用RT-PCR法检测食品中诺沃克样病毒

The Detection of Norwalk Virus in Food by RT-PCR

使用RTPCR法对贝类样品中的诺沃克样病毒进行了检测。对食品中不同的病毒富集方法进行了研究,评价了CHCl3和Frezon去除RTPCR抑制剂的效果,筛选出最佳的病毒富集方法即2次PEG6000(终浓度12%PEG6000,03mol/LNaCl)沉降病毒,并使用CHCl3除去RTPCR抑制剂。此富集方法的回收率为135%。另外,对不同的RNA提取方法的灵敏度进行了比较,最终确认硅胶膜法为简便、快速、有效的RNA提取方法。并且,使用了Superscript反转录酶进行反转录,提高了检测的灵敏度。选用了289和290混合引物进行食品中NLVs的检测,这一引物组合可同时检测Ⅰ型和Ⅱ型NLVs,扩增出1个319bp的特异性片段。通过以上条件的优化,建立了通过RTPCR检测贝类中NLVs的有效方法。该方法的NLVs最低检出限为10×103RT-PCR50/15g。对32件贝类样品进行了NLVs的检测,其中2份毛蚶中被检出NLVs。

The detection of Norwalk Virus (NLVs) in shellfish by RT-PCR was studied in this paper. Different methods for the recovery of virus from foods were studied and the effect of using chloroform and Frezon to remove RT-PCR inhibitor were evaluated. The best viral concentration method, which included twice PEG6000 precipitations (the finial concentration was 12% PEG 6 000, 0.3?mol/L NaCl ) and chloroform extraction to remove RT-PCR inhibitor, was selected. The virus recovery followed this processing procedure is 13.5%. In addition, we compared different methods for RNA extraction and decided to use the silica gel membrane to extraction, which was simple, rapid and effective. Moreover, the detection limit had been improved by the use of Superscript reverse transcriptase. We selected a broad primer pair (289 and 290), which was designed for the detection of both NLVs genogroupⅠand genogroup Ⅱ, to detect NLVs in foods and produced specific products of 319bp. Under the optimal of condition, we established an effective method of detecting NLVs in shellfish. The detection limit is 1.0×103 RT-PCR_~50 /1.5?g. 32 shellfish samples were detected for NLVs and two of them were found positive.