双重荧光逆转录-聚合酶链式反应与荧光逆转录-重组酶介导等温扩增快速检测食源性GI型、GII型诺如病毒

Rapid detection of food-borne GI and GII norovirus by dual fluorescent reverse transcription polymerase chain reaction and fluorescent reverse transcription recombinase-aided amplification

中文关键词:  荧光RT-PCR  恒温荧光RT-RAA  GI型诺如病毒  GII型诺如病毒

英文关键词:fluorescent reverse transcription polymerase chain reaction  isothermal fluorescence reverse transcription recombinase-aided amplification  GI norovirus  GII norovirus

中文摘要:

      目的 建立双重荧光逆转录-聚合酶链式反应(reverse transcription polymerase chain reaction, RT-PCR)与荧光逆转录-重组酶介导等温扩增(reverse transcription recombinase-aided amplification, RT-RAA)快速有效地检测食品中食源性GI型和GII型诺如病毒的方法。方法 根据GI型、GII型诺如病毒基因组保守序列设计引探, 经过一系列引探浓度筛选, 建立了双重荧光RT-PCR和恒温荧光RT-RAA两种检测方法, 分别从敏感性、特异性、稳定性、重复性等方面进行了比较研究; 同时将这两种方法应用于实际临床样本检测中, 并与GB 4789.42—2016《食品安全国家标准 食品微生物学检验 诺如病毒检验》进行比对验证。结果 建立的两种方法特异性良好, 敏感性均可达10 copies/μL; 双重荧光RT-PCR方法质粒梯度变异系数(coefficient of variation, CV)值在0.1%~1.5%区间, 恒温荧光RT-RAA方法CV值在1.0%~10.0%区间, 稳定性和重复性显示双重荧光RT-PCR优于恒温荧光RT-RAA检测方法; 31份诺如病毒阳性食品核酸样本均能检出, 且50份食品盲样检测结果与国标一致。结论 本研究成功建立了双重荧光RT-PCR与恒温荧光RT-RAA快速检测食源性GI型、GII型诺如病毒方法, 两种方法各有优缺点, 双重荧光RT-PCR稳定性和重复性好, 恒温荧光RT-RAA检测时间短, 仅20 min就能完成扩增, 可根据不同需求选择一种或两种作为食源性GI型、GII型诺如病毒的检测方法。

英文摘要:

      Objective To establish a rapid and effective method for the detection of food-borne GI and GII norovirus in food by double fluorescent reverse transcription polymerase chain reaction (RT-PCR) and fluorescent reverse transcription recombinase-aided amplification (RT-RAA). Methods Based on GI and GII norovirus genome conserved sequence design, two detection methods, dual fluorescence RT-PCR and constant temperature fluorescence RT-RAA, were established through a series of detection concentration screening, and the sensitivity, specificity, stability and repeatability were compared. At the same time, the two methods were applied to the actual clinical sample detection, and the comparison was verified with GB 4789.42—2016 National standard for food safety-Microbiology inspection-Norovirus inspection. Results The established two methods had good specificity and sensitivity of 10 copies/μL. The coefficient of variation (CV) of dual-fluorescence RT-PCR method was in the range of 0.1%-1.5%, and the CV of isothermal fluorescence RT-RAA method was in the range of 1.0%-10.0%. The stability and repeatability of the double fluorescence RT-PCR method were superior to the constant temperature fluorescence RT-RAA method. All 31 norovirus-positive food nucleic acid samples were detected, and the test results of 50 blind food samples were consistent with the national standard. Conclusion Dual fluorescence RT-PCR and thermostatic fluorescence RT-RAA methods for rapid detection of food-derived GI and GII norovirus are successfully established. Both methods have advantages and disadvantages. Dual fluorescence RT-PCR has good stability and repeatability, and the detection time of thermostatic fluorescence RT-RAA is short, and the amplification can be completed in only 20 min. According to different needs, one or two methods can be selected as the detection methods of food-borne GI and GII norovirus.