%0 Journal Article
%T 肠道病毒71型荧光定量PCR方法的比较与评价*<br>Comparison and evaluation of different fluorescent quantitative PCR methods for enterovirus 71
%A 赵伊珂
%A 卫海燕
%A 黄学勇
%A 程宁宁
%A 潘静静
%A 王若琳
%A 许汴利
%A 郭万申
%J 郑州大学学报（医学版）
%D 2017
%R 10.13705/j.issn.1671-6825.2017.05.006
%X 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TaqMan探针荧光定量PCR方法。方法:将VP1全序连接到克隆质粒pMAL-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bioPerfectus 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(P<0.001),方法C测得的CT值小于方法A和B(P<0.05); 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。<br>Aim: To determine copy number of the enterovirus 71(EV71)in clinical specimens by quantitative analysis, and compare 3 commercial TaqMan fluorescence quantitative PCR methods systematically and comprehensively.Methods: VP1 sequence of EV71 was inserted into cloning vector pMAL-c2X. The purified plasmid was used as the standard sample for quantitative detection of 105 clinical specimens collected from the special monitoring system of HFMD in Henan Province. Positive rate and CT value among the 3 commercial TaqMan fluorescence quantitative PCR methods from bioPerfectus Technologies(method A), KINGHAWK(method B), and Beijing ABT(method C)were compared, and consistency was analyzed.Results: The recombinant plasmid pMAL-c2X was constructed successfully. Standard curve equation was Y=-3.35X+47.49.For 105 specimens, positive rate of EV71 measured by method A, B, and C was 41.90%(44/105), 42.86%(45/105), and 41.90%(44/105), respectively, and the difference was not significant(P=0.987). Among positive specimens, copy number by method C(14.17±3.19)was less than method A(24.34±3.92)and B(25.08±3.94), and the difference was significant(P<0.05). For 3 methods, the limit of detection was 102, 10, and 102 copies; sensitivity was 93.62%, 95.74%, and 93.62%; specificity was 100.00%, 75.00%, and 100.00%. The result of 3 methods showed poor consistency.Conclusion: EV71 fluorescence quantitative PCR kit for commercialization is suitable for the qualitative detection of clinical samples; however, it needs further study for the quantitative detection of EV71
%K 肠道病毒71型
%K 荧光定量PCR
%K TaqMan 探针&lt
%K br&gt
%K enterovirus 71
%K fluorescent quantitative PCR
%K TaqMan probe
%U http://jms.zzu.edu.cn/oa/darticle.aspx?type=view&id=201705006