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目的探讨采用叠氮溴化乙锭(EMA)联合聚合酶链反应(PCR)快速鉴别死活腺病毒的方法。方法将不同稀释度的病毒接种至生长良好的单层细胞上,通过细胞病变效应(CPE)的发生情况计算病毒滴度;将3种腺病毒和新城疫鸡瘟病毒分别制备成106 PFU/mL的母液并梯度稀释备用,提取DNA后用PCR扩增后进行凝胶电泳,观察目的片段的扩增结果;分别用0μg/mL、70μg/mL、120μg/mL和150μg/mL的EMA处理灭活后的腺病毒,提取DNA进行PCR,观察目的片段的电泳结果;用120μg/mL的EMA处理107 PFU/mL、106 PFU/mL、105PFU/mL、104 PFU/mL、103 PFU/mL的腺病毒,观察PCR和凝胶电泳结果。结果 104 PFU/mL及以上滴度的病毒DNA PCR后得到阳性条带,其他滴度的病毒DNA PCR后未检测到阳性条带;3个型别的腺病毒(共8个分离株)的DNA均扩增出目的条带,新城疫鸡瘟病毒的DNA未扩增出目的条带;120μg/mL及150μg/mL的EMA抑制灭活腺病毒DNA扩增,未得到阳性条带;120μg/mL EMA不影响107 PFU/mL、106 PFU/mL、105 PFU/mL的腺病毒活病毒DNA扩增,得到阳性条带。结论本研究证实EMA-PCR方法可快速鉴别死活腺病毒,能有效避免单纯PCR检测腺病毒产生的假阳性结果。
Objective To investigate the rapid identification of live and living adenovirus by ethidium bromide ethidium bromide (EMA) combined with polymerase chain reaction (PCR). Methods Different dilutions of virus were inoculated into well-grown monolayer cells and virus titers were calculated by the occurrence of cytopathic effect (CPE). Three adenovirus and Newcastle disease virus were prepared respectively at 106 PFU / mL The mother liquor was diluted and stored in a gradient manner. DNA was extracted and amplified by PCR. The PCR products were analyzed by gel electrophoresis to observe the amplification results. After inactivation with 0, 70, 120, and 150 μg / mL EMA, Adenovirus, DNA was extracted for PCR, and electrophoresis results of the target fragment were observed. Adenovirus of 107 PFU / mL, 106 PFU / mL, 105 PFU / mL, 104 PFU / mL and 103 PFU / mL was treated with 120 μg / Observe PCR and gel electrophoresis results. Results Positive DNA bands were obtained after PCR with 104 PFU / mL titer DNA and no positive bands were detected after PCR with other titer DNAs. DNA of 3 types of adenovirus (8 isolates) The target bands were amplified and the bands of the newcastle disease virus were not amplified. EMA of 120μg / mL and 150μg / mL inhibited the amplification of inactivated adenovirus DNA, and no positive bands were obtained. 120μg / mL EMA The positive bands were obtained by amplification of live virus DNA without affecting 107 PFU / mL, 106 PFU / mL, 105 PFU / mL. Conclusion The present study confirms that EMA-PCR can rapidly identify live and dead adenovirus and can effectively prevent false positive results of adenovirus detection by simple PCR.