RT-PCR方法检测贝类中诺沃克样病毒的研究  

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

作　　者：张颖[1] 吴风亮[2] 

机构地区：[1]中国环境管理干部学院生态学系,河北秦皇岛066001 [2]河北农业大学农村发展学院,河北保定071001

出　　处：《食品科学》2008年第5期347-351,共5页Food Science

摘　　要：目的:本研究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技术检测贝类中诺沃克样病毒(NLVs)的方法。方法:利用脊髓灰质炎病毒作为参照毒株,优化了甘氨酸缓冲液-聚乙二醇(PEG)病毒浓缩方法;同时比较了异硫氰酸胍法、SDS-蛋白酶K法、Trizol-异丙醇法、试剂盒法四种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:本研究采用的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:本研究建立了一个灵敏度较高、较为有效的RT-PCR技术检测贝类中诺沃克样病毒的方法。Objective： To explore the methods of both virus enrichment and nucleic acid extraction for establishing a Norwalklike viruses （NL Vs） detection method by RT-PCR. Method： In this study, with poliovirus as the model to optimize glycine-PEG concentration method; The effects of 4 methods of extracting RNA from NLVs in sheUfish were compared; Detection of Norwalk- like viruses in commercial shellfish samples was performed, and positive samples were demonstrated by DNA sequencing. Results： This study conducted virus enrichment with pH 9.5 glycine buffer-16% polyethylene glycol （PEG） and RNA extraction with trizol-isopropanol. The recovery of viruses was 16.8% from seeded shellfish; and the detection limit was 8.1 ×10^2 RT-PCR50/ 5 g shellfish. NLVs in 3 of 25 samples were found by RT-PCR and validated by DNA sequencing. Conclusion： In this study, a NLVs detection method from shellfish is established, which is sensitive and available.

关 键 词：诺沃克样病毒 反转录PCR 贝类 检测 

分 类 号：TS254.7[轻工技术与工程—水产品加工及贮藏工程]