摘要
建立了一种快速、灵敏、高度特异的检测变形杆菌属(Proteus)的方法——环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)。通过对GenBank中变形杆菌属atpD基因序列(AX109601)进行分析,设计了6条引物(2条内引物、2条外引物、2条环引物),在Bst大片段聚合酶的作用下,对模板DNA进行梯状等温扩增,产生白色沉淀,并且优化LAMP的反应体系,检验其灵敏性与特异性。扩增产物用限制性内切酶Psp1406Ⅰ(AclⅠ)酶切,观察酶切片段大小,验证方法的正确性。结果表明,该方法在61℃保温50 min即可完成,最低检测限为5.4 CFU/mL,灵敏度高于常规PCR 10倍。对其它食品病原菌进行检测,结果均未出现目的条带。表明LAMP法检测变形杆菌属灵敏度高、特异性好,操作简便,无需特殊的仪器设备,有恒温加热设备就可以满足检测条件,极适合在我国广大基层实验室开展应用。
This established a rapid,sensitive,highly specific detection of Proteus method-loop-mediated isothermal amplification（loop-mediated isothermal amplification,LAMP）.Through analysis for Proteus atpD gene sequence（AX109601） in the GenBank,design of the six primers（two inner primers,two outer primers,two loop primers）,under the influence of the Bst large fragment polymerase,templateisothermal amplification of DNA for laddering,produce a white precipitate,and optimize the LAMP reaction system,to test its sensitivity and specificity.Amplification products were digested with restriction endonuclease Psp1406Ⅰ（AclⅠ） digested fragment size observed to verify this method.The results show that the insulation in the 61 ℃ 50 min to complete,and the minimum detection limit was 5.4 CFU/mL,more sensitive than conventional PCR 10 times.For other food pathogens,and the results were not there target band.That Proteus LAMP assay sensitivity,specificity,simple,no special equipment,constant temperature heating equipment to meet the test conditions,is very suitable for the majority of the grassroots in our laboratory on the application.
出处
《食品工业》
北大核心
2011年第10期106-109,共4页
The Food Industry
基金
河北省自然科学基金C2008000216
