【摘要】：目的:定量檢測腸道病毒71型(EV71)臨床標(biāo)本中病毒拷貝數(shù),評價3種商業(yè)化Taq Man探針熒光定量PCR方法。方法:將VP1全序連接到克隆質(zhì)粒p MAL-c2X上,純化的質(zhì)粒作為標(biāo)準(zhǔn)品。選取河南開封手足口病監(jiān)測試點(diǎn)的105份手足口病患兒糞便標(biāo)本,分別應(yīng)用bio Perfectus Technologies(方法 A)、KINGHAWK(方法 B)和Beijing ABT(方法 C)公司的熒光定量PCR方法進(jìn)行檢測,比較EV71陽性檢出率和定量分析結(jié)果的差異,分析3種方法的一致性。結(jié)果:成功構(gòu)建重組質(zhì)粒并繪制標(biāo)準(zhǔn)曲線,方程為Y=-3.35X+47.49;分別用方法 A、B、C對105份標(biāo)本進(jìn)行檢測,EV71陽性檢出率分別為41.90%(44/105)、42.86%(45/105)和41.90%(44/105),差異無統(tǒng)計(jì)學(xué)意義(P=0.987);CT值分別為(24.34±3.92)、(25.08±3.94)和(14.17±3.19),差異有統(tǒng)計(jì)學(xué)意義(P0.001),方法 C測得的CT值小于方法 A和B(P0.05);3種方法最低檢測限值分別為102、10和102拷貝,靈敏度分別為93.62%、95.74%和93.62%,特異度分別為100.00%、75.00%和100.00%;一致性分析結(jié)果顯示,3種方法一致性較差。結(jié)論:商業(yè)化的EV71熒光定量PCR試劑盒適用于定性檢測臨床標(biāo)本,定量檢測EV71方法需要進(jìn)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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