反转录PCR技术检测贝类中诺瓦克样病毒的研究

Studies on Reverse Transcription-Polymerase Chain Reaction Assay for Detection of Norwalk-like Virus in Shellfish

目的:从病毒富集和核酸提取两方面进行探索,旨在建立一个反转录(RT)PCR技术检测贝类中诺瓦克样病毒(NLVs)的方法。方法:利用脊髓灰质炎病毒作为参照毒株,优化甘氨酸缓冲液-聚乙二醇(PEG)病毒浓缩方法;同时比较异硫氰酸胍法、SDS-蛋白酶K法、Trizol-异丙醇法、试剂盒法4种RNA提取方法;对市售贝类样品进行了检测,并利用基因测序对阳性样品进行验证。结果:采用pH9.5甘氨酸缓冲液-16%聚乙二醇病毒浓缩法,病毒的回收率为16.8%;利用Trizol-异丙醇法提取RNA,检出限为8.1×102RT-PCR50/5g贝肉;实际检测贝类样品25件,其中3件样品为阳性,基因测序结果亦证实为阳性。结论:建立了一个灵敏度较高的RT-PCR检测贝类中诺瓦克样病毒的方法。

Objective： To investigate of both virus concentration and nucleic acid extraction, and to establish a detection method for determination of NLVs in shellfish by RT-PCR. Method：In this study, using Poliovirus as the model to optimize glycine-PEG concentration method; 4 methods of extracting RNA from NLVs in shellfish were compared; Norwalk-like viruses in commercial shellfish samples was detected, positive samples were also demonstrated by DNA sequencing. Results： This study involves virus concentration using pH9.5 glycine buffer-16% polyethylene glycol （PEG）, RNA extracting with Trizol-isopropanol. This method can receive 16.8% viruses from seeded shellfish; the detection limit was 8.1×10^2 RT-PCR50/5g shellfish. NLVs were found by using RT-PCR and DNA sequencing in 3 of 25 samples. Conclusion：In this study, a NLVs detection method for shellfish sample was established, which was sensitive and available.