人微小病毒B19荧光定量聚合酶链式反应检测方法的建立

许良云; 张其刚; 范广来; 赵建华

doi:10.7619/jcmp.20210246

人微小病毒B19荧光定量聚合酶链式反应检测方法的建立

Establishment of fluorescent quantitative polymerase chain reaction detection method for human parvovirus B19

摘要

摘要:
目的建立可同时检测各型人微小病毒B19（HPV B19）的荧光定量聚合酶链式反应（PCR）方法。
方法运用在线工具MAFFT比对分析3种基因型的HPV B19代表病毒株NS1区域序列，针对保守区序列设计特异性的引物和探针。建立检测HPV B19实时荧光定量PCR方法，并对该方法的敏感性和特异性进行评估。
结果 1×10⁷~1×10²拷贝/μL以及20拷贝/μL质粒DNA模板绘制的标准曲线，反应效率E为0.946，R²值为0.996 6；本方法的检测下限为10拷贝/μL，定量下限为20拷贝/μL。单纯疱疹病毒（HSV）2型、巨细胞病毒（CMV）、B族链球菌（GBS）、乙型肝炎病毒（HBV）、EB病毒（EBV）的阳性DNA病毒检测结果均为阴性，参考质粒及HPV B19阳性样本正常扩增。
结论建立的荧光定量PCR检测方法具有较好的敏感性和特异性，可运用于HPV B19感染的筛查和诊断。

Abstract:
Objective To establish a fluorescent quantitative polymerase chain reaction detection method (PCR) method for simultaneous detection of human parvovirus B19(HPV B19).
Methods The online tool MAFFT was used to compare and analyze the sequence NS1 gene conserved region of three genotypes HPV B19, and specific primers and probes were designed for conserved region sequences. The fluorescent quantitative PCR method for detecting HPV B19 was established, and the sensitivity and specificity of the method were evaluated.
Results A standard curve drawn with a plasmid DNA template at a concentration of 1×10⁷ to 1×10² copies/μL and 20 copies/μL, the reaction efficiency E was 0.946 and R² was 0.996 6; the detection limit of this method was 10 copies/μL and the lower limit of quantification was 20 copies/μL. Herpes simplex virus (HSV) type 2, cytomegalovirus (CMV), group B streptococcus (GBS), hepatitis B virus (HBV), Epstein-Barr virus (EBV) positive DNA virus test results were negative, and the reference plasmid and HPV B19 positive samples were amplified.
Conclusion The established fluorescent quantitative PCR detection method can be applied for the screening and diagnosis of HPV B19 infection as high sensitivity and specificity.

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