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建立了粪便中诺如病毒逆转录PCR-毛细管电泳-激光诱导荧光快速检测方法。根据诺如病毒核酸保守序列选择引物,扩增出特异的PCR产物;采用响应曲面法进行毛细管电泳条件优化,以含有DNA荧光染料SYBR Gold的0.5%甲基纤维素为筛分介质,通过激光诱导荧光法检测诺如病毒的PCR产物。在优化的毛细管电泳条件下,9 min内可完成诺如病毒PCR产物的检测。扩增产物测序后,与基因库中诺如病毒的序列进行同源性比对,一致性达99%。迁移时间的日内和日间相对标准偏差分别为1.1%~1.3%和1.8%~2.6%,已用于粪便中诺如病毒的快速检测。
A rapid reverse transcription PCR-capillary electrophoresis-laser-induced fluorescence detection method was developed for stool samples. The specific PCR products were amplified according to the conservative sequence of Norovirus nucleic acid. The response surface methodology was used to optimize the conditions of capillary electrophoresis. The medium containing 0.5% methylcellulose containing SYBR Gold DNA as screening medium was induced by laser Fluorescence detection of Norovirus PCR products. Under optimized capillary electrophoresis, the detection of Norovirus PCR product was completed within 9 min. After sequencing of the amplified product, homology comparison with Norovirus in the GenBank was performed, and the identity was 99%. The relative standard deviations (RSDs) of intra-day and inter-day migration were 1.1% -1.3% and 1.8% -2.6% respectively, which were used for the rapid detection of norovirus in stool.