本文選題：EMA + PCR��； 參考：《四川大學學報(醫(yī)學版)》2017年01期

【摘要】：目的探討采用疊氮溴化乙錠(EMA)聯(lián)合聚合酶鏈反應(PCR)快速鑒別死活腺病毒的方法。方法將不同稀釋度的病毒接種至生長良好的單層細胞上,通過細胞病變效應(CPE)的發(fā)生情況計算病毒滴度;將3種腺病毒和新城疫雞瘟病毒分別制備成106 PFU/mL的母液并梯度稀釋備用,提取DNA后用PCR擴增后進行凝膠電泳,觀察目的片段的擴增結果;分別用0μg/mL、70μg/mL、120μg/mL和150μg/mL的EMA處理滅活后的腺病毒,提取DNA進行PCR,觀察目的片段的電泳結果;用120μg/mL的EMA處理107 PFU/mL、106 PFU/mL、105PFU/mL、104 PFU/mL、103 PFU/mL的腺病毒,觀察PCR和凝膠電泳結果。結果 104 PFU/mL及以上滴度的病毒DNA PCR后得到陽性條帶,其他滴度的病毒DNA PCR后未檢測到陽性條帶;3個型別的腺病毒(共8個分離株)的DNA均擴增出目的條帶,新城疫雞瘟病毒的DNA未擴增出目的條帶;120μg/mL及150μg/mL的EMA抑制滅活腺病毒DNA擴增,未得到陽性條帶;120μg/mL EMA不影響107 PFU/mL、106 PFU/mL、105 PFU/mL的腺病毒活病毒DNA擴增,得到陽性條帶。結論本研究證實EMA-PCR方法可快速鑒別死活腺病毒,能有效避免單純PCR檢測腺病毒產生的假陽性結果。
[Abstract]:Objective to study the method of rapid identification of dead and living adenovirus by EMA combined with polymerase chain reaction (PCR).Methods the virus with different dilution was inoculated into the monolayer cells with good growth, and the titer of the virus was calculated by CPE (cytopathic effect).Three kinds of adenovirus and Newcastle disease chicken plague virus were prepared into 106 PFU/mL mother liquor and diluted by gradient dilution. The DNA was extracted and amplified by PCR. The result of the amplification of the target fragment was observed.The inactivated adenovirus was treated with 0 渭 g / mL 70 渭 g / mL EMA and 150 渭 g/mL respectively. The DNA was extracted to observe the electrophoresis results of the target fragment, and the 107PFU / mL105PFUmL105PFUmL104PFUmL103pmL10 PFU/mL adenovirus was treated with 120 渭 g/mL EMA, and the results of PCR and gel electrophoresis were observed.Results positive bands were obtained after DNA PCR of 104 PFU/mL and above, but no positive bands were detected after DNA PCR of other titers, and the target bands were amplified from the DNA of 3 types of adenovirus (8 isolates).The DNA of Newcastle disease chicken plague virus did not amplify the target band of 120 渭 g/mL and 150 渭 g/mL EMA to inhibit the DNA amplification of inactivated adenovirus. The positive band of 120 渭 g/mL EMA did not affect the DNA amplification of 107PFU / mL 106PFU / mL 105 PFU/mL adenovirus live virus, and the positive band was obtained.Conclusion EMA-PCR method can be used to identify adenovirus rapidly and avoid the false positive results of PCR alone.
【作者單位】： 四川大學華西公共衛(wèi)生學院衛(wèi)生檢驗與檢疫系;
【基金】：大學生創(chuàng)新訓練計劃(No.201410610119)資助
【分類號】：R440

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