肠道病毒71型荧光定量PCR方法的比较与评价

发布时间：2019-06-03 06:34

【摘要】：目的:定量检测肠道病毒71型(EV71)临床标本中病毒拷贝数,评价3种商业化Taq Man探针荧光定量PCR方法。方法:将VP1全序连接到克隆质粒p MAL-c2X上,纯化的质粒作为标准品。选取河南开封手足口病监测试点的105份手足口病患儿粪便标本,分别应用bio Perfectus Technologies(方法 A)、KINGHAWK(方法 B)和Beijing ABT(方法 C)公司的荧光定量PCR方法进行检测,比较EV71阳性检出率和定量分析结果的差异,分析3种方法的一致性。结果:成功构建重组质粒并绘制标准曲线,方程为Y=-3.35X+47.49;分别用方法 A、B、C对105份标本进行检测,EV71阳性检出率分别为41.90%(44/105)、42.86%(45/105)和41.90%(44/105),差异无统计学意义(P=0.987);CT值分别为(24.34±3.92)、(25.08±3.94)和(14.17±3.19),差异有统计学意义(P0.001),方法 C测得的CT值小于方法 A和B(P0.05);3种方法最低检测限值分别为102、10和102拷贝,灵敏度分别为93.62%、95.74%和93.62%,特异度分别为100.00%、75.00%和100.00%;一致性分析结果显示,3种方法一致性较差。结论:商业化的EV71荧光定量PCR试剂盒适用于定性检测临床标本,定量检测EV71方法需要进一步研究。
[Abstract]:Objective: to quantitatively detect the copy number of enterovirus 71 (EV71) in clinical specimens and evaluate three commercial Taq Man probe fluorescence quantitative PCR methods. Methods: the VP1 was ligated to the cloned plasmid p MAL-c2X and the purified plasmid was used as the standard. 105 fecal specimens of children with HFMD in Kaifeng, Henan Province were detected by fluorescence quantitative PCR (bio Perfectus Technologies (method A), KINGHAWK (method B) and Beijing ABT (method C (Beijing ABT (method C), respectively. The difference between the positive rate of EV71 and the results of quantitative analysis was compared, and the consistency of the three methods was analyzed. Results: the recombinant plasmid was successfully constructed and the standard curve was drawn. The equation was Y 鈮，

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