改良的qPCR检测水痘-带状疱疹病毒的临床应用*

The clinical application of modified qPCR in detection of varicella-zoster virus*

摘要:

目的?分析改良的qPCR与水痘-带状疱疹病毒（VZV）DNA试剂盒检测VZV DNA的检出率，并进一步探讨其临床应用。方法?选取2017年5月—2017年7月在西南医科大学附属医院就诊的带状疱疹患者31例。利用患者疱液提取标准品，并根据VZV DNA保守序列ORF59制备引物以设计一种改良的qPCR。对比该方法与试剂盒检测VZV DNA的效果后，用该方法测定伐昔洛韦抗病毒治疗前后外周血单个核细胞（PBMC）和血浆VZV DNA的阳性率。结果?患者疱液提取的标准品大小为395?bp。经测序并与VZV DNA进行比对，标准品被匹配上的碱基数占比99%。该qPCR的标准曲线决定系数R2=0.9999，回归方程Y=-3.9367X+45.07，其中X为Log值，Y为Ct值。改良的qPCR与VZV DNA试剂盒检测PBMC中VZV DNA阳性率均为73.3%。治疗前后PBMC与血浆的VZV DNA阳性率比较，差异均无统计学意义（P?>0.05）。PBMC和血浆在治疗前与治疗后的VZV DNA阳性率比较，差异无统计学意义（P?>0.05）。 结论?改良的qPCR与VZV DNA试剂盒的检测效果相近，且血浆可替代PBMC作为检测标本。该方法具有经济、快捷的优势，值得临床推广应用。

Abstract:

Objective To compare the efficacy of the modified quantitative polymerase chain reaction (qPCR) and varicella-zoster virus (VZV) DNA Kit in detecting VZV DNA, and to explore the clinical application of the modified qPCR.?Methods?Thirty-one patients with shingles who were admitted to the Affiliated Hospital of Southwest Medical University from May 2017 to July 2017 were included in the study. The standard sample was extracted from the blister fluid of the patients, and primers for the modified qPCR were designed according to the conservative sequence of VZV DNA ORF59. After comparing the efficacy of the two methods in detecting VZV DNA, we detected the VZV DNA in peripheral blood mononuclear cells (PBMC) and plasma with the modified qPCR before and after the antiviral therapy with valaciclovir.?Results?The standard sample extracted from the blister fluid of the patients was 395 bp in size. The matched bases of the standard sample account for 99% after being sequenced and compared with VZV DNA. In the standard curve of the qPCR assay, the coefficient of determination (R2) was 0.9999 and the regression equation was Y?=?-3.9367X?+?45.07, in which X referred to the logarithm of initial copy number of template DNA and Y was the cycle threshold (Ct) value. To compare the efficacy of modified qPCR and VZV DNA kit in detecting VZV DNA extracted from PBMC, the positive rates were both found to be 73.3%. In the comparison of positive rates in detecting VZV DNA before and after antiviral therapy, no significant difference was observed both in PBMC specimens and in plasma specimens (P > 0.05). Besides, in the comparison of positive rates in detecting VZV DNA between PBMC specimens and plasma specimens, there was no significant difference found both before antiviral therapy and after antiviral therapy (P > 0.05).?Conclusions?The modified qPCR and the VZV DNA kit showed similar efficacy in detecting VZV DNA, and plasma may replace PBMC as the test specimen. Thus, the modified qPCR is economical and time-saving, and worthy of clinical application.