Field-deployable Porcine Epidemic Diarrhea Virus Diagnostics Utilizing CRISPR-Cas13a

Overview

Journal Virulence

Specialty Microbiology

Date 2024 Nov 19

PMID 39560197

Authors

Yuanyuan Wang

Dalin He

Weihua Li

Yaqin Dong

Linlin Fang

Deju Liu

Yi Tang

Shaobo Xiao

Affiliations

Soon will be listed here.

Abstract

Porcine epidemic diarrhoea virus (PEDV), a pathogenic microorganism that induces epidemic diarrhoea in swine, causes substantial economic damage to swine-farming nations. To prevent and control PEDV infections, the availability of upgraded and rapid virus detection techniques is crucial. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)13a system, namely, programmability of CRISPR RNA (crRNA) and "collateral" promiscuous RNase activity of Cas13a after target RNA identification. In this study, we aimed to develop a recombinase polymerase amplification (RPA)-based CRISPR-Cas13a approach for PEDV diagnosis for the first time. The results showed that up to 10 copies of the target PEDV DNA standard/µL were detected after 40 min at 37 °C. PEDV detection exhibited remarkable specificity compared to that of other selected pathogens. Additionally, this RPA-based CRISPR-Cas13a approach could be used to clinical samples, with similar performance to that of reverse transcription-quantitative polymerase chain reaction (RT - qPCR). The results of our proposed approach were visualized using either lateral flow strips or fluorescence for field-deployable viral diagnostics, thereby facilitating its use in endemic regions. Overall, our proposed approach showed good reliability, sensitivity, and specificity, suggesting that it is applicable for detecting other viruses in diagnosing diseases and inspecting food safety.

References

Kim S, Lee J, Jung J, Kim I, Hyun B, Kim H . Genetic characterization of porcine epidemic diarrhea virus in Korea from 1998 to 2013. Arch Virol. 2015; 160(4):1055-64. PMC: 7086719. DOI: 10.1007/s00705-015-2353-y. View

Sun R, Cai R, Chen Y, Liang P, Chen D, Song C . Outbreak of porcine epidemic diarrhea in suckling piglets, China. Emerg Infect Dis. 2012; 18(1):161-3. PMC: 3381683. DOI: 10.3201/eid1801.111259. View

Abudayyeh O, Gootenberg J, Konermann S, Joung J, Slaymaker I, Cox D . C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 2016; 353(6299):aaf5573. PMC: 5127784. DOI: 10.1126/science.aaf5573. View

Kellner M, Koob J, Gootenberg J, Abudayyeh O, Zhang F . SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc. 2019; 14(10):2986-3012. PMC: 6956564. DOI: 10.1038/s41596-019-0210-2. View

Smargon A, Cox D, Pyzocha N, Zheng K, Slaymaker I, Gootenberg J . Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28. Mol Cell. 2017; 65(4):618-630.e7. PMC: 5432119. DOI: 10.1016/j.molcel.2016.12.023. View

Shan Y, Zhou X, Huang R, Xing D . High-Fidelity and Rapid Quantification of miRNA Combining crRNA Programmability and CRISPR/Cas13a trans-Cleavage Activity. Anal Chem. 2019; 91(8):5278-5285. DOI: 10.1021/acs.analchem.9b00073. View

Liu L, Li X, Ma J, Li Z, You L, Wang J . The Molecular Architecture for RNA-Guided RNA Cleavage by Cas13a. Cell. 2017; 170(4):714-726.e10. DOI: 10.1016/j.cell.2017.06.050. View

Sun X, Wang Y, Zhang L, Liu S, Zhang M, Wang J . CRISPR-Cas9 Triggered Two-Step Isothermal Amplification Method for O157:H7 Detection Based on a Metal-Organic Framework Platform. Anal Chem. 2020; 92(4):3032-3041. DOI: 10.1021/acs.analchem.9b04162. View

Abudayyeh O, Gootenberg J, Essletzbichler P, Han S, Joung J, Belanto J . RNA targeting with CRISPR-Cas13. Nature. 2017; 550(7675):280-284. PMC: 5706658. DOI: 10.1038/nature24049. View

10.

Lee C . Porcine epidemic diarrhea virus: An emerging and re-emerging epizootic swine virus. Virol J. 2015; 12:193. PMC: 4687282. DOI: 10.1186/s12985-015-0421-2. View

11.

Peng Q, Fang L, Ding Z, Wang D, Peng G, Xiao S . Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology. J Virol Methods. 2019; 276:113772. PMC: 7113866. DOI: 10.1016/j.jviromet.2019.113772. View

12.

Gilbert L, Horlbeck M, Adamson B, Villalta J, Chen Y, Whitehead E . Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014; 159(3):647-61. PMC: 4253859. DOI: 10.1016/j.cell.2014.09.029. View

13.

Li Y, Zheng F, Fan B, Muhammad H, Zou Y, Jiang P . Development of an indirect ELISA based on a truncated S protein of the porcine epidemic diarrhea virus. Can J Microbiol. 2015; 61(11):811-7. DOI: 10.1139/cjm-2015-0213. View

14.

Ding Z, Fang L, Jing H, Zeng S, Wang D, Liu L . Porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between IRF3 and TBK1. J Virol. 2014; 88(16):8936-45. PMC: 4136253. DOI: 10.1128/JVI.00700-14. View

15.

Barnes K, Lachenauer A, Nitido A, Siddiqui S, Gross R, Beitzel B . Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases in real-time. Nat Commun. 2020; 11(1):4131. PMC: 7431545. DOI: 10.1038/s41467-020-17994-9. View

16.

He D, Liu G, Yang J, Jiang X, Wang H, Fan Y . Specific High-Sensitivity Enzymatic Molecular Detection System Termed RPA-Based CRISPR-Cas13a for Duck Tembusu Virus Diagnostics. Bioconjug Chem. 2022; 33(6):1232-1240. DOI: 10.1021/acs.bioconjchem.2c00200. View

17.

Diel D, Lawson S, Okda F, Singrey A, Clement T, Fernandes M . Porcine epidemic diarrhea virus: An overview of current virological and serological diagnostic methods. Virus Res. 2016; 226:60-70. PMC: 7172987. DOI: 10.1016/j.virusres.2016.05.013. View

18.

Wei N, Zheng B, Niu J, Chen T, Ye J, Si Y . Rapid Detection of Genotype II African Swine Fever Virus Using CRISPR Cas13a-Based Lateral Flow Strip. Viruses. 2022; 14(2). PMC: 8879322. DOI: 10.3390/v14020179. View

19.

Ma L, Lian K, Zhu M, Tang Y, Zhang M . Visual detection of porcine epidemic diarrhea virus by recombinase polymerase amplification combined with lateral flow dipstrip. BMC Vet Res. 2022; 18(1):140. PMC: 9013981. DOI: 10.1186/s12917-022-03232-5. View

20.

Song D, Park B . Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. Virus Genes. 2012; 44(2):167-75. PMC: 7089188. DOI: 10.1007/s11262-012-0713-1. View