Selective endpoint visualized detection of Vibrio parahaemolyticus with CRISPR/Cas12a assisted PCR using thermal cycler for on-site application

Mengyao Zhang; Chengzhi Liu; Ya Shi; Jun Wu; Jian Wu; Huan Chen

doi:10.1016/j.talanta.2020.120818

Selective endpoint visualized detection of Vibrio parahaemolyticus with CRISPR/Cas12a assisted PCR using thermal cycler for on-site application

Talanta. 2020 Jul 1:214:120818. doi: 10.1016/j.talanta.2020.120818. Epub 2020 Feb 11.

Authors

Mengyao Zhang¹, Chengzhi Liu², Ya Shi², Jun Wu³, Jian Wu⁴, Huan Chen⁵

Affiliations

¹ College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China.
² Key Laboratory of Microbiol Technology and Bioinformatics of Zhejiang Province, Zhejiang Institute of Microbiology, Hangzhou, 310012, China; NMPA Key Laboratory for Testing and Risk Warning of Pharmaceutical Microbiology, Zhejiang Institute of Microbiology, Hangzhou, 310012, China.
³ Lin'an Center for Disease Control and Prevention, Lin'an, 311300, China.
⁴ College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China. Electronic address: wujian69@zju.edu.cn.
⁵ Key Laboratory of Microbiol Technology and Bioinformatics of Zhejiang Province, Zhejiang Institute of Microbiology, Hangzhou, 310012, China; NMPA Key Laboratory for Testing and Risk Warning of Pharmaceutical Microbiology, Zhejiang Institute of Microbiology, Hangzhou, 310012, China. Electronic address: chenhuan7809@gmail.com.

PMID: 32278427
DOI: 10.1016/j.talanta.2020.120818

Abstract

Vibrio parahaemolyticus is a major cause of seafood-associated food poisoning. It is of great significance to develop an accurate, simple and cost-effective method to identify infected seafood, especially for on-site application. Polymerase chain reaction (PCR) remains the golden standard for nucleic acid detection. But traditional methods heavily reply on sophisticated instrument and specialized operators, which limits the application for on-site detections. Here we developed a novel, specific and visualized detection method for PCR based on CRISPR/Cas12a system. On a low-cost thermal cycler, amplification reaction can be conducted easily. The CRISPR/Cas12a system was specifically designed to evaluate amplicons, eliminating false positive results. Besides the negative samples remained colorless, the positive samples generated obvious green fluorescence, which could be easily distinguished by the naked eye using a homemade UV device. The presented detection method was verified by detecting shrimp samples. The limit of detection is 1.02 × 10² copies/μL. This presented method provided a new strategy for specific endpoint detection of PCR and advanced its application in field for food safety assurance.

Keywords: CRISPR/Cas12a; On-site detection; Polymerase chain reaction; Thermal cycler; Vibrio parahaemolyticus; Visualized detection.

MeSH terms

Animals
Biosensing Techniques
CRISPR-Cas Systems / genetics*
Electrochemical Techniques
Food Contamination / analysis
Particle Size
Penaeidae / microbiology*
Polymerase Chain Reaction*
RNA, Bacterial / genetics*
Surface Properties
Temperature*
Vibrio parahaemolyticus / genetics
Vibrio parahaemolyticus / isolation & purification*

Substances

RNA, Bacterial