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目的建立基于SYBR Green和Taqman探针的2种实时荧光定量PCR(qPCR)方法,用于检测人单纯疱疹病毒(HSV)并评估2种方法检测血浆中HSV的有效性。方法根据GenBank中HSV的UL30基因保守序列设计引物和探针,并构建含有UL30基因保守序列的重组质粒,以该重组质粒为标准品,通过对144(人)份献浆者HSV的检测,建立并评估基于SYBR Green和Taqman探针的2种qPCR检测方法。结果所建立的2种qPCR方法对于HSV均具有高度特异性,对HBV和梅毒无交叉反应;检测灵敏度高,检测下限均为5×101cp/反应体系,并都有较好的批间和批内重复性;基于SYBR Green的qPCR检测方法得到的熔解曲线峰单一。应用该2种qPCR方法对献浆人群血浆标本HSV检测:检出率均为2.08%(3/144),二者都能用于HSV载量的测定。结论成功建立了2种qPCR反应体系,二者对HSV的检测特异性强、灵敏度高、均一性好,可应用于献血(浆)者血液检测。
Objective To establish two real-time quantitative PCR (qPCR) methods based on SYBR Green and Taqman probes for the detection of human herpes simplex virus (HSV) and evaluate the effectiveness of the two methods in detecting plasma HSV. Methods Primers and probes were designed according to the conserved sequence of UL30 gene of HSV in GenBank. A recombinant plasmid containing UL30 gene sequence was constructed. The recombinant plasmid was used as a standard to detect HSV in 144 human plasma samples. Two qPCR assays based on SYBR Green and Taqman probes were also evaluated. Results The two qPCR methods were highly specific to HSV and had no cross-reaction with HBV and syphilis. The detection sensitivity was high and the detection limit was 5 × 101cp / reaction system. Both of them had good inter-assay and intra-assay Repeatability. The SYBR Green-based qPCR detection showed a single melting curve. The application of these two qPCR methods on HSV detection of plasma samples from donors: The detection rates were 2.08% (3/144), both of which could be used for the determination of HSV load. Conclusion Two kinds of qPCR reaction systems were successfully established. Both of them are specific to HSV and have high sensitivity and good homogeneity. They can be used in the blood testing of blood donors (plasma).