PCR-焦磷酸测序检测金黄色葡萄球菌研究  

A Research on Detecting Staphylococcus Aureus by PCR-Pyrosequencing

作　　者：许龙岩[1] 袁慕云[1] 唐勤[2] 邹志飞[1] 相大鹏[1] 

机构地区：[1]广东出入境检验检疫局,广东广州510623 [2]南方医科大学

出　　处：《检验检疫学刊》2013年第1期29-33,共5页Journal of Inspection and Quarantine

基　　金：广东省科技项目计划(2010B020316007)

摘　　要：[目的]研究从DNA碱基序列水平检测金黄色葡萄球菌(Staphylococcus aureus)的方法。[方法]根据金黄色葡萄球菌凝固酶基因设计扩增引物和测序引物,先用PCR特异性地扩增目的片段,再制备单链模板,在测序引物引导下用焦磷酸测序法检测DNA碱基序列,检测的DNA碱基序列为GCCTGTGGGTACT-TCTCCTGCCAGTATAAT,则判断为金黄色葡萄球菌。[结果]20株金黄色葡萄球菌均扩增出大小215bp的DNA片段,而其他对照菌株未扩增出DNA条带。焦磷酸测序结果,20株金黄色葡萄球菌均测出与预期相符的DNA碱基序列,而其他对照菌株未测出DNA碱基序列。[结论]建立的方法特异性高,整个试验可在21-27h完成,是快速检测金黄色葡萄球菌的有效手段,可用于食品中金黄色葡萄球菌的检测。A research is performed on detecting Staphylococcus aureus by DNA base sequence. First, we design a pair of PCR primers and a sequencing primer according to the Coagulase gene of Staphylococcus aureus. Then the target fragment was specifically amplified by PCR and a single - stranded DNA templates from the PCR products was prepared. Finally, under the guidance of the sequencing primer, pyrosequencing was used to sequence DNA base sequences. Once we obtained the DNA base sequences GCCTGTGGGTACTTCTCCTGCCAGTATAAT, Staphy-lococcus aureus was judged. PCR results showed that 215bp DNA fragments of 20 Staphylococcus aureus strains had be amplified, while the other control strains had not. Furthermore, pyrosequencing results of 20 Staphylococcus aureus were in line with the expected DNA base sequence, while the other control strains did not. This newly established method is highly accurate and the whole process can be completed within 21-27hrs. The method is an effective means for rapid detection of Staphylococcus aureus and can be used in the detection of Staphylococcus aureus in food.

关 键 词：金黄色葡萄球菌 PCR 焦磷酸测序 DNA碱基序列 凝固酶 

分 类 号：Q939.97[生物学—微生物学]