食品中金黄色葡萄球菌DNA环介导恒温扩增快速检测方法的建立与应用  

Establishment of Rapid Method for Detecting Staphylococcus aureus in Food by Loop-Mediated Isothermal Amplification

作　　者：徐义刚 李苏龙 李丹丹 姜艳春 谢晓峰 

机构地区：[1]黑龙江省出入境检验检疫局检验检疫技术中心,哈尔滨150001 [2]海南省出入境检验检疫局检验检疫技术中心,海口570125

出　　处：《中国农业科学》2010年第8期1655-1663,共9页Scientia Agricultura Sinica

基　　金：国家质检总局科技计划项目(2008IK162);黑龙江省博士后基金项目(LBH-Z09)

摘　　要：【目的】金黄色葡萄球菌是一种人畜共患病的重要致病菌,主要经食品传播,引起食物中毒、组织及器官的化脓性炎症,建立快速、准确的检测方法对预防和控制金黄色葡萄球菌的传染具有重要意义。【方法】DNA环介导恒温扩增(loop-mediatedisothermalamplification,LAMP)是一种新颖的快速、高灵敏核酸扩增技术,本研究利用该技术建立金黄色葡萄球菌LAMP检测方法。基于金黄色葡萄球菌高度保守的Sa442基因序列,设计4条特异性LAMP扩增引物,两条外引物F3和B3及两条内引物FIP和BIP,在BstDNAPolymerase作用下,65℃恒温水浴中,对26个种类共45株细菌进行扩增,所试的20株金黄色葡萄球菌均为LAMP阳性,说明所设计的LAMP引物具有高度特异性。【结果】本LAMP方法对纯培养的金黄色葡萄球菌检测灵敏度为25CFU/mL,对污染食品中金黄色葡萄球菌的检测灵敏度为42CFU/g,40—60min内即可完成检测。检验检疫实践证明,利用本LAMP方法对958份肉类、蛋奶及其制品、人工污染样品等进行检测,检出115份LAMP阳性,与国标法(GB)检测结果一致。【结论】本LAMP方法操作简便、特异性强、灵敏度高,具有良好的实用性。[ Objective ] Staphylococcus aureus is an important pathogen of zoonose, mainly initiates its transmission through food contamination and causes food poisoning, suppurative inflammation of tissues and organs. Thus, the development of a technique that can identify the microbe quickly and accurately is of great importance. [Method] Loop-mediated isothermal amplification （LAMP） for DNA is a novel nucleic acid amplification method with rapidity and high sensitivity. In this study, a LAMP method for detecting Staphylococcus aureus was developed. Four primers including two outer primers F3, B3 and two inner primers FIP, BIP were designed according to highly conserved Sa442 gene of Staphylococcus aureus. Genomic DNA extracted from 45 bacteria strains including 20 Staphylococcus aureus strains and 25 other bacterial strains were amplified using the primers with Bst DNA polymerase at 65℃, only Staphylococcus aureus strains were LAMP positive result, which indicated that the LAMP primers designed were highly specific for target bacteria. [ Result] The detection sensitivity of the LAMP method for Staphylococcus aureus purely cultured was 25 CFU/mL and for Staphylococcus aureus in contaminated food was 42 CFU/g, which could complete the test in 40-60 min. In practice, 115 pieces of samples detected from 958 pieces of meat samples, egg, dairy products and artificial contamination samples were positive using the LAMP method, which accorded with the detection result by GB [ Conclusion ] The LAMP assay established in this study is a sensitive, rapid and simple tool for detecting Staphylococcus aureus.

关 键 词：金黄色葡萄球菌 Sa442基因 环介导恒温扩增 快速检测 

分 类 号：TS207.4[轻工技术与工程—食品科学]