两种实时荧光定量PCR方法的建立及其用于献浆者血液单纯疱疹病毒检测的有效性比较

发布时间：2018-04-18 06:52

本文选题：单纯疱疹病毒 + 实时荧光定量PCR　；参考：《中国输血杂志》2014年06期

【摘要】：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。
[Abstract]:Objective to establish two real-time fluorescent quantitative PCR methods based on SYBR Green and Taqman probes for the detection of human herpes simplex virus (HSV) and to evaluate the effectiveness of these two methods in detecting HSV in plasma.Methods Primer and probe were designed according to the conserved sequence of UL30 gene of HSV in GenBank, and the recombinant plasmid containing conserved sequence of UL30 gene was constructed. Using the recombinant plasmid as standard, the HSV of 144 (human) donors was detected.Two qPCR detection methods based on SYBR Green and Taqman probe were established and 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, the detection limit was 5 脳 101cp/ reaction system, and both had good reproducibility between batches and within batches.The melting curve peak obtained by qPCR detection method based on SYBR Green is single.The two qPCR methods were used to detect HSV in plasma samples: the detectable rate was 2.08 / 144%, both of which could be used for the determination of HSV load.Conclusion two kinds of qPCR reaction systems were successfully established. The two methods have the advantages of high specificity, high sensitivity and good homogeneity for detection of HSV, and can be used for blood detection in blood donors.
【作者单位】：四川大学生命科学学院;成都生物制品研究所有限责任公司;
【分类号】：R373.11;R3416

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