Establishment and preliminary application of RAA assay for varicella-zoster virus
10.3760/cma.j.cn112866-20230927-00034
- VernacularTitle:水痘-带状疱疹病毒RAA检测方法的建立及初步应用
- Author:
Haoze LIU
1
;
Ruichen WANG
;
Weijia ZHANG
;
Xiaohui YAO
;
Shihong FU
;
Kai NIE
;
Fan LI
;
Qikai YIN
;
Ying HE
;
Huanyu WANG
;
Ruiping HU
;
Songtao XU
Author Information
1. 内蒙古医科大学基础医学院生物化学与分子生物学教研室，呼和浩特 010110
- Keywords:
Recombinase-aid amplification;
Varicella-zoster virus;
Testing
- From:
Chinese Journal of Experimental and Clinical Virology
2023;37(6):631-636
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a rapid method for the detection of varicella-zoster virus (VZV) by recombinase-aid amplification (RAA).Methods:The whole genome sequences of VZV were downloaded from the global shared database for comparison and analysis. Specific primers and probe were designed for the four conserved genes respectively and the optimal combination was selected. The optimal reaction system was selected through the concentration gradient of primers and probes, and a fluorescence RAA detection method was established. The sensitivity of the method was evaluated with VZV positive plasmid standard and clinical samples with gradient dilution, the repeatability of the method was evaluated with the lowest detectable limit concentration of positive plasmid standard, and the specificity of other viral nucleic acid method was evaluated. At the same time, this method and quantitative real-time PCR (qPCR) were used to detect clinical samples and the result were compared.Results:The optimal combination of primer pair F2/R2 and probe P2 targeting open reading frame (ORF) 28 gene was selected. Considering the cost factor, the optimal primer concentration was set at 500 nmol/L and the optimal probe concentration was 280 nmol/L. The minimum detection limit was 10 1 copies/μL, and the minimum clinical positive samples with a Ct value of 36.027 could be detected, and the result of repeated experiments were consistent. The method has no cross-reaction with other viral nucleic acids. The detection rate of clinical positive samples was 93.33%, which was almost identical to that of qPCR. Conclusions:This method is simple to operate with high sensitivity, strong specificity, low requirements for experimental conditions, visual detection result, and can detect VZV nucleic acid in samples within 20 minutes, which is a rapid VZV detection method that can be considered for clinical use for detection.
