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目的本研究比较了多种可能适用于生食水产鱼肉中诺如病毒(No V)的提取方法。方法人工污染GII型No V三文鱼肉样品,通过病毒洗脱、浓缩、核酸提取,以及实时荧光定量反转录-聚合酶链式反应(RT-q PCR)检测等步骤的比较和优化,提高样品中No V的检测成功率和回收率。结果采用Tri/Glycine/Beef extract缓冲溶液洗脱、PEG/Na Cl溶液沉降、增加氯仿-正丁醇(1∶1,V/V)溶液萃取等步骤,可以有效富集样品中的No V,并有效去除样品中的PCR抑制成分。优化后的检测方案可以将人工污染GII型No V的鱼肉样品的回收率由不足3%提高至15.15%,检测灵敏度可以达到17.3 RT-PCRU/25 g。结论优化后的病毒提取方法具有良好的耐用性和重现性,适合于生食水产鱼肉中No V的快速检测。
Aims This study compared a number of extraction methods that may be suitable for Norovirus (No V) in raw fish. Methods Samples of GV No V salmon meat were artificially contaminated by the steps of virus elution, concentration, nucleic acid extraction and RT-qPCR. No V in the detection of success and recovery rate. The results showed that No V was effectively enriched by using Tri / Glycine / Beef extract as elution buffer and PEG / Na Cl as a solution, and adding chloroform-n-butanol (1: 1, V / V) And effectively remove PCR inhibitors from the sample. The optimized detection scheme can increase the recovery rate of fish samples with artificial contamination type GII No V from less than 3% to 15.15% and the detection sensitivity can reach 17.3 RT-PCRU / 25 g. Conclusion The optimized virus extraction method has good durability and reproducibility and is suitable for the rapid detection of No V in raw fish.