精准医学杂志2025，Vol.40Issue(2)：127-130,4.DOI:10.13362/j.jpmed.202540038
诺如病毒GⅡ.4型荧光RT-RPA检测方法的建立
Establishment of a fluorescent reverse transcription-recombinase polymerase amplification assay for norovirus type G Ⅱ.4
摘要
Abstract
Objective To establish a fluorescent reverse transcription-recombinase polymerase amplification(RT-RPA)assay for norovirus(NoV)type G Ⅱ.4.Methods The NoV G Ⅱ.4 gene sequence was obtained from NCBI,and the primers and probes were designed by selecting the conserved regions after sequence alignment.With NoV G Ⅱ.4 standards as the detection ob-ject,the temperature,primer concentration,and probe concentration of the fluorescent RT-RPA reaction system were optimized to establish a fluorescent RT-RPA assay for NoV G Ⅱ.4.This method was assessed in terms of sensitivity,specificity,and clinical validation by detecting different concentrations of NoV G Ⅱ.4 standards and the nucleic acids of NoV G Ⅰ,other subtypes of NoV G Ⅱ(NoV G Ⅱ.2,NoV G Ⅱ.3,NoV G Ⅱ.7,NoV G Ⅱ.8,and NoV G Ⅱ.17),rotavirus,adenovirus,parechovirus,astrovirus,and sapovirus.Results The fluorescent RT-RPA assay had an optimal reaction temperature of 41 ℃,an optimal primer concentra-tion of 630 nmol/L,and an optimal probe concentration of 240 nmol/L for detecting NoV G Ⅱ.4.And the limit of detection was 1 × 101 copies/μL for NoV G Ⅱ.4,the other nucleic acids did not produce the fluorescent signal that could be detected by the instru-ment.Conclusion This study establishes a fluorescent RT-RPA assay for NoV G Ⅱ.4,which is characterized by high sensitivi-ty,good specificity,and rapid reaction speed,and it is expected to provide a new method for the rapid detection of NoV G Ⅱ.4 at ports.关键词
诺如病毒/重组酶聚合酶扩增/核酸扩增技术/敏感性与特异性Key words
Norovirus/Recombinase polymerase amplification/Nucleic acid amplification techniques/Sensitivity and specificity分类
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郑志坤,徐翮飞,丁伟,张娟,杜建森,侯琳,张瑾..诺如病毒GⅡ.4型荧光RT-RPA检测方法的建立[J].精准医学杂志,2025,40(2):127-130,4.基金项目
国家"十四五"科技部研发计划(2022YFC2302800) （2022YFC2302800）
