双重PCR-DHPLC技术快速检测水产品中金黄色葡萄球菌

根据编码金黄色葡萄球菌肠毒素A的sea基因、编码耐热核酸酶的nuc基因为目的基因，设计两对特异性引物，利用聚合酶链式反应结合变性高效液相色谱技术，建立水产品中金黄色葡萄球菌的双重PCR-DHPLC检测方法。以30株参考菌株进行特异性实验，除所试8株金葡菌出现目的片段和阳性吸收峰外，其余菌株均未检测到目的片段与阳性吸收峰，表明该方法具有良好的特异性。灵敏性实验结果表明，检测灵敏度达到菌液浓度50CFU/mL，比普通的凝胶电泳高一个数量级。利用建立的双重PCR-DHPLC检测方法对240份水产品进行检测，共检出22株金黄色葡萄球菌，检出率为9.2%，其中含有肠毒素基因有8株，占总样本的3.3%，与国标法检出阳性率比较无显著差异，证明该方法具有良好的实用性。实验证明，本研究建立的双重PCR-DHPLC方法不仅可以特异、灵敏、简便快速且高通量的实现对水产品中金葡菌的检测，而且也可通过对肠毒素基因的分析，方便地判断出该菌株致病性的强弱。

Rapid Detection of Staphylococcus aureus in Aquatic Products by Duplex PCR-DHPLC

Staphylococcus aureus is an important pathogen that can cause food poisoning. Two pairs of specific primers were designed according to the sequences of the sea gene (staphylococcal enterotoxin A) and the nuc gene from Staphylococcus aureus for the development of a duplex polymerase chain reaction-denaturing high performance liquid chromatography (PCRDHPLC) to detect Staphylococcus aureus strains in aquatic products. The positive and negative detection using this duplex PCR-DHPLC method was observed in 8 and 22 out of 30 strains, respectively. This method had high sensitivity with the detection limit of 50 CFU/mL, which revealed an improvement by 10 fold compared with the ordinary gel electrophoresis. Based on the duplex PCR-DHPLC method, 240 aquatic products were analyzed and showed a positive detection rate of 9.2% for Staphylococcus aureus contamination, which did not exhibit significant difference in positive detection rate from national standard approach. Therefore, duplex PCR-DHPLC can be used for specific, sensitive, rapid and high throughput detection of Staphylococcus aureus in aquatic products, and also can be used to determine the strength of pathogenicity of Staphylococcus aureus through analysis of the staphylococcal enterotoxin gene.