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