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