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目的建立水产品中5种常见致病菌的快速高分辨检测手段,提高检测的效率和准确性。方法筛选沙门菌、金黄色葡萄球菌、单核细胞增生李斯特菌、副溶血性弧菌、霍乱弧菌的特异基因,通过参阅文献和自行设计,分别确定invA、nuc、prfA、tlh、owpW为检测基因,优化引物浓度和引物组合,进行多重PCR扩增,产物经DHPLC进行快速检测。优化确定的方法通过重现性试验,特异性试验,灵敏度试验,以及人工污染水产品样品的检测。结果 DHPLC检测出峰顺序依次为invA、nuc、prfA、tlh、owpW,扩增片段大小为284 bp、329 bp、388 bp、450 bp、588 bp,此方法具有良好的重现性和特异性,灵敏度分别可达188 CFU/ml、670 CFU/ml、280 CFU/ml、240 CFU/ml、580 CFU/ml,不同水产品的状态、成分以及增菌液类型对后续检测不存在影响。结论本方法可以很好的满足实际食品微生物检测的要求。
Objective To establish a rapid high-resolution detection method for five common pathogens in aquatic products to improve the efficiency and accuracy of detection. Methods The specific genes of Salmonella, Staphylococcus aureus, Listeria monocytogenes, Vibrio parahaemolyticus and Vibrio cholerae were screened. The invA, nuc, prfA, tlh and owpW were determined by referring to the literature and self-designed Detecting the gene, optimizing the primer concentration and primer combination, performing multiplex PCR amplification, and the product was detected rapidly by DHPLC. Optimization and determination of the method through the reproducibility test, specificity test, sensitivity test, as well as artificial pollution aquatic product samples. Results The peak sequence of DHPLC was invA, nuc, prfA, tlh and owpW. The amplified fragments were 284 bp, 329 bp, 388 bp, 450 bp and 588 bp in length. This method has good reproducibility and specificity, Sensitivity of 188 CFU / ml, 670 CFU / ml, 280 CFU / ml, 240 CFU / ml, 580 CFU / ml, respectively, the status of aquatic products, composition and enrichment broth type did not affect the follow-up test. Conclusion This method can well meet the requirements of actual food microbiological testing.