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