摘要
为设计一种快速、高灵敏度检测诺如病毒(Norovirus,NV)的方法.本文通过检索参考NCBI数据库中收录的国内典型流行的NV毒株BJSMQ的基因序列NC_039476.1,合成了NV的标准重组质粒,根据NV的ORF1基因保守序列设计一组特异性引物和荧光探针;通过正交试验进行程序及试剂反应体系的优化,设计TaqMan荧光定量PCR检测方法.结果表明该检测方法具有高灵敏度,最低检测限可达1.2 copies/μL,且在标准重组质粒浓度1.2×10^(5)~1.2×10^(0) copies/μL范围内具有较好的线性关系,其R^(2)=0.9907;该方法特异性强,在病毒的混合重组质粒样液中仅与NV病毒反应;组内和组间的重复性良好,变异系数(CV)值均小于2%;在数字PCR试验中进一步验证了该方法的可行性.本文设计的TaqMan荧光定量PCR检测方法可用于NV毒株的快速准确检测,有望成为NV诊断的有效工具.
To design a rapid and highly sensitive method for detecting Norovirus(NV),this study synthesized a standard recombinant plasmid of NV by retrieving gene sequences of the domestically prevalent NV strain BJSMQ from the NCBI database(Accession number NC_039476.1).Specific primers and fluorescent probes were designed based on the conserved sequence of the NV ORF1 gene.An orthogonal experiment was conducted to optimize the procedure and a reagent reaction system for Taq Man fluorescent quantitative PCR detection.The results showed that this detection method exhibited high sensitivity,with a detection limit as low as 1.2 copies/μL.It demonstrated good linearity within the range of standard recombinant plasmid concentrations from 1.2×10^(5)to 1.2×10^(0)copies/μL,with a linear correlation coefficient(R^(2))of 0.9907.The method showed strong specificity,reacting only with NV virus in mixed recombinant plasmid samples.Both intra-assay and inter-assay repeatability were excellent,with coefficient of variation(CV)values all less than 2%.Further validation of this method was conducted using digital PCR experiments.In summary,the designed Taq Man fluorescent quantitative PCR detection method can be used for the rapid and accurate detection of NV strains,potentially serving as an effective tool for NV diagnosis.
作者
陈波
李家奇
付立申
吴文明
CHEN Bo;LI Jia-qi;FU Li-shen;WU Wen-ming(School of Applied Physics and Materials,Wuyi University,Jiangmen 529020,China;School of Environmental and Chemical Engineering,Wuyi University,Jiangmen 529020,China;Institute of Biological and Medical Engineering,Guangdong Academy of Sciences,Guangzhou 510316,China)
出处
《五邑大学学报（自然科学版）》
CAS
2024年第3期1-6,共6页
Journal of Wuyi University(Natural Science Edition)
基金
国家科技计划人才项目(0523129001)。
