金黄色葡萄球菌的多重PCR快速鉴定方法

Rapid detection of Staphylococcus aureus by a multiplex PCR assay

目的建立多重PCR快速鉴定金黄色葡萄球菌检测方法,并了解nuc和nucA基因在食源性金黄色葡萄球菌中的分布状况。方法设计nuc、nucA和16S rDNA基因的引物,利用单重PCR方法和测序验证引物的特异性;建立多重PCR快速检测方法,并应用此方法对23株食源性金黄色葡萄球菌、15株其他种属细菌以及一起食物中毒样品的增菌液进行检验,以评价该方法的特异性和灵敏性。结果利用单对引物对实验室的4个菌株进行盲筛,出现的条带均为单一条带,且与目的片段长度一致,测序结果显示扩增片段为目的基因片段。建立的多重PCR方法对金黄色葡萄球菌有很好的特异性和灵敏性,灵敏度达100 cfu/μl,研究所涉及的23株食源性金黄色葡萄球菌nuc和nucA基因均为阳性。结论该方法简便、快速、特异性好、灵敏度高,适用于金黄色葡萄球菌的快速鉴定。

Objective To establish a multiplex PCR method for identifying Staphylococcus aureus and to study the distribution of tbermonuclease genes in foodborne Staphylococcus aureus. Methods Three primers were designed for detecting nuc, nucA and 16S rDNA genes. The specificities of three primers were tested by single PCR method and sequencing. The specificity and sensitivity of the multiplex PCR method were tested. Results The specificities of three primers met the test requirement. The specificity met the test requirement,and the detection limits for DNA template were 100 cfu/μl. All the Staphylococcus aureus strains had nuc and nucA genes. Conclusion The multiplex PCR method is fast,specific,rigorous, sensitive and can be used for identifying a large number of Staphylococcus aureus strains simultaneously.