method. The real-time RPA assay has adequate sensitivity and specificity for the diagnosis of pulmonary tuberculosis and could be a viable diagnostic tool in resource-limited settings, but the lateral flow assay did not perform as well, perhaps due to the fact we used stored sputum specimens from a biorepository. More work is needed to optimize the RPA-lateral flow assay, to get a more accurate estimate of its specificity and sensitivity using prospectively collected specimens, and to develop both assays into point-of-care tests that can be easily deployed in the field.

PrimerBankID	Target Pathogen	Target Gene
RPB0354	Mycobacterium tuberculosis	IS1081
RPB0355	Mycobacterium tuberculosis	IS6110

Rapid detection of H5 subtype avian influenza virus using CRISPR Cas13a based-lateral flow dipstick

Author(s):

Yang Li,Jiajing Shang,Juan Luo,Fuyou Zhang,Ge Meng,Yingjie Feng,Wenming Jiang,Xiaohui Yu,Chunran Deng,Guanhui Liu,Hualei Liu

Journal:

Frontiers in Microbiology

Year:

2023

Abstract:

Due to its high mortality rate, highly pathogenic avian influenza (HPAI), a notifiable animal illness designated by the World Organisation for Animal Health (WOAH), has caused enormous financial losses to the poultry sector. The H5 subtype of avian influenza virus (H5-AIV) is regarded as the most common highly pathogenic avian influenza virus (HPAIV) that threatens public health and safety. Virus isolation and reverse transcription quantitative PCR (RT-qPCR) are usually used to detect H5-AIV and are important for the timely diagnosis and control of H5-AIV. However, these methods are time-consuming and require a significant amount of effort. In this study, we established a recombinase-aided amplification (RAA) combined with CRISPR-Cas13a and lateral flow dipstick (LFD) assay for the detection of H5-AIV. The results showed that the process can be completed within 40 min at 37°C. The method had a detection limit of 0.1 copy/μL, which was comparable to the RT-qPCR. There was no cross-reactivity with H3-AIV, H7-AIV, H9-AIV, H10-AIV, IBV, NDV, RVA and DAstV. The kappa value of RT-RAA-Cas13a-LFD and RT-qPCR in 380 clinical samples was 0.89 (κ>0.75). In conclusion, we established a convenient, efficient and accurate method to detect H5-AIV, and the results can be visualized and interpreted using LFD, which can be adapted to the needs of grassroots laboratories and field-deployable assays. This approach provides a new perspective for clinical H5-AIV diagnosis and has great potential for application in clinical quarantine of the poultry farming.

PrimerBankID	Target Pathogen	Target Gene
RPB0431	Influenza A virus (H5)	HA gene

Isothermal Amplification and CRISPR\Cas12a-System-Based Assay for Rapid, Sensitive and Visual Detection of Staphylococcus aureus

Author(s):

Danhong Xu,Haijuan Zeng,Wenhui Wu,Hua Liu,Jinbin Wang

Journal:

Foods

Year:

2023

Abstract:

Staphylococcus aureus exists widely in the natural environment and is one of the main food-borne pathogenic microorganisms causing human bacteremia. For safe food management, a rapid, high-specificity, sensitive method for the detection of S. aureus should be developed. In this study, a platform for detecting S. aureus (nuc gene) based on isothermal amplification (loop-mediated isothermal amplification-LAMP, recombinase polymerase amplification-RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas12a) proteins system (LAMP, RPA-CRISPR/Cas12a) was proposed. In this study, the LAMP, RPA-CRISPR/Cas12a detection platform and immunochromatographic test strip (ICS) were combined to achieve a low-cost, simple and visualized detection of S. aureus. The limit of visual detection was 57.8 fg/µL of nuc DNA and 6.7 × 102 CFU/mL of bacteria. Moreover, the platform could be combined with fluorescence detection, namely LAMP, RPA-CRISPR/Cas12a-flu, to establish a rapid and highly sensitive method for the detection of S. aureus. The limit of fluorescence detection was 5.78 fg/µL of genomic DNA and 67 CFU/mL of S. aureus. In addition, this detection platform can detect S. aureus in dairy products, and the detection time was ~40 min. Consequently, the isothermal amplification CRISPR/Cas12a platform is a useful tool for the rapid and sensitive detection of S. aureus in food.

PrimerBankID	Target Pathogen	Target Gene
RPB0356	Staphylococcus aureus	nuc gene

Rapid detection of human adenovirus subgroup B using recombinase polymerase amplification assay

Author(s):

Yongzhe Zhu,Binghui Xia,Haizhou Xu,Zengxin Liu,Ru Wang,Qingqing Cai,Ping Zhao,Zhongtian Qi

Journal:

Virus Genes

Year:

2024

Abstract:

Human adenovirus subgroup B (HAdV B) is one of the major pathogens of human respiratory virus infections, which has considerable transmission and morbidity in a variety of populations. Therefore, rapid and specific detection of HAdV B in clinical samples is essential for diagnosis. This study aimed to develop a product for rapid nucleic acid detection of HAdV B using recombinase polymerase amplification assay (RPA) and validate the performance of this method by using clinical samples. Results showed that this method achieved a lower limit of detection (LOD) of 10 copies/μL and had no cross-reactivity with other adenovirus subgroups or respiratory pathogens. In addition to high sensitivity, it can be completed within 30 min at 40 °C. There is no need to perform nucleic acid extraction on clinical samples. Taking qPCR as the gold standard, the RPA assay possessed a high concordance (Cohen's kappa, 0.896; 95% CI 0.808-0.984; P < 0.001), with a sensitivity of 87.80% and a specificity of 100.00%. The RPA assay developed in this study provided a simple and highly specific method, making it an important tool for rapid adenovirus nucleic acid detection and facilitating large-scale population screening in resource-limited settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0236	HAdV	HAdV B 1A（E1A）gene

Establishment of portable Pseudomonas aeruginosa detection platform based on one-tube CRISPR\Cas12a combined with recombinase polymerase amplification technology

Author(s):

Haitao Yang,Aibo Liu,Fenfen Ma,Xuzhu Gao,Kun Wang,Yan Wang

Journal:

Clinica Chimica Acta

Year:

2024

Abstract:

Pseudomonas aeruginosa, a common Gram-negative bacterium, is associated with diverse diseases. Its increasing resistance to antibiotics presents challenges in clinical treatment. The predominant diagnostic approach involves conventional biochemical cultures, known for their time and labor intensiveness. Despite progress in isothermal amplification studies, limitations persist, including reliance on specialized equipment, intricate primer design, and aerosol contamination. Therefore, there is a demand for enhanced clinical assays. This study successfully combined RPA and CRISPR/Cas12a techniques. Through a series of experiments involving the design and screening of lasB crRNA, the creation of lasB RPA primers, and the establishment of a streamlined RPA-CRISPR/Cas12a assay, the study developed a one-tube detection method targeting P. aeruginosa's lasB gene. The assay demonstrated inclusive behavior across standard and 21 isolates, while specifically discerning P. aeruginosa from diverse strains. Sensitivity reached 15.9 CFU/reaction. Clinical validation revealed a 97.62% concordance with traditional methods. The one-tube assay's protocol mitigated aerosol contamination. Offering precision, specificity, and sensitivity, this method shows promise for field applications in resource-scarce regions, enabling early detection and improved management of P. aeruginosa infections.

PrimerBankID	Target Pathogen	Target Gene
RPB0268	Pseudomonas aeruginosa	lasB gene

Efficient magnetic enrichment cascade single-step RPA-CRISPR\Cas12a assay for rapid and ultrasensitive detection of Staphylococcus aureus in food samples

Author(s):

Yujie Ma,Hongjuan Wei,Yunxiang Wang,Xiaodan Cheng,Hong Chen,Xingsheng Yang,Hongsheng Zhang,Zhen Rong,Shengqi Wang

Journal:

Journal Of Hazardous Materials

Year:

2024

Abstract:

Staphylococcus aureus (S. aureus) is a prevalent foodborne pathogen that could cause severe food poisoning. Thus, rapid, efficient, and ultrasensitive detection of S. aureus in food samples is urgently needed. Here, we report an efficient magnetic enrichment cascade single-step recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay for the ultrasensitive detection of S. aureus. Magnetic beads (MBs) functionalized with S. aureus-specific antibodies were initially used for S. aureus enrichment from the complex matrix, with 98% capture efficiency in 5 min and 100-fold sensitivity improvement compared with unenriched S. aureus. Next, a single-step RPA-CRISPR/Cas12a-based diagnostic system with optimized extraction-free bacteria lysis was constructed. This assay could detect as low as 1 copy/μL (five copies/reaction) of extracted DNA template and 10 CFU/mL of S. aureus within 40 min. Furthermore, the assay could effectively detect S. aureus in real food samples such as lake water, orange juice, pork, and lettuce, with concordant results to qPCR assays. The proposed cascade signal-amplification assay eliminates the need for lengthy bacterial culture and complex sample preparation steps. Hence, the proposed assay shows great application potential for rapid, efficient, and ultrasensitive detection of pathogens in real food samples.

PrimerBankID	Target Pathogen	Target Gene
RPB0276	Staphylococcus aureus	nuc gene

detecting RSV and ADV is rapid, sensitive, and specific.

Author(s):

Gao Hongdan,Du Yao,Chai Qiang,Huang Meng,Liu Xiaorong,Xing Zhihao,Ma Dongli

Journal:

Journal Of International Medical Research

Year:

2024

Abstract:

Objective: Respiratory syncytial virus (RSV) and respiratory adenovirus (ADV) are two common pathogens that cause acute respiratory tract infections in children. We aimed to develop a rapid method for detecting both pathogens simultaneously. Methods: The recombinase polymerase isothermal amplification (RPA) method was combined with the CRISPR/Cas detection system. The assay's specificity and sensitivity were explored by designing RPA primers and CRISPR RNAs (crRNAs) through multi-sequence comparisons, optimizing the reaction conditions, and using a fluorescent reading device. The consistency of the test results of 160 clinical pharyngeal swab samples was studied using quantitative polymerase chain reaction (qPCR) results as a comparative control. Results: RSV and ADV could be detected at levels as low as 104 copies/mL and 103 copies/mL, respectively, within 50 minutes with no cross-reactivity with other similar pathogens. For the clinical samples, compared with the qPCR method, the sensitivities for RSV and ADV were 98.1% and 91.4%, respectively, and the detection specificities were both 100%. The Kappa values were greater than 0.95, suggesting a high degree of consistency. Conclusion: This method for detecting RSV and ADV is rapid, sensitive, and specific. It can accurately detect mixed infections in a timely manner, making it suitable for use in areas with scarce healthcare resources.

PrimerBankID	Target Pathogen	Target Gene
RPB0229	RSV	N protein gene
RPB0230	AdV	Hexon protein gene

Based on CRISPR-Cas13a system, to establish a rapid visual detection method for avian influenza viruses

Author(s):

Zongshu Zhang,Chunguang Wang,Xi Chen,Zichuang Zhang,Guoqiang Shi,Xianghe Zhai,Tie Zhang

Journal:

Frontiers in Veterinary Science

Year:

2024

Abstract:

To rapidly, specifically, and sensitively detect avian influenza virus (AIV), this research established a visual detection method of recombinase-aided amplification (RAA) based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated proteins 13a (Cas13a) system. In this study, specific primers and CRISPR RNA (crRNA) were designed according to the conservative sequence of AIV Nucleprotein (NP) gene. RAA technology was used to amplify the target sequence, and the amplification products were visually detected by lateral flow dipstick (LFD). The specificity, sensitivity, and reproducibility of RAA-CRISPR-Cas13a-LFD were evaluated. At the same time, this method and polymerase chain reaction (PCR)-agarose electrophoresis method were used to detect clinical samples, and the coincidence rate of the two detection methods was calculated. The results showed that the RAA-CRISPR-Cas13a-LFD method could achieve specific amplification of the target gene fragments, and the detection results could be visually observed through the LFD. Meanwhile, there was no cross-reaction with infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), and Newcastle disease virus (NDV). The sensitivity reached 100 copies/μL, which was 1,000-fold higher than that of PCR-agarose electrophoresis method. The coincidence rate of clinical tests was 98.75 %, and the total reaction time was ~1 h. The RAA-CRISPR-Cas13a-LFD method established in this study had the advantages of rapid, simple, strong specificity, and high sensitivity, which provided a new visual method for AIV detection.

PrimerBankID	Target Pathogen	Target Gene
RPB0296	Influenza A virus	Nucleprotein (NP) gene

Rapid and sensitive detection of nucleic acids using an RAA-CRISPR\Cas12b one-pot detection assay (Rcod)

Author(s):

Kangfeng Lin,Kaihu Yao,Xiao Li,Qinghan Li,Xiangju Guo,Weixin You,Wenjing Ren,Ya Bian,Jianguang Guo,Zhen Sun,Rui Zhang,Xiaoqing Yang,Zhiyong Li,Boan Li

Journal:

Talanta

Year:

2024

Abstract:

Rapid, sensitive and specific methods are crucial for nucleic acid detection. CRISPR/Cas12b has recently been widely used in nucleic acid detection. However, due to its thermophagic property, DNA isothermal recombinase-aided amplification (RAA) and subsequent CRISPR/Cas12b detection require two separate reactions, which is cumbersome and inconvenient and may cause aerosol pollution. In this study, we propose an RAA-CRISPR/Cas12b one-pot detection assay (Rcod) for Bordetella pertussis detection without additional amplification product transfer steps. The time from sample processing to response time was less than 30 min using nucleic acid extraction-free method, and the sensitivity reached 0.2 copies/μL. In this system, Alicyclobacillus acidoterrestris Cas12b protein (AacCas12b) exhibited strong and specific trans-cleavage activity at a constant temperature of 37 °C, while the cis-cleavage activity was weak. This characteristic reduces the interference of AacCas12b with nucleic acids in the system. Compared with real-time PCR, our Rcod system detected B. pertussis in 221 clinical samples with a sensitivity and specificity of 97.96 % and 99.19 %, respectively, with nucleic acid extraction-free method. The rapid, sensitive and specific Rcod system provides ideas for the establishment of CRISPR-based one-step nucleic acid detection and may aid the development of reliable point-of-care nucleic acid tests. IMPORTANCE: Pertussis is an acute respiratory infection caused by B. pertussis that is highly contagious and potentially fatal, and early diagnosis is essential for the treatment of whooping cough. In this study, we found that AacCas12b has high and strongly specific trans-cleavage activity at lower temperatures. A RAA-CRISPR/Cas12b one-step detection platform (Rcod) without interference with amplification was developed. In addition, the combination of Rcod and nucleic acid extraction-free method can quickly and accurately detect the qualitative detection of B. pertussis, and the detection results are visualized, which makes the pathogen nucleic acid detection and analysis process simpler, and provides a new method for the rapid clinical diagnosis of B. pertussis.

PrimerBankID	Target Pathogen	Target Gene
RPB0340	Bordetella pertussis CS	IS1663 gene

Multiplex solid-phase RPA coupled CRISPR-based visual detection of SARS-CoV-2

Author(s):

Xiaochen Qin,Ratul Paul,Yuyuan Zhou,Yue Wu,Xuanhong Cheng,Yaling Liu

Journal:

Biosens Bioelectron X

Year:

2023

Abstract:

The COVID-19 pandemic has presented a significant challenge to the world's public health and led to over 6.9 million deaths reported to date. A rapid, sensitive, and cost-effective point-of-care virus detection device is essential for the control and surveillance of the contagious severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. The study presented here aimed to demonstrate a solid-phase isothermal recombinase polymerase amplification coupled CRISPR-based (spRPA-CRISPR) assay for on-chip multiplexed, sensitive and visual COVID-19 DNA detection. The assay targets the SARS-CoV-2 structure protein encoded genomes and can simultaneously detect two specific genes without cross-interaction. The amplified target sequences were immobilized on the one-pot device surface and detected using the mixed Cas12a-crRNA collateral cleavage of reporter-released fluorescent signal when specific genes were recognized. The endpoint signal can be directly visualized for rapid detection of COVID-19. The system was tested with samples of a broad range of concentrations (20 to 2 × 104 copies) and showed analytical sensitivity down to 20 copies per microliter. Furthermore, a low-cost blue LED flashlight (~$12) was used to provide a visible SARS-CoV-2 detection signal of the spRPA-CRISPR assay which could be purchased online easily. Thus, our platform provides a sensitive and easy-to-read multiplexed gene detection method that can specifically identify low concentration genes.

PrimerBankID	Target Pathogen	Target Gene
RPB0394	SARS-CoV-2	E gene
RPB0395	SARS-CoV-2	N gene

A photocontrolled one-pot isothermal amplification and CRISPR-Cas12a assay for rapid detection of SARS-CoV-2 Omicron variants

Author(s):

Qian Sun,Hongqing Lin,Yuan Li,Liping Yuan,Baisheng Li,Yunan Ma,Haiying Wang,Xiaoling Deng,Hongliang Chen,Shixing Tang

Journal:

Microbiology Spectrum

Year:

2024

Abstract:

CRISPR-Cas technology has widely been applied to detect single-nucleotide mutation and is considered as the next generation of molecular diagnostics. We previously reported the combination of nucleic acid amplification (NAA) and CRISPR-Cas12a system to distinguish major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. However, the mixture of NAA and CRISPR-Cas12a reagents in one tube could interfere with the efficiency of NAA and CRISPR-Cas12a cleavage, which in turn affects the detection sensitivity. In the current study, we employed a novel photoactivated CRISPR-Cas12a strategy integrated with recombinase polymerase amplification (RPA) to develop one-pot RPA/CRISPR-Cas12a genotyping assay for detecting SARS-CoV-2 Omicron sub-lineages. The new system overcomes the potential inhibition of RPA due to early CRISPR-Cas12a activation and cleavage of the target template in traditional one-pot assay using photocleavable p-RNA, a complementary single-stranded RNA to specifically bind crRNA and precisely block Cas12a activation. The detection can be finished in one tube at 39℃ within 1 h and exhibits a low limit of detection of 30 copies per reaction. Our results demonstrated that the photocontrolled one-pot RPA/CRISPR-Cas12a assay could effectively identify three signature mutations in the spike gene of SARS-CoV-2 Omicron variant, namely, R346T, F486V, and 49X, and distinguish Omicron BA.1, BA.5.2, and BF.7 sub-lineages. Furthermore, the assay achieved a sensitivity of 97.3% and a specificity of 100.0% and showed a concordance of 98.3% with Sanger sequencing results.IMPORTANCEWe successfully developed one-pot recombinase polymerase amplification/CRISPR-Cas12a genotyping assay by adapting photocontrolled CRISPR-Cas technology to optimize the conditions of nucleic acid amplification and CRISPR-Cas12a-mediated detection. This innovative approach was able to quickly distinguish severe acute respiratory syndrome coronavirus 2 Omicron variants and can be readily modified for detecting any nucleic acid mutations. The assay system demonstrates excellent clinical performance, including rapid detection, user-friendly operations, and minimized risk of contamination, which highlights its promising potential as a point-of-care testing for wide applications in resource-limiting settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0252	SARS-CoV-2 (Omicron)	R346T
RPB0253	SARS-CoV-2 (Omicron)	F486V;49X

A CRISPR\Cas12 trans-cleavage reporter enabling label-free colorimetric detection of SARS-CoV-2 and its variants

Author(s):

Hansol Kim,Hyowon Jang,Jayeon Song,Sang Mo Lee,Seoyoung Lee,Hyung-Jun Kwon,Sunjoo Kim,Taejoon Kang,Hyun Gyu Park

Journal:

Biosensors and Bioelectronics

Year:

2024

Abstract:

We present a label-free colorimetric CRISPR/Cas-based method enabling affordable molecular diagnostics for SARS-CoV-2. This technique utilizes 3,3'-diethylthiadicarbocyanine iodide (DISC2(5)) which exhibits a distinct color transition from purple to blue when it forms dimers by inserting into the duplex of the thymidine adenine (TA) repeat sequence. Loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA) was used to amplify target samples, which were subsequently subjected to the CRISPR/Cas12a system. The target amplicons would activate Cas12a to degrade nearby TA repeat sequences, preserving DISC2(5) in its free form to display purple as opposed to blue in the absence of the target. Based on this design approach, SARS-CoV-2 RNA was colorimetrically detected very sensitively down to 2 copies/μL, and delta and omicron variants of SARS-CoV-2 were also successfully identified. The practical diagnostic utility of this method was further validated by reliably identifying 179 clinical samples including 20 variant samples with 100% clinical sensitivity and specificity. This technique has the potential to become a promising CRISPR-based colorimetric platform for molecular diagnostics of a wide range of target pathogens.

PrimerBankID	Target Pathogen	Target Gene
RPB0264	SARS-CoV-2	S gene

Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification

Author(s):

Xiao-Xuan Zhu,Ying-Si Wang,Su-Juan Li,Ru-Qun Peng,Xia Wen,Hong Peng,Qing-Shan Shi,Gang Zhou,Xiao-Bao Xie,Jie Wang

Journal:

Front. Microbiol

Year:

2024

Abstract:

The principal pathogen responsible for chronic urinary tract infections, immunocompromised hosts, and cystic fibrosis patients is Pseudomonas aeruginosa, which is difficult to eradicate. Due to the extensive use of antibiotics, multidrug-resistant P. aeruginosa has evolved, complicating clinical therapy. Therefore, a rapid and efficient approach for detecting P. aeruginosa strains and their resistance genes is necessary for early clinical diagnosis and appropriate treatment. This study combines recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats-association protein 13a (CRISPR-Cas13a) to establish a one-tube and two-step reaction systems for detecting the mexX gene in P. aeruginosa. The test times for one-tube and two-step RPA-Cas13a methods were 5 and 40 min (including a 30 min RPA amplification reaction), respectively. Both methods outperform Quantitative Real-time Polymerase Chain Reactions (qRT-PCR) and traditional PCR. The limit of detection (LoD) of P. aeruginosa genome in one-tube and two-step RPA-Cas13a is 10 aM and 1 aM, respectively. Meanwhile, the designed primers have a high specificity for P. aeruginosa mexX gene. These two methods were also verified with actual samples isolated from industrial settings and demonstrated great accuracy. Furthermore, the results of the two-step RPA-Cas13a assay could also be visualized using a commercial lateral flow dipstick with a LoD of 10 fM, which is a useful adjunt to the gold-standard qRT-PCR assay in field detection. Taken together, the procedure developed in this study using RPA and CRISPR-Cas13a provides a simple and fast way for detecting resistance genes.

PrimerBankID	Target Pathogen	Target Gene
RPB0249	Pseudomonas aeruginosa	mexX gene

Efficient detection of Streptococcus pyogenes based on recombinase polymerase amplification and lateral flow strip

Author(s):

Xu-Zhu Gao,Yu-Die Cao,Yu-Zhi Gao,Juan Hu,Tuo Ji

Journal:

European Journal of Clinical Microbiology & Infectious Diseases

Year:

2024

Abstract:

Purpose: This article aims to establish a rapid visual method for the detection of Streptococcus pyogenes (GAS) based on recombinase polymerase amplification (RPA) and lateral flow strip (LFS). Methods: Utilizing speB of GAS as a template, RPA primers were designed, and basic RPA reactions were performed. To reduce the formation of primer dimers, base mismatch was introduced into primers. The probe was designed according to the forward primer, and the RPA-LFS system was established. According to the color results of the reaction system, the optimum reaction temperature and time were determined. Thirteen common clinical standard strains and 14 clinical samples of GAS were used to detect the selectivity of this method. The detection limit of this method was detected by using tenfold gradient dilution of GAS genome as template. One hundred fifty-six clinical samples were collected and compared with qPCR method and culture method. Kappa index and clinical application evaluation of the RPA-LFS were carried out. Results: The enhanced RPA-LFS method demonstrates the ability to complete the amplification process within 6 min at 33 °C. This method exhibits a high analytic sensitivity, with the lowest detection limit of 0.908 ng, and does not exhibit cross-reaction with other pathogenic bacteria. Conclusions: The utilization of RPA and LFS allows for efficient and rapid testing of GAS, thereby serving as a valuable method for point-of-care testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0221	Streptococcus pyogenes	sepB
RPB0321	Streptococcus pyogenes M1 GAS	speB

Establishing a pulmonary aspergillus fumigatus infection diagnostic platform based on RPA-CRISPR-Cas12a

Author(s):

Chunhui Lin,Jing Zhou,Nana Gao,Runde Liu,Ge Li,Jinyu Wang,Guoping Lu,Jilu Shen

Journal:

World Journal of Microbiology and Biotechnology

Year:

2024

Abstract:

In this study, we devised a diagnostic platform harnessing a combination of recombinase polymerase amplification (RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. Notably, this platform obviates the need for intricate equipment and finds utility in diverse settings. Two result display methods were incorporated in this investigation: the RPA-Cas12a-fluorescence method and the RPA-Cas12a-LFS (lateral flow strip). Upon validation, both display platforms exhibited no instances of cross-reactivity, with seven additional types of fungal pathogens responsible for respiratory infections. The established detection limit was ascertained to be as low as 102 copies/µL. In comparison to fluorescence quantitative PCR, the platform demonstrated a sensitivity of 96.7%, a specificity of 100%, and a consistency rate of 98.0%.This platform provides expeditious, precise, and on-site detection capabilities, thereby rendering it a pivotal diagnostic instrument amenable for deployment in primary healthcare facilities and point-of-care settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0349	Aspergillus fumigatus	Beta-tubulin gene

Rapid Detection of Measles Virus Using Reverse Transcriptase\Recombinase Polymerase Amplification Coupled with CRISPR\Cas12a and a Lateral Flow Detection: A Proof-of-Concept Study

Author(s):

Elena Pinchon,Steven Henry,Fanny Leon,Chantal Fournier-Wirth,Vincent Foulongne,Jean-François Cantaloube

Journal:

Diagnostics

Year:

2024

Abstract:

The measles virus is highly contagious, and efforts to simplify its diagnosis are essential. A reverse transcriptase/recombinase polymerase amplification assay coupled with CRISPR/Cas12a and an immunochromatographic lateral flow detection (RT-RPA-CRISPR-LFD) was developed for the simple visual detection of measles virus. The assay was performed in less than 1 h at an optimal temperature of 42 °C. The detection limit of the assay was 31 copies of an RNA standard in the reaction tube. The diagnostic performances were evaluated on a panel of 27 measles virus RT-PCR-positive samples alongside 29 measles virus negative saliva samples. The sensitivity and specificity were 96% (95% CI, 81-99%) and 100% (95% CI, 88-100%), respectively, corresponding to an accuracy of 98% (95% CI, 94-100%; p < 0.0001). This method will open new perspectives in the development of the point-of-care testing diagnosis of measles.

PrimerBankID	Target Pathogen	Target Gene
RPB0243	Measles morbillivirus	N region
RPB0497	Measles Virus	Meas_NS1， Meas_NR1

SARS-CoV-2 recombinase polymerase amplification assay with lateral flow readout and duplexed full process internal control

Author(s):

Coleman D Martin,Andrew T Bender,Benjamin P Sullivan,Lorraine Lillis,David S Boyle,Jonathan D Posner

Journal:

Sensors & Diagnostics

Year:

2024

Abstract:

Nucleic acid amplification tests for the detection of SARS-CoV-2 have been an important testing mechanism for the COVID-19 pandemic. While these traditional nucleic acid diagnostic methods are highly sensitive and selective, they are not suited to home or clinic-based uses. Comparatively, rapid antigen tests are cost-effective and user friendly but lack in sensitivity and specificity. Here we report on the development of a one-pot, duplexed reverse transcriptase recombinase polymerase amplification SARS-CoV-2 assay with MS2 bacteriophage as a full process control. Detection is carried out with either real-time fluorescence or lateral flow readout with an analytical sensitivity of 50 copies per reaction. Unlike previously published assays, the RNA-based MS2 bacteriophage control reports on successful operation of lysis, reverse transcription, and amplification. This SARS-CoV-2 assay features highly sensitive detection, visual readout through an LFA strip, results in less than 25 minutes, minimal instrumentation, and a useful process internal control to rule out false negative test results.

PrimerBankID	Target Pathogen	Target Gene
RPB0247	SARS-CoV-2	(N) gene

Novel methods for the rapid and sensitive detection of Nipah virus based on a CRISPR\Cas12a system

Author(s):

Xi Yang,Kexin Xu,Siying Li,Jiangnian Zhang,Yinli Xie,Yongliang Lou,Xingxing Xiao

Journal:

Analyst

Year:

2024

Abstract:

Nipah virus (NiV), a bat-borne zoonotic viral pathogen with high infectivity and lethality to humans, has caused severe outbreaks in several countries of Asia during the past two decades. Because of the worldwide distribution of the NiV natural reservoir, fruit bats, and lack of effective treatments or vaccines for NiV, routine surveillance and early detection are the key measures for containing NiV outbreaks and reducing its influence. In this study, we developed two rapid, sensitive and easy-to-conduct methods, RAA-CRISPR/Cas12a-FQ and RAA-CRISPR/Cas12a-FB, for NiV detection based on a recombinase-aided amplification (RAA) assay and a CRISPR/Cas12a system by utilizing dual-labeled fluorophore-quencher or fluorophore-biotin ssDNA probes. These two methods can be completed in 45 min and 55 min and achieve a limit of detection of 10 copies per μL and 100 copies per μL of NiV N DNA, respectively. In addition, they do not cross-react with nontarget nucleic acids extracted from the pathogens causing similar symptoms to NiV, showing high specificity for NiV N DNA detection. Meanwhile, they show satisfactory performance in the detection of spiked samples from pigs and humans. Collectively, the RAA-CRISPR/Cas12a-FQ and RAA-CRISPR/Cas12a-FB methods developed by us would be promising candidates for the early detection and routine surveillance of NiV in resource-poor areas and outdoors.

PrimerBankID	Target Pathogen	Target Gene
RPB0352	Nipah virus	N gene

A multiplex RPA coupled with CRISPR-Cas12a system for rapid and cost-effective identification of carbapenem-resistant Acinetobacter baumannii

Author(s):

Zihan Zhou,Lina Liang,Chuan Liao,Lele Pan,Chunfang Wang,Jiangmei Ma,Xueli Yi,Meiying Tan,Xuebin Li,Guijiang Wei

Journal:

Frontiers in Microbiology

Year:

2024

Abstract:

Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a severe nosocomial threat, prompting a need for efficient detection methods. Traditional approaches, such as bacterial culture and PCR, are time-consuming and cumbersome. The CRISPR-based gene editing system offered a potential approach for point-of-care testing of CRAB. Methods: We integrated recombinase polymerase amplification (RPA) and CRISPR-Cas12a system to swiftly diagnose CRAB-associated genes, OXA-51 and OXA-23. This multiplex RPA-CRISPR-Cas12a system eliminates bulky instruments, ensuring a simplified UV lamp-based outcome interpretation. Results: Operating at 37°C to 40°C, the entire process achieves CRAB diagnosis within 90 minutes. Detection limits for OXA-51 and OXA-23 genes are 1.3 × 10-6 ng/μL, exhibiting exclusive CRAB detection without cross-reactivity to common pathogens. Notably, the platform shows 100% concordance with PCR when testing 30 clinical Acinetobacter baumannii strains. Conclusion: In conclusion, our multiplex RPA coupled with the CRISPR-Cas12a system provides a fast and sensitive CRAB detection method, overcoming limitations of traditional approaches and holding promise for efficient point-of-care testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0350	Acinetobacter baumannii	OXA-51
RPB0351	Acinetobacter baumannii	OXA-23

Detection of Staphylococcus aureus virulence gene pvl based on CRISPR strip

Author(s):

Li Jin,XiaoFeng Hu,Yuan Tian,MengYa Fang,Xue Dong,YaXuan Jiang,Yao Han,Hao Li,Yansong Sun

Journal:

Frontiers in Immunology

Year:

2024

Abstract:

Introduction:Staphylococcus aureus (S. aureus) is a prominent pathogen responsible for both hospital-acquired and community-acquired infections. Among its arsenal of virulence factors, Panton-Valentine Leucocidin (PVL) is closely associated with severe diseases such as profound skin infections and necrotizing pneumonia. Patients infected with pvl-positive S. aureus often exhibit more severe symptoms and carry a substantially higher mortality risk. Therefore, it is crucial to promptly and accurately detect pvl-positive S. aureus before initiating protective measures and providing effective antibacterial treatment. Methods: In this study, we propose a precise identification and highly sensitive detection method for pvl-positive S. aureus based on recombinase-assisted amplification and the CRISPR-ERASE strip which we previously developed. Results: The results revealed that this method achieved a detection limit of 1 copy/μL for pvl-positive plasmids within 1 hour. The method successfully identified all 25 pvl-positive and 51 pvl-negative strains among the tested 76 isolated S. aureus samples, demonstrating its concordance with qPCR. Discussion: These results show that the CRISPR-ERASE detection method for pvl-positive S. aureus has the advantages of high sensitivity and specificity, this method combines the characteristics of recombinase-assisted amplification at room temperature and the advantages of ERASE test strip visualization, which can greatly reduce the dependence on professional laboratories. It is more suitable for on-site detection than PCR and qPCR, thereby providing important value for rapid on-site detection of pvl.

PrimerBankID	Target Pathogen	Target Gene
RPB0266	Staphylococcus aureus	pvl
RPB0267	Staphylococcus aureus	clfA

Simple, Visual, Point-of-Care SARS-CoV-2 Detection Incorporating Recombinase Polymerase Amplification and Target DNA–Protein Crosslinking Enhanced Chemiluminescence

Author(s):

Hui Chen,Zhiyuan Zhuang,Naihan Xu,Ying Feng,Kaixin Fang,Chunyan Tan,Ying Tan

Journal:

Biosensors

Year:

2024

Abstract:

The ongoing COVID-19 pandemic, driven by persistent SARS-CoV-2 transmission, threatens human health worldwide, underscoring the urgent need for an efficient, low-cost, rapid SARS-CoV-2 detection method. Herein, we developed a point-of-care SARS-CoV-2 detection method incorporating recombinase polymerase amplification (RPA) and DNA-protein crosslinking chemiluminescence (DPCL) (RPADPCL). RPADPCL involves the crosslinking of biotinylated double-stranded RPA DNA products with horseradish peroxidase (HRP)-labeled streptavidin (SA-HRP). Modified products are captured using SA-labeled magnetic beads, and then analyzed using a chemiluminescence detector and smartphone after the addition of a chemiluminescent substrate. Under optimal conditions, the RPADPCL limit of detection (LOD) was observed to be 6 copies (within the linear detection range of 1-300 copies) for a plasmid containing the SARS-CoV-2 N gene and 15 copies (within the linear range of 10-500 copies) for in vitro transcribed (IVT) SARS-CoV-2 RNA. The proposed method is convenient, specific, visually intuitive, easy to use, and does not require external excitation. The effective RPADPCL detection of SARS-CoV-2 in complex matrix systems was verified by testing simulated clinical samples containing 10% human saliva or a virus transfer medium (VTM) spiked with a plasmid containing a SARS-CoV-2 N gene sequence or SARS-CoV-2 IVT RNA. Consequently, this method has great potential for detecting targets in clinical samples.

PrimerBankID	Target Pathogen	Target Gene
RPB0240	SARS-CoV-2	SARS-CoV-2 N gene

A Multiplex Recombinase-Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC-2, and blaNDM-1 Genes in Klebsiella pneumoniae

Author(s):

Shao-Wei Hua,Jie Wang,Zi-Jin Zhao,Feng-Yu Tian,Meng Zhao,Yu-Xin Wang,Rui-Qing Zhang,Zhi-Qiang Han,Shi-Jue Gao,Xiao-Na Lv,Hong-Yi Li,Xin-Xin Shen,Xue-Jun Ma,Zhi-Shan Feng

Journal:

Clinical Laboratory Analysis

Year:

2024

Abstract:

Objective: This study aimed to establish a highly sensitive and rapid single-tube, two-stage, multiplex recombinase-aided qPCR (mRAP) assay to specifically detect the khe, blaKPC-2, and blaNDM-1 genes in Klebsiella pneumoniae. Methods: mRAP was carried out in a qPCR instrument within 1 h. The analytical sensitivities of mRAP for khe, blaKPC-2, and blaNDM-1 genes were tested using recombinant plasmids and dilutions of reference strains. A total of 137 clinical isolates and 86 sputum samples were used to validate the clinical performance of mRAP. Results: mRAP achieved the sensitivities of 10, 8, and 14 copies/reaction for khe, blaKPC-2, and blaNDM-1 genes, respectively, superior to qPCR. The Kappa value of qPCR and mRAP for detecting khe, blaKPC-2, and blaNDM-1 genes was 1, 0.855, and 1, respectively (p < 0.05). Conclusion: mRAP is a rapid and highly sensitive assay for potential clinical identification of khe, blaKPC-2, and blaNDM-1 genes in K. pneumoniae.

PrimerBankID	Target Pathogen	Target Gene
RPB0317	Klebsiella pneumoniae	khe gene
RPB0318	Klebsiella pneumoniae	blaKPC-2 gene
RPB0319	Klebsiella pneumoniae	blaNDM-1gene

Automatic Microfluidic Harmonized RAA-CRISPR Diagnostic System for Rapid and Accurate Identification of Bacterial Respiratory Tract Infections

Author(s):

Xinran Xiang,Xiaoqing Ren,Qianyu Wen,Gaowa Xing,Yuting Liu,Xiaowei Xu,Yuhuan Wei,Yuhan Ji,Tingting Liu,Huwei Song,Shenghang Zhang,Yuting Shang,Minghui Song

Journal:

Analytical Chemistry

Year:

2024

Abstract:

Respiratory tract infections (RTIs) pose a grave threat to human health, with bacterial pathogens being the primary culprits behind severe illness and mortality. In response to the pressing issue, we developed a centrifugal microfluidic chip integrated with a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats (CRISPR) system to achieve rapid detection of respiratory pathogens. The limitations of conventional two-step CRISPR-mediated systems were effectively addressed by employing the all-in-one RAA-CRISPR detection method, thereby enhancing the accuracy and sensitivity of bacterial detection. Moreover, the integration of a centrifugal microfluidic chip led to reduced sample consumption and significantly improved the detection throughput, enabling the simultaneous detection of multiple respiratory pathogens. Furthermore, the incorporation of Chelex-100 in the sample pretreatment enabled a sample-to-answer capability. This pivotal addition facilitated the deployment of the system in real clinical sample testing, enabling the accurate detection of 12 common respiratory bacteria within a set of 60 clinical samples. The system offers rapid and reliable results that are crucial for clinical diagnosis, enabling healthcare professionals to administer timely and accurate treatment interventions to patients.

PrimerBankID	Target Pathogen	Target Gene
RPB0269	Acinetobacter baumannii	\
RPB0270	Klebsiella pneumoniae	\
RPB0271	Pseudomonas aeruginosa	\
RPB0272	Stenotrophomonas maltophilia	\
RPB0273	Staphylococcus aureus	\
RPB0274	Haemophilus influenzae	\
RPB0275	Streptococcus pneumoniae	\

SARS-CoV-2 and Its Omicron Variants Detection with RT-RPA -CRISPR\Cas13a-Based Method at Room Temperature

Author(s):

Jia Li,Xiaojun Wang,Liujie Chen,Lili Duan,Fenghua Tan,Kai Li,Zheng Hu

Journal:

Reports of Biochemistry & Molecular Biology logo

Year:

2023

Abstract:

Background: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global health crisis, with genetic mutations and evolution further creating uncertainty about epidemic risk. It is imperative to rapidly determine the nucleic acid sequence of SARS-CoV-2 and its variants to combat the coronavirus pandemic. Our goal was to develop a rapid, room-temperature, point-of-care (POC) detection system to determine the nucleic acid sequences of SARS-CoV-2 isolates, especially omicron variants. Methods: Based on the conserved nucleotide sequence of SARS-CoV-2, bioinformatics software was used to analyze, design, and screen optimal enzymatic isothermal amplification primers and efficient CRISPR RNAs (crRNAs) of CRISPR/Cas13a to the target sequences. Reverse transcription-recombinase polymerase amplification (RT-RPA) was used to amplify the virus, and CRISPR/Cas13a-crRNA was used to cleave the SARS-CoV-2 target sequence. The sensitivity of nucleic acid detection was assessed by serial dilution of plasmid templates. All reactions were performed at room temperature. Results: RT-RPA, combined with CRISPR/Cas13a, can detect the SARS-CoV-2 with a minimum content of 102 copies/μL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 103 copies/μL. Conclusions: The method developed in this study has potential application in clinical detection of SARS-CoV-2 and its omicron variants.

PrimerBankID	Target Pathogen	Target Gene
RPB0370	SARS-CoV-2	S gene
RPB0371	SARS-CoV-2	ORF1ab gene
RPB0372	SARS-CoV-2	N gene
RPB0373	SARS-CoV-2 (Omicron)	Spike gene

PACRAT: pathogen detection with aptamer-observed cascaded recombinase polymerase amplification–in vitro transcription

Author(s):

Pavana Khan,Lauren M Aufdembrink,Katarzyna P Adamala,Aaron E Engelhart

Journal:

RNA

Year:

2024

Abstract:

The SARS-CoV-2 pandemic underscored the need for early, rapid, and widespread pathogen detection tests that are readily accessible. Many existing rapid isothermal detection methods use the recombinase polymerase amplification (RPA), which exhibits polymerase chain reaction (PCR)-like sensitivity, specificity, and even higher speed. However, coupling RPA to other enzymatic reactions has proven difficult. For the first time, we demonstrate that with tuning of buffer conditions and optimization of reagent concentrations, RPA can be cascaded into an in vitro transcription reaction, enabling detection using fluorescent aptamers in a one-pot reaction. We show that this reaction, which we term PACRAT (pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription) can be used to detect SARS-CoV-2 RNA with single-copy detection limits, Escherichia coli with single-cell detection limits, and 10-min detection times. Further demonstrating the utility of our one-pot, cascaded amplification system, we show PACRAT can be used for multiplexed detection of the pathogens SARS-CoV-2 and E. coli, along with multiplexed detection of two variants of SARS-CoV-2.

PrimerBankID	Target Pathogen	Target Gene
RPB0234	SARS-CoV-2	Nucleocapsid N3 gene

Rapid, sensitive, and user-friendly detection of Pseudomonas aeruginosa using the RPA\CRISPR\Cas12a system

Author(s):

Wenjing Zhang,Hai Qu,Xin Wu,Jingjing Shi,Xinling Wang

Journal:

BMC microbiology

Year:

2024

Abstract:

Background: Pseudomonas aeruginosa (P. aeruginosa) is a life-threatening bacterium known for its rapid development of antibiotic resistance, posing significant challenges in clinical treatment, biosecurity, food safety, and environmental monitoring. Early and accurate identification of P. aeruginosa is crucial for effective intervention. Methods: The lasB gene of P. aeruginosa was selected as the target for the detection. RPA primers for recombinase polymerase amplification (RPA) and crRNA for CRISPR/Cas12a detection were meticulously designed to target specific regions within the lasB gene. The specificity of the RPA/CRISPR/Cas12a detection platform was assessed using 15 strains. The detection limit of RPA/CRISPR/Cas12a detection platform was determined by utilizing a pseudo-dilution series of the P. aeruginosa DNA. The practical applicability of the RPA/CRISPR/Cas12a detection platform was validated by comparing it with qPCR on 150 samples (35 processed meat product samples, 55 cold seasoned vegetable dishes, 60 bottled water samples). Results: The RPA/CRISPR/Cas12a detection platform demonstrates high specificity, with no cross-reactivity with non-P. aeruginosa strains. This assay exhibits remarkable sensitivity, with a limit of detection (LOD) of 100 copies/µL for fluorescence assay and 101 copies/µL for the LFTS method. Furthermore, the performance of the RPA/CRISPR/Cas12a detection platform is comparable to that of the well-established qPCR method, while offering advantages such as shorter reaction time, simplified operation, and reduced equipment requirements. Conclusions: The RPA/CRISPR/Cas12a detection platform presents a straightforward, accurate, and sensitive approach for early P. aeruginosa detection and holds great promise for diverse applications requiring rapid and reliable identification.

PrimerBankID	Target Pathogen	Target Gene
RPB0228	Pseudomonas aeruginosa	lasB gene

A Method for Detecting Five Carbapenemases in Bacteria Based on CRISPR-Cas12a Multiple RPA Rapid Detection Technology

Author(s):

Huaming Xu,Chunhui Lin,Hao Tang,Rongrong Li,Zhaoxin Xia,Yi Zhu,Zhen Liu,Jilu Shen

Journal:

Infection and Drug Resistance

Year:

2024

Abstract:

Introduction: As the last line of defense for clinical treatment, Carbapenem antibiotics are increasingly challenged by multi-drug resistant bacteria containing carbapenemases. The rapid spread of these multidrug-resistant bacteria is the greatest threat to severe global health problems. Methods: To solve the problem of rapid transmission of this multidrug-resistant bacteria, we have developed a rapid detection technology using CRPSPR-Cas12a gene editing based on multiple Recombinase polymerase amplification. This technical method can directly isolate the genes of carbapenemase-containing bacteria from samples, with a relatively short detection time of 30 minutes. The instrument used for the detection is relatively inexpensive. Only a water bath can complete the entire experiment of Recombinase polymerase amplification and trans cleavage. This reaction requires no lid during the entire process while reducing a large amount of aerosol pollution. Results: The detection sensitivity of this method is 1.5 CFU/mL, and the specificity is 100%. Discussion: This multi-scene detection method is suitable for screening populations in wild low-resource environments and large-scale indoor crowds. It can be widely used in hospital infection control and prevention and to provide theoretical insights for clinical diagnosis and treatment.

PrimerBankID	Target Pathogen	Target Gene
RPB0297	Klebsiella pneumoniae	KPC gene
RPB0298	Klebsiella pneumoniae	NDM gene
RPB0299	Klebsiella pneumoniae	OXA gene
RPB0300	Klebsiella pneumoniae	IMP gene
RPB0301	Klebsiella pneumoniae	VIM gene

Detection method for reverse transcription recombinase-aided amplification of avian influenza virus subtypes H5, H7, and H9

Author(s):

Zongshu Zhang,Zichuang Zhang,Chunguang Wang,Xianghe Zhai,Wenjing Wang,Xi Chen,Tie Zhang

Journal:

BMC Veterinary Research

Year:

2024

Abstract:

Background: Avian influenza virus (AIV) not only causes huge economic losses to the poultry industry, but also threatens human health. Reverse transcription recombinase-aided amplification (RT-RAA) is a novel isothermal nucleic acid amplification technology. This study aimed to improve the detection efficiency of H5, H7, and H9 subtypes of AIV and detect the disease in time. This study established RT-RAA-LFD and real-time fluorescence RT-RAA (RF-RT-RAA) detection methods, which combined RT-RAA with lateral flow dipstick (LFD) and exo probe respectively, while primers and probes were designed based on the reaction principle of RT-RAA. Results: The results showed that RT-RAA-LFD could specifically amplify H5, H7, and H9 subtypes of AIV at 37 °C, 18 min, 39 °C, 20 min, and 38 °C, 18 min, respectively. The sensitivity of all three subtypes for RT-RAA-LFD was 102 copies/µL, which was 10 ∼100 times higher than that of reverse transcription polymerase chain reaction (RT-PCR) agarose electrophoresis method. RF-RT-RAA could specifically amplify H5, H7, and H9 subtypes of AIV at 40 °C, 20 min, 38 °C, 16 min, and 39 °C, 17 min, respectively. The sensitivity of all three subtypes for RF-RT-RAA was 101 copies/µL, which was consistent with the results of real-time fluorescence quantification RT-PCR, and 100 ∼1000 times higher than that of RT-PCR-agarose electrophoresis method. The total coincidence rate of the two methods and RT-PCR-agarose electrophoresis in the detection of clinical samples was higher than 95%. Conclusions: RT-RAA-LFD and RF-RT-RAA were successfully established in this experiment, with quick response, simple operation, strong specificity, high sensitivity, good repeatability, and stability. They are suitable for the early and rapid diagnosis of Avian influenza and they have positive significance for the prevention, control of the disease, and public health safety.

PrimerBankID	Target Pathogen	Target Gene
RPB0341	Influenza A virus (H5)	HA gene
RPB0342	Influenza A virus (H7)	HA gene
RPB0343	Influenza A virus (H9)	HA gene
RPB0344	Influenza A virus (H5)	HA gene
RPB0345	Influenza A virus (H7)	HA gene
RPB0346	Influenza A virus (H9)	HA gene

FARPA-based tube array coupled with quick DNA extraction enables ultra-fast bedside detection of antibiotic-resistant pathogens

Author(s):

Jinling Huang,Huijie Yue,Wei Wei,Jingwen Shan,Yue Zhu,Liying Feng,Yi Ma,Bingjie Zou,Haiping Wu,Guohua Zhou

Journal:

Analyst

Year:

2024

Abstract:

Rapid and accurate detection of pathogens and antimicrobial-resistant (AMR) genes of the pathogens are crucial for the clinical diagnosis and effective treatment of infectious diseases. However, the time-consuming steps of conventional culture-based methods inhibit the precise and early application of anti-infection therapy. For the prompt treatment of pathogen-infected patients, we have proposed a novel tube array strategy based on our previously reported FARPA (FEN1-aided recombinase polymerase amplification) principle for the ultra-fast detection of antibiotic-resistant pathogens on site. The entire process from "sample to result" can be completed in 25 min by combining quick DNA extraction from a urine sample with FARPA to avoid the usually complicated DNA extraction step. Furthermore, a 36-tube array made from commercial 384-well titre plates was efficiently introduced to perform FARPA in a portable analyser, achieving an increase in the loading sample throughput (from several to several tens), which is quite suitable for the point-of-care testing (POCT) of multiple pathogens and multiple samples. Finally, we tested 92 urine samples to verify the performance of our proposed method. The sensitivities for the detection of E. coli, K. pneumoniae, E. faecium, and E. faecalis were 92.7%, 93.8%, 100% and 88.9%, respectively. The specificities for the detection of the four pathogens were 100%. Consequently, our rapid, low-cost and user-friendly POCT method holds great potential for guiding the rational use of antibiotics and reducing bacterial resistance.

PrimerBankID	Target Pathogen	Target Gene
RPB0327	Klebsiella pneumoniae	KPC
RPB0328	Klebsiella pneumoniae	NDM
RPB0329	Klebsiella pneumoniae	K.pneumoniae

Sensitive and visual detection of SARS-CoV-2 using RPA-Cas12a one-step assay with ssDNA-modified crRNA

Author(s):

Qinlong Zeng,Miaojin Zhou,Weiheng Deng,Qian Gao,Zhuo Li,Lingqian Wu,Desheng Liang

Journal:

Analytica Chimica Acta

Year:

2024

Abstract:

Background: CRISPR-Cas12a based one-step assays are widely used for nucleic acid detection, particularly for pathogen detection. However, the detection capability of the one-step assay is reduced because the Cas12a protein competes with the isothermal amplification enzymes for the target DNA and cleaves it. Therefore, the key to improving the sensitivity of the one-step assay is to address the imbalance between isothermal amplification and CRISPR detection. In previous study, we developed a Cas12a one-step assay using single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA) and applied this method for the detection of pathogenic DNA. Results: Here, we utilized mD-crRNA to establish a sensitive one-step assay that enables the visual detection of SARS-CoV-2 under ultraviolet light, achieving a detection limit of 5 aM without cross-reactivity. The sensitivity of mD-crRNA in the one-step assay was 100-fold higher than that of wild-type crRNA. Mechanistic studies revealed that the addition of ssDNA at the 3' end of mD-crRNA attenuates the binding affinity between the Cas12a-mD-crRNA complex and the target DNA. Consequently, this reduction in binding affinity decreases the cis-cleavage activity of Cas12a, mitigating its cleavage of the target DNA in the one-step assay. As a result, there is an augmentation in the amplification and accumulation of target DNA, thereby enhancing detection sensitivity. In the clinical testing of 40 SARS-CoV-2 RNA samples, the concordance between the results of the one-step assay and known qPCR results was 97.5 %. Significance: The one-step assay using mD-crRNA proves to be highly sensitive and specificity and visually effective for the detection of SARS-CoV-2. Our study delves into the application of the mD-crRNA-mediated one-step assay in nucleic acid detection and its associated reaction mechanism. This holds great significance in addressing the inherent incompatibility issues between isothermal amplification and CRISPR detection.

PrimerBankID	Target Pathogen	Target Gene
RPB0339	SARS-CoV-2	N gene

Cas12a\Guide RNA-Based Platform for Rapidly and Accurately Detecting blaKPC Gene in Carbapenem-Resistant Enterobacterales

Author(s):

Keke Li,Yaozhou Wu,Meng Liu,Junwen Yan,Lianhua Wei

Journal:

Infection and Drug Resistance

Year:

2024

Abstract:

Purpose: Accurate detection and identification of pathogens and their associated resistance mechanisms are essential prerequisites for implementing precision medicine in the management of Carbapenem-resistant Enterobacterales (CRE). Among the various resistance mechanisms, the production of KPC carbapenemase is the most prevalent worldwide. Consequently, this study aims to develop a convenient and precise nucleic acid detection platform specifically for the blaKPC gene. Methods: The initial phase of our research methodology involved developing a CRISPR/Cas12a detection framework, which was achieved by designing highly specific single-guide RNAs (sgRNAs) targeting the blaKPC gene. To enhance the sensitivity of this system, we incorporated three distinct amplification techniques-polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA)-into the CRISPR/Cas12a framework. Subsequently, we conducted a comparative analysis of the sensitivity and specificity of these three amplification methods when used in combination with the CRISPR/Cas12a system. Additionally, we assessed the clinical applicability of the methodologies by evaluating fluorescence readouts from 80 different clinical isolates. Furthermore, we employed lateral flow assay technology to provide a visual representation of the results, facilitating point-of-care testing. Results: Following a comparative analysis of the sensitivity and specificity of the three methods, we identified the RPA-Cas12a approach as the optimal detection technique. Our findings demonstrated that the limit of detection (LoD) of the RPA-Cas12a platform was 1 aM (~1 copy/µL) for plasmid DNA and 5 × 10³ fg/µL for genomic DNA. Furthermore, both the sensitivity and specificity of the platform achieved 100% upon validation with 80 clinical isolates. Conclusion: These findings suggest that the developed RPA-Cas12a platform represents a promising tool for the cost-effective, convenient, and accurate detection of the blaKPC gene.

PrimerBankID	Target Pathogen	Target Gene
RPB0302	Klebsiella pneumoniae	blaKPC gene

Detection and identification of SARS-CoV-2 and influenza a based on microfluidic technology

Author(s):

Yujie Liu,Guanliu Yu,Hongkun Liang,Wenbo Sun,Lulu Zhang,Michael G Mauk,Hua Li,Lei Chen

Journal:

Analytical Methods

Year:

2024

Abstract:

As of now, the global COVID-19 pandemic caused by SARS-CoV-2, which began in 2019, has been effectively controlled. However, the symptoms of influenza A virus infection were similar to those of SARS-CoV-2 infection, but they required different treatment approaches. To make the detection more accurate and the treatment more targeted. We developed a system that integrates RPA and CRISPR assays, allowing for the rapid, highly specific, and sensitive detection and differentiation of SARS-CoV-2, H1N1, and H3N2. Under isothermal amplification conditions, the RPA-CRISPR Cas12a detection system achieved a detection limit as low as 5 copies per μL, demonstrating excellent specificity. The measurement time was approximately 30 minutes. The RPA-CRISPR Cas12a detection system combined with the microfluidic chip we designed to simultaneously detect three viruses, providing a potential solution for efficient and reliable diagnosis.

PrimerBankID	Target Pathogen	Target Gene
RPB0231	Influenza A virus (H1N1)	\
RPB0232	Influenza A virus (H3N2)	\
RPB0233	SARS-CoV-2	\

Dual Gene Detection of H5N1 Avian Influenza Virus Based on Dual RT-RPA

Author(s):

Qian Wang,Shiwen Wu,Jiangbing Shuai,Ye Li,Xianshu Fu,Mingzhou Zhang,Xiaoping Yu,Zihong Ye,Biao Ma

Journal:

Molecules

Year:

2024

Abstract:

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10-7 ng/μL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 (p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus.

PrimerBankID	Target Pathogen	Target Gene
RPB0294	Influenza A virus (H5N1)	HA gene
RPB0295	Influenza A virus (H5N1)	M2 gene

A rapid and ultra-sensitive dual readout platform for Klebsiella pneumoniae detection based on RPA-CRISPR\Cas12a

Author(s):

Meiying Tan,Lina Liang,Chuan Liao,Zihan Zhou,Shaoping Long,Xueli Yi,Chunfang Wang,Caiheng Wei,Jinyuan Cai,Xuebin Li,Guijiang Wei

Journal:

Frontiers in Cellular and Infection Microbiology

Year:

2024

Abstract:

The bacterium Klebsiella pneumoniae (Kp) was the primary pathogen of hospital-acquired infection, but the current detection method could not rapidly and conveniently identify Kp. Recombinase polymerase amplification (RPA) was a fast and convenient isothermal amplification technology, and the clustered regularly interspaced short palindromic repeats (CRISPR) system could rapidly amplify the signal of RPA and improve its limit of detection (LOD). In this study, we designed three pairs of RPA primers for the rcsA gene of Kp, amplified the RPA signal through single-strand DNA reporter cleavage by CRISPR/Cas12a, and finally analyzed the cleavage signal using fluorescence detection (FD) and lateral flow test strips (LFTS). Our results indicated that the RPA-CRISPR/Cas12a platform could specifically identify Kp from eleven common clinical pathogens. The LOD of FD and LFTS were 1 fg/μL and 10 fg/μL, respectively. In clinical sample testing, the RPA-CRISPR/Cas12a platform was consistent with the culture method and qPCR method, and its sensitivity and specificity were 100% (16/16) and 100% (9/9), respectively. With the advantages of detection speed, simplicity, and accuracy, the RPA-CRISPR/Cas12a platform was expected to be a convenient tool for the early clinical detection of Kp.

PrimerBankID	Target Pathogen	Target Gene
RPB0250	Klebsiella pneumoniae	rcsA gene

CRISPR-Cas12a-based ultrasensitive assay for visual detection of SARS-CoV-2 RNA

Author(s):

Shaohua Gong,Kexin Song,Wei Pan,Na Li,Bo Tang

Journal:

Analyst

Year:

2024

Abstract:

The development of ultrasensitive and visual methods is of great significance for molecular diagnosis at the point-of-care. In this study, we have integrated recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system to design an ultrasensitive strategy for visual nucleic acid testing. RPA is utilized to amplify the target nucleic acid, producing amplicons that activate the single-stranded DNase property of CRISPR-Cas12a. The activated CRISPR-Cas12a then degrades the single-stranded DNA on magnetic nanoparticles (MNPs), releasing immobilized GOx from the MNPs which catalyses the chromogenic substrate. The developed method exhibits remarkable sensitivity, successfully detecting as low as 10 aM (∼6 copies per μL) of the target nucleic acid by visual colour changes in solution. The instrumental limit of detection is calculated to be 2.86 aM (∼2 copies per μL), comparable to the sensitivity of polymerase chain reaction (PCR). Importantly, this approach only requires isothermal incubation operation and does not involve costly instruments. The method has been validated by visually detecting the SARS-CoV-2 RNA gene fragment within 50 minutes. With its ultrasensitivity, simplicity of operation, and potential for integration into a point-of-care detection kit, this strategy holds great promise for nucleic acid testing in various settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0315	SARS-CoV-2	orflab gene

Rapid and one-tube detection of human metapneumovirus using the RT-RPA and CRISPR\Cas12a

Author(s):

Yao Du,Xiaorong Liu,Hongdan Gao,Xiaoqian Liu,Meng Huang,Qiang Chai,Zhihao Xing,Tao Zhang,Dongli Ma

Journal:

Journal of Virological Methods

Year:

2024

Abstract:

Human metapneumovirus (HMPV) is a common pathogen that can cause acute respiratory tract infections and is prevalent worldwide. There is yet no effective vaccine or specific treatment for HMPV. Early, rapid, and accurate detection is essential to treat the disease and control the spread of infection. In this study, we created the One-tube assay by combining Reverse Transcription-Recombinase Polymerase Amplification (RT-RPA) with the CRISPR/Cas12a system. By targeting the nucleoprotein (N) gene of HMPV to design specific primers and CRISPR RNAs (crRNAs), combining RT-RPA and CRISPR/Cas12a, established the One-tube assay. Meanwhile, the reaction conditions of the One-tube assay were optimized to achieve rapid and visual detection of HMPV. This assay could detect HMPV at 1 copy/μL in 30 min, without cross-reactivity with nine other respiratory pathogens. We validated the detection performance using clinical specimens and showed that the coincidence rate was 98.53 %，compared to the quantitative reverse-transcription polymerase chain reaction. The One-tube assay reduced the detection time and simplified the manual operation, while maintaining the detection performance and providing a new platform for HMPV detection.

PrimerBankID	Target Pathogen	Target Gene
RPB0235	HMPV	nucleoprotein (N) gene

CRISPR\Cas13a-based genome editing for establishing the detection method of H9N2 subtype avian influenza virus

Author(s):

Sha-Sha Chen,Yong-Lei Yang,Hong-Yun Wang,Tian-Kui Guo,Riaz-M Azeem,Chun-Wei Shi,Gui-Lian Yang,Hai-Bin Huang,Yan-Long Jiang,Jian-Zhong Wang,Xin Cao,Nan Wang,Yan Zeng,Wen-Tao Yang,Chun-Feng Wang

Journal:

Poultry Science

Year:

2024

Abstract:

Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA (recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimize the constructed assay. The sensitivity of the optimized detection system is excellent, with a minimum detection limit of 10° copies/μL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV.

PrimerBankID	Target Pathogen	Target Gene
RPB0290	Influenza A virus (H9N2)	HA gene

RT-RPA- Pf Ago detection platform for one-tube simultaneous typing diagnosis of human respiratory syncytial virus

Author(s):

Jia-Yu Liao,Xue-Yong Feng,Jie-Xiu Zhang,Tian-Dan Yang,Min-Xuan Zhan,Yong-Mei Zeng,Wei-Yi Huang,Hao-Bin Lian,Lin Ke,Si-Si Cai,Nan-Fei Zhang,Jin-Wen Fang,Xiao-Ying Cai,Jun-Duo Chen,Guang-Yu Lin,Li-Yun Lin,Wei-Zhong Chen,Yu-Yan Liu,Fei-Fei Huang,Chuang-Xing Lin,Min Lin

Journal:

Front. Cell. Infect. Microbiol.

Year:

2024

Abstract:

Human respiratory syncytial virus (HRSV) is the most prevalent pathogen contributing to acute respiratory tract infections (ARTI) in infants and young children and can lead to significant financial and medical costs. Here, we developed a simultaneous, dual-gene and ultrasensitive detection system for typing HRSV within 60 minutes that needs only minimum laboratory support. Briefly, multiplex integrating reverse transcription-recombinase polymerase amplification (RT-RPA) was performed with viral RNA extracted from nasopharyngeal swabs as a template for the amplification of the specific regions of subtypes A (HRSVA) and B (HRSVB) of HRSV. Next, the Pyrococcus furiosus Argonaute (PfAgo) protein utilizes small 5'-phosphorylated DNA guides to cleave target sequences and produce fluorophore signals (FAM and ROX). Compared with the traditional gold standard (RT-qPCR) and direct immunofluorescence assay (DFA), this method has the additional advantages of easy operation, efficiency and sensitivity, with a limit of detection (LOD) of 1 copy/μL. In terms of clinical sample validation, the diagnostic accuracy of the method for determining the HRSVA and HRSVB infection was greater than 95%. This technique provides a reliable point-of-care (POC) testing for the diagnosis of HRSV-induced ARTI in children and for outbreak management, especially in resource-limited settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0347	HRSV A	\
RPB0348	HRSV B	\

Establishment of a rapid detection method for Mycoplasma pneumoniae based on RPA-CRISPR-Cas12a technology

Author(s):

Ge Li,Jing Zhou,Nana Gao,Runde Liu,Jilu Shen

Journal:

Clinica Chimica Acta

Year:

2024

Abstract:

Mycoplasma pneumoniae can cause respiratory infections and pneumonia, posing a serious threat to the health of children and adolescents. Early diagnosis of Mycoplasma pneumoniae infection is crucial for clinical treatment. Currently, diagnostic methods for Mycoplasma pneumoniae infection include pathogen detection, molecular biology techniques, and bacterial culture, all of which have certain limitations. Here, we developed a rapid, simple, and accurate detection method for Mycoplasma pneumoniae that does not rely on large equipment or complex operations. This technology combines the CRISPR-Cas12a system with recombinase polymerase amplification (RPA), allowing the detection results to be observed through fluorescence curves and immunochromatographic lateral flow strips.It has been validated that RPA-CRISPR/Cas12a fluorescence analysis and RPA-CRISPR/Cas12-immunochromatographic exhibit no cross-reactivity with other common pathogens, and The established detection limit was ascertained to be as low as 102 copies/µL.Additionally, 49 clinical samples were tested and compared with fluorescence quantitative polymerase chain reaction, demonstrating a sensitivity and specificity of 100%. This platform exhibits promising clinical performance and holds significant potential for clinical application, particularly in settings with limited resources, such as clinical care points or resource-constrained areas.

PrimerBankID	Target Pathogen	Target Gene
RPB0245	Klebsiella pneumoniae	P1 gene

Fluorescent and colorimetric dual-readout platform for tuberculosis point-of-care detection based on dual signal amplification strategy and quantum dot nanoprobe

Author(s):

Ou Hu,Yingyu Gong,Yuexiang Chang,Yaoju Tan,Zuanguang Chen,Wei Bi,Zhengjin Jiang

Journal:

Biosensors and Bioelectronics

Year:

2024

Abstract:

Rapid and accurate diagnosis of tuberculosis (TB) is of great significance to control the spread of this devastating infectious disease. In this work, a sensitive and low-cost point-of-care testing (POCT) detection platform for TB was developed based on recombinase polymerase amplification (RPA)-catalytic hairpin assembly (CHA)-assisted dual signal amplification strategy. This platform could achieve homogeneous fluorescent and visual diagnosis of TB by using CdTe quantum dots (QDs) signal reporter. In the presence of target DNA (IS1081 gene fragment), RPA amplicons blocked by short oligonucleotide strands could trigger CHA signal amplification, leading to the Ag+ releasing from C-Ag+-C structure and the fluorescence quenching of CdTe QDs by the released Ag+. Furthermore, the detection performance of CdTe QDs modified by 3-mercaptopropionic acid (MPA) or thiomalic acid (TMA) (MPA-capped QDs and TMA-capped QDs) was systematically compared. Experimental results demonstrated that TMA-capped QDs exhibited better detection sensitivity due to their stronger interaction with Ag+. The limits of detection (LODs) of fluorescence and visual analysis were as low as 0.13 amol L-1 and 0.33 amol L-1. This method was successfully applied to the clinical sputum samples from 36 TB patients and 20 healthy individuals, and its quantitative results were highly consistent with those obtained by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The proposed approach has the advantages of high sensitivity and specificity, simple operation and low cost, and is expected to be applied in clinical TB screening and diagnosis.

PrimerBankID	Target Pathogen	Target Gene
RPB0287	Mycobacterium tuberculosis	IS10181 gene

A compact microfluidic platform for rapid multiplex detection of respiratory viruses via centrifugal polar-absorbance spectroscopy

Author(s):

Ya Su,Xiangyu Jin,Fan Yang,Xuekai Liu,Fenggang Li,Qingchen Zhao,Jialu Hou,Shuailong Zhang,Hang Li,Guoliang Huang,Rongxin Fu

Journal:

Talanta

Year:

2024

Abstract:

Nucleic acid detection technology has become a crucial tool in cutting-edge research within the life sciences and clinical diagnosis domains. Its significance is particularly highlighted during the respiratory virus pandemic, where nucleic acid testing plays a pivotal role in accurately detecting the virus. Isothermal amplification technologies have been developed and offer advantages such as rapidity, mild reaction conditions and excellent stability. Among these methods, recombinase polymerase amplification (RPA) has gained significant attention due to its simple primer design and resistance to multiple reaction inhibitors. However, the detection of RPA amplicons hinders the widespread adoption of this technology, leading to a research focus on cost-effective and convenient detection methods for RPA nucleic acid testing. In this study, we propose a novel computational absorption spectrum approach that utilizes the polar GelRed dye to efficiently detect RPA amplicons. By exploiting the asymmetry of GelRed molecules upon binding with DNA, polar electric dipoles are formed, leading to precipitate formation through centrifugal vibration and electrostatic interaction. The quantification of amplicon content is achieved by measuring the residual GelRed concentration in the supernatant. Our proposed portable and integrated microfluidic device successfully detected five respiratory virus genes simultaneously. The optimized linear detection was achieved and the sensitivity for all the targets reached 100 copies/μL. The total experiment could be finished in 27 min. The clinical experiments demonstrated the practicality and accuracy. This cost-effective and convenient detection scheme presents a promising biosensor for rapid virus detection, contributing to the advancement of RPA technology.

PrimerBankID	Target Pathogen	Target Gene
RPB0309	SARS-CoV-2	orflab gene
RPB0310	SARS-CoV-2	N gene
RPB0311	RSV A	F gene
RPB0312	Influenza A virus	M1 gene
RPB0313	Influenza B virus	NS1 gene

Development of RPA-Cas12a assay for rapid and sensitive detection of Pneumocystis jirovecii

Author(s):

Qiming Liu,Hao Zeng,Ting Wang,HongXia Ni,Yongdong Li,Weidong Qian,Ting Fang,Guozhang Xu

Journal:

BMC Microbiology

Year:

2024

Abstract:

Pneumocystis jirovecii is a prevalent opportunistic fungal pathogen that can lead to life-threatening Pneumocystis pneumonia in immunocompromised individuals. Given that timely and accurate diagnosis is essential for initiating prompt treatment and enhancing patient outcomes, it is vital to develop a rapid, simple, and sensitive method for P. jirovecii detection. Herein, we exploited a novel detection method for P. jirovecii by combining recombinase polymerase amplification (RPA) of nucleic acids isothermal amplification and the trans cleavage activity of Cas12a. The factors influencing the efficiency of RPA and Cas12a-mediated trans cleavage reaction, such as RPA primer, crRNA, the ratio of crRNA to Cas12a and ssDNA reporter concentration, were optimized. Our RPA-Cas12a-based fluorescent assay can be completed within 30-40 min, comprising a 25-30 min RPA reaction and a 5-10 min trans cleavage reaction. It can achieve a lower detection threshold of 0.5 copies/µL of target DNA with high specificity. Moreover, our RPA-Cas12a-based fluorescent method was examined using 30 artificial samples and demonstrated high accuracy with a diagnostic accuracy of 93.33%. In conclusion, a novel, rapid, sensitive, and cost-effective RPA-Cas12a-based detection method was developed and demonstrates significant potential for on-site detection of P. jirovecii in resource-limited settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0248	Pneumocystis jirovecii	5.8 S ribosomal RNA

Rapid and Highly Sensitive Detection of Mycobacterium tuberculosis Utilizing the Recombinase Aided Amplification-Based CRISPR-Cas13a System

Author(s):

Qiao Li,Nenhan Wang,Mengdi Pang,Honghao Miao,Xiaowei Dai,Bo Li,Xinyu Yang,Chuanyou Li,Yi Liu

Journal:

Microorganisms

Year:

2024

Abstract:

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (MTB) infection, remains a major threat to global public health. To facilitate early TB diagnosis, an IS6110 gene-based recombinase aided amplification (RAA) assay was coupled to a clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas13a fluorescence assay to create a rapid MTB detection assay (named RAA-CRISPR-MTB). Its diagnostic efficacy was evaluated for sensitivity and specificity through sequential testing of recombinant plasmids, mycobacterium strains, and clinical specimens. RAA-CRISPR detected IS6110 genes at levels approaching 1 copy/μL with pUC57-6110 as the template and 10 copies/μL with H37Rv as the template. There was no observed cross detection of non-tuberculosis mycobacteria (NTM) with either template. Furthermore, RAA-CRISPR testing of 151 clinical specimens yielded a diagnostic specificity rate of 100% and a diagnostic sensitivity rate of 69% that exceeded the corresponding Xpert MTB/RIF assay rate (60%). In conclusion, we established a novel RAA-CRISPR assay that achieved highly sensitive and specific MTB detection for use as a clinical TB diagnostic tool in resource-poor settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0237	Mycobacterium tuberculosis	IS6110 genes

Engineering the bacteriophage 80 alpha endolysin as a fast and ultrasensitive detection toolbox against Staphylococcus aureus

Author(s):

Feng Zhao,Yixi Yang,Wenyao Zhan,Zhiqi Li,Hui Yin,Jingjing Deng,Waner Li,Rui Li,Qi Zhao,Jian Li

Journal:

Biosensors and Bioelectronics

Year:

2024

Abstract:

The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 102 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply.

PrimerBankID	Target Pathogen	Target Gene
RPB0314	Staphylococcus aureus	nuc gene

Rapid and sensitive detection of methicillin-resistant Staphylococcus aureus through the RPA- Pf Ago system

Author(s):

Weizhong Chen,Jiexiu Zhang,Huagui Wei,Jie Su,Jie Lin,Xueyan Liang,Jiangtao Chen,Rong Zhou,Lin Li,Zefang Lu,Guangyu Sun

Journal:

Front Microbiol

Year:

2024

Abstract:

Introduction: Both the incidence and mortality rates associated with methicillin-resistant Staphylococcus aureus (MRSA) have progressively increased worldwide. A nucleic acid testing system was developed in response, enabling swift and precise detection of Staphylococcus aureus (S. aureus) and its MRSA infection status. This facilitates improved prevention and control of MRSA infections. Methods: In this work, we introduce a novel assay platform developed by integrating Pyrococcus furiosus Argonaute (PfAgo) with recombinase polymerase amplification (RPA), which was designed for the simultaneous detection of the nuc and mecA genes in MRSA. Results: This innovative approach enables visual MRSA detection within 55 mins, boasting a detection limit of 102 copies/μL. Characterized by its high specificity, the platform accurately identifies MRSA infections without cross-reactivity to other clinical pathogens, highlighting its unique capability for S. aureus infection diagnostics amidst bacterial diversity. Validation of this method was performed on 40 clinical isolates, demonstrating a 95.0% accuracy rate in comparison to the established Vitek2-COMPACT system. Discussion: The RPA-PfAgo platform has emerged as a superior diagnostic tool, offering enhanced sensitivity, specificity, and identification efficacy for MRSA detection. Our findings underscore the potential of this platform to significantly improve the diagnosis and management of MRSA infection.

PrimerBankID	Target Pathogen	Target Gene
RPB0241	Staphylococcus aureus	nuc gene
RPB0242	Staphylococcus aureus	mecA gene

An extraction-free one-pot assay for rapid detection of Klebsiella pneumoniae by combining RPA and CRISPR\Cas12a

Author(s):

Jinyu Fu,Rurong Mo,Ziyao Li,Shijie Xu,Xiyu Cheng,Binghuai Lu,Shuobo Shi

Journal:

Biosensors and Bioelectronics

Year:

2025

Abstract:

Klebsiella pneumoniae poses a significant threat to global public health. Traditional clinical diagnostic methods, such as bacterial culture and microscopic identification, are not suitable for point-of-care testing. In response, based on the suboptimal protospacer adjacent motifs, this study develops an extraction-free one-pot assay, named EXORCA (EXtraction-free One-pot RPA-CRISPR/Cas12a assay), designed for the immediate, sensitive and efficient detection of K. pneumoniae. The EXORCA assay can be completed within approximately 30 min at a constant temperature and allows for the visualization of results either through a fluorescence reader or directly by the naked eye under blue light. The feasibility of the assay was evaluated using twenty unextracted clinical samples, achieving a 100% (5/5) positive predictive value and a 100% (15/15) negative predictive value in comparison to qPCR. These results suggest that the EXORCA assay holds significant potential as a point-of-care testing tool for the rapid identification of pathogens, such as K. pneumonia.

PrimerBankID	Target Pathogen	Target Gene
RPB0251	Klebsiella pneumoniae	rcsA gene

A specific and ultrasensitive Cas12a\crRNA assay with recombinase polymerase amplification and lateral flow biosensor technology for the rapid detection of Streptococcus pyogenes

Author(s):

Yu Cheng,Jiawen Lyu,Jiangfeng Han,Long Feng,Xiangmei Li,Pei Li,Shanfeng Zhang,Wenqiao Zang

Journal:

Clinical Microbiology

Year:

2024

Abstract:

The potential of CRISPR/Cas systems for nucleic acid detection in novel biosensing applications is remarkable. The current clinical diagnostic detection of Streptococcus pyogenes (S. pyogenes) is based on serological identification, culture, and PCR. We report a rapid, simple, and sensitive method for detecting and screening for S. pyogenes. This novel method is a promising supplemental test. After 10 min of the sample processing and 10 min of recombinase polymerase amplification, followed by 10 min of Cas12 reaction and 3 min of lateral flow biosensor (LFB) readout, a visible outcome can be observed without the need for magnification within 33 min. This platform is robust, inexpensive, and appropriate for on-site testing. A new technique for detection was created using CRISPR-Cas12a technology, which includes two measurements: a fluorescent-CRISPR-S. pyogenes test and a LFB-CRISPR-S. pyogenes test. An approach utilizing CRISPR Cas12a was developed, and the accuracy and precision of this technique were assessed. The LoD for the fluorescence-CRISPR- S. pyogenes assay was 1 copy/μL, and the technique effectively differentiated S. pyogenes from other microorganisms. Moreover, the detection outcomes were presented in a user-friendly manner using lateral flow biosensor strips. Conclusion: A rapid and sensitive Cas12a/crRNA assay using recombinase RPA and LFB was developed to detect S. pyogenes. The Cas12a/crRNA-based assay exhibited high specificity among different bacteria strains and extremely high sensitivity. The accuracy and rapidity of this method make it a promising tool for S. pyogenes detection and screening. Importance: Patients may experience a range of symptoms due to Streptococcus pyogenes infections, including superficial skin infections, pharyngitis, and invasive diseases in subcutaneous tissues like streptococcal toxic shock syndrome. At present, the clinical diagnostic detection of S. pyogenes is based on serological identification, culture, and PCR. These detection methods are time-consuming and require sophisticated equipment, making these methods challenging for routine laboratories. Thus, there is a need for a detection platform that is capable of quickly and accurately identifying S. pyogenes. In this study, a rapid and sensitive Cas12a/crRNA assay using recombinase RPA and LFB was developed to detect S. pyogenes. The Cas12a/crRNA-based assay exhibited high specificity among different bacteria strains and extremely high sensitivity. This method probably plays an important role for S. pyogenes detection and screening.

PrimerBankID	Target Pathogen	Target Gene
RPB0244	Streptococcus pyogenes M1 GAS	sdaB gene
RPB0511	Streptococcus pyogenes	sdaB

A Rapid Detection Method for H3 Avian Influenza Viruses Based on RT-RAA

Author(s):

Jiaqi Li,Huan Cui,Yuxin Zhang,Xuejing Wang,Huage Liu,Yingli Mu,Hongwei Wang,Xiaolong Chen,Tongchao Dong,Cheng Zhang,Ligong Chen

Journal:

Animals

Year:

2024

Abstract:

The continued evolution of H3 subtype avian influenza virus (AIV)-which crosses the interspecific barrier to infect humans-and the potential risk of genetic recombination with other subtypes pose serious threats to the poultry industry and human health. Therefore, rapid and accurate detection of H3 virus is highly important for preventing its spread. In this study, a method based on real-time reverse transcription recombinase-aided isothermal amplification (RT-RAA) was successfully developed for the rapid detection of H3 AIV. Specific primers and probes were designed to target the hemagglutinin (HA) gene of H3 AIV, ensuring highly specific detection of H3 AIV without cross-reactivity with other important avian respiratory viruses. The results showed that the detection limit of the RT-RAA fluorescence reading method was 224 copies/response within the 95% confidence interval, while the detection limit of the RT-RAA visualization method was 1527 copies/response within the same confidence interval. In addition, 68 clinical samples were examined and the results were compared with those of real-time quantitative PCR (RT-qPCR). The results showed that the real-time fluorescence RT-RAA and RT-qPCR results were completely consistent, and the kappa value reached 1, indicating excellent correlation. For visual detection, the sensitivity was 91.43%, the specificity was 100%, and the kappa value was 0.91, which also indicated good correlation. In addition, the amplified products of RT-RAA can be visualized with a portable blue light instrument, which enables rapid detection of H3 AIV even in resource-constrained environments. The H3 AIV RT-RAA rapid detection method established in this study can meet the requirements of basic laboratories and provide a valuable reference for the early diagnosis of H3 AIV.

PrimerBankID	Target Pathogen	Target Gene
RPB0353	Influenza A virus (H3N2)	HA gene

Recombinase-aided amplification assay for rapid detection of imipenem-resistant Pseudomonas aeruginosa and rifampin-resistant Pseudomonas aeruginosa

Author(s):

Yao Zhou,Ruiqing Shi,Liang Mu,Linlin Tian,Mengshan Zhou,Wenhan Lyu,Yaodong Chen

Journal:

Frontiers In Cellular And Infection Microbiology

Year:

2024

Abstract:

The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr, in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr, the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/μL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.

PrimerBankID	Target Pathogen	Target Gene
RPB0219	Pseudomonas aeruginosa	oprD gene
RPB0220	Pseudomonas aeruginosa	arr gene

Isothermal recombinase polymerase amplification and silver nanoparticle assay: a sustainable approach for ultrasensitive detection of Klebsiella pneumoniae

Author(s):

Naresh Patnaik,Nidhi Orekonday,Ruchi Jain Dey

Journal:

Analytical Methods

Year:

2024

Abstract:

Our study addresses the urgent need for effective detection of Klebsiella pneumoniae, a recognized threat by the World Health Organization (WHO). Current challenges in managing K. pneumoniae infections include the lack of rapid and affordable detection tools, particularly in resource-limited point-of-care (POC) settings. To tackle this, we developed an innovative molecular detection pipeline combining three POC-compatible methods. Firstly, we employed Insta DNA™ card-based sample collection and DNA extraction for simplicity and ease of use. Next, we utilized recombinase polymerase amplification (RPA) targeting the Klebsiella hemolysin gene, khe, specific to the K. pneumoniae species complex (KpSC). Finally, we integrated a silver nanoparticle (AgNP) aggregation assay for visual detection, offering a rapid, sensitive, and specific method capable of detecting as few as ∼3 bacteria of K. pneumoniae within ∼45 minutes. This approach eliminates the need for complex equipment, making it highly suitable for field and resource-limited POC applications. Moreover, our method introduces an environmentally significant detection strategy. The method developed minimizes chemical reagent usage and reduces the carbon footprint associated with sample transportation. Furthermore, our method reduces waste compared to the traditional detection techniques, offering a safer alternative to ethidium bromide or other DNA dyes which are often genotoxic and mutagenic in nature. Silver nanoparticles, being environmentally safer, can also be recycled from the waste, contributing to sustainability in nanoparticle production and disposal. Overall, our technique presents a promising solution for detecting K. pneumoniae in various settings, including environmental, water, and food samples, as well as industrial or hospital effluents. By aligning with global efforts to improve public health and environmental sustainability, our approach holds significant potential for enhancing disease management and reducing environmental impact.

PrimerBankID	Target Pathogen	Target Gene
RPB0227	Klebsiella pneumoniae	khe gene