Establishment of a nucleic acid detection method for varicella-zoster virus based on RAA-CRISPR/Cas12a
10.3760/cma.j.cn112866-20241022-00155
- VernacularTitle:基于RAA-CRISPR/Cas12a水痘-带状疱疹病毒核酸检测方法的建立
- Author:
Ziyi LI
1
;
Ruichen WANG
;
Haoze LIU
;
Tianzi ZHANG
;
Tianxin SHI
;
Qianqian CUI
;
Qikai YIN
;
Fan LI
;
Kai NIE
;
Shihong FU
;
Huanyu WANG
;
Canlei SONG
;
Qiufang XU
;
Songtao XU
Author Information
1. 中国医科大学，沈阳 110122
- Publication Type:Journal Article
- Keywords:
Varicella-zoster virus;
RAA;
CRISPR/CAS12a;
Detection method
- From:
Chinese Journal of Experimental and Clinical Virology
2025;39(2):242-249
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a method for the rapid detection of varicella-zoster virus (VZV) by recombinase-aid amplification (RAA) combined with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system.Methods:Clinical samples of suspected herpes zoster in Shandong province and Shanghai from 2023 to 2024 were collected, nucleic acids of positive samples were extracted, RAA-specific primers and crRNA (CRISPR RNA, crRNA) were designed for the conserved region of VZV, and the fluorescence intensity generated by Cas12a non-specific cleavage of single-stranded fluorescent probes was used to screen highly sensitive crRNAs and optimize the concentrations of crRNA, Cas12a and ssDNA probes. The sensitivity and reproducibility of the RAA-CRISPR/Cas12a detection method were evaluated by using synthesized plasmids and clinical samples, and the specificity of the method was evaluated by using other viral nucleic acids. The method was used to detect clinical samples by using the method and quantitative real-time PCR (qPCR) method, and the detection rate and consistency of the two method were compared.Results:The highly sensitive crRNA-4 was screened from the four crRNAs designed, and a VZV detection method for RAA-CRISPR/CAS12a based on fluorescence intensity measurement was established, which could be detected at 37℃ in 45 min, and the sensitivity of the detection could reach 10 copies/μL, a minimum clinical sample with a Ct value of 38.980 can be detected. It has high specificity and no cross-reactivity with Adenovirus 7, Herpes simplex virus type I, Herpes simplex virus type II, Coxsackieviruses A16, Cytomegalovirus, Epstein-Barr virus, Measles virus, Mumps virus, Enterovirus 71, Japanese encephalitis virus genotype 5. It has good stability, and can be successfully detected in low, medium and high concentrations of viral positive plasmids with good consistency. The detection rate of the clinically positive samples was 100%, which was completely consistent with the qPCR test result.Conclusions:RAA isothermal amplification technology combined with CRISPR-CAS12a technology was used to establish an accurate method for the detection of VZV virus, which was highly sensitive, specific, and had low requirements for experimental conditions, and could be completed within 45 min, which could provide strong technical support for the early detection of VZV.
