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

Author(s):

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

Journal:

Biosensors and Bioelectronics

Year:

2024

Abstract:

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

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

CRISPR-Cas13a-based detection method for avian influenza virus

Author(s):

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

Journal:

Frontiers in Microbiology

Year:

2023

Abstract:

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

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

A Blood Drying Process for DNA Amplification

Author(s):

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

Journal:

small

Year:

2023

Abstract:

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

PrimerBankID	Target Pathogen	Target Gene
RPB0412	Staphylococcus aureus	vicK gene

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

Author(s):

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

Journal:

Scientific Reports

Year:

2023

Abstract:

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

PrimerBankID	Target Pathogen	Target Gene
RPB0366	Pseudomonas aeruginosa	\

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

Author(s):

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

Journal:

Journal of Medical Virology

Year:

2023

Abstract:

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

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

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

Author(s):

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

Journal:

BMC Microbiology

Year:

2023

Abstract:

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

PrimerBankID	Target Pathogen	Target Gene
RPB0364	HRV	VP4 gene

Rapid detection of avian influenza virus based on CRISPR-Cas12a

Author(s):

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

Journal:

Virology Journal

Year:

2023

Abstract:

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

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

CRISPR\Cas13-assisted carbapenem-resistant Klebsiella pneumoniae detection

Author(s):

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

Journal:

Journal of Microbiology,Immunology and Infection

Year:

2024

Abstract:

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

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

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

Author(s):

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

Journal:

AMB Express

Year:

2023

Abstract:

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

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

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

Author(s):

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

Journal:

Frontiers in Microbiology

Year:

2023

Abstract:

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

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

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