Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification.
- Author:
Han Ji JIANG
1
;
Rong TAN
1
;
Min JIN
1
;
Jing YIN
1
;
Zhi Xian GAO
1
;
Hai Bei LI
1
;
Dan Yang SHI
1
;
Shu Qing ZHOU
1
;
Tian Jiao CHEN
1
;
Dong YANG
1
;
Jun Wen LI
1
Author Information
- Publication Type:Journal Article
- Keywords: CRISPR/Cas12a-VD; Cross-reactivity; Isothermal amplification; Recombinase polymerase amplification; Vibrio parahaemolyticus; Visual detection
- MeSH: CRISPR-Cas Systems; Nucleic Acid Amplification Techniques/methods*; Recombinases/genetics*; Vibrio parahaemolyticus/genetics*
- From: Biomedical and Environmental Sciences 2022;35(6):518-527
- CountryChina
- Language:English
-
Abstract:
Objective:To establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP) .
Methods:We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD).
Results:CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10 -18 M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10 2 CFU/g.
Conclusion:The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.
