PCR和毛细管电泳技术检测沙门氏菌等5种病原菌的方法研究  

Investigation on the multiplex PCR in detection of five types of bacteria

作　　者：肖勇[1] 沙丹[1] 凌霞[1] 孙纳[1] 管红霞[1] 张敬平[1] 

机构地区：[1]无锡市疾病预防控制中心,江苏无锡214023

出　　处：《现代预防医学》2012年第14期3611-3614,共4页Modern Preventive Medicine

基　　金：国家卫生部资助项目(WKJ2006-2-10)

摘　　要：目的采用多重PCR技术检测结合毛细管电泳成像技术建立快速检测沙门氏菌、志贺氏菌、副溶血性弧菌金黄色葡萄球菌和大肠杆菌O157的方法。方法根据沙门氏菌hilA基因、志贺氏菌ipaH基因、副溶血性弧菌TDH基因、金黄色葡萄球菌的pSa-442 Sau3AI fragment基因及大肠杆菌O157的rfbE基因设计异性特PCR引物,被检样品经4 h振荡培养后金属浴裂解制备DNA模板,使用全自动毛细管电泳核酸检测系统分析PCR扩增产物。结果在580 bp、423bp、257 bp、245 bp和194 bp处分别出现预期的特异性DNA条带,且无非特异扩增条带出现。敏感性试验显示在模拟标本中的检测灵敏度,沙门氏菌为101-2cfu/ml、志贺氏菌为101cfu/ml、副溶血性弧菌为102cfu/ml、金黄色葡萄球菌为102cfu/ml、大肠杆菌O157为101cfu/ml。结论该方法操作方便、特异性和灵敏度高、分辨率高,可同时完成5种病原菌的检测,在公共卫生突发事件中具有实用价值。OBJECTIVE To establish a multiplex polymerase chain reaction（PCR）assay in detection of Salmonella,Shigella,Vibrio parahaemolyticus,Staphylococcus aureus and Escherichia coli O157.METHODS Based on the gene sequences of hilA gene in Salmonella,ipaH gene in Shigella,TDH gene in Vibrio parahaemolyticus,pSa-442 Sau3AI fragment gene Staphylococcus aureus and rfbE gene Escherichia coli O157,three pairs of primers were designed.Genomic DNA was extracted by metal bath after shaking culture for 4 hours,and the PCR-amplified products were analyzed by automatic capillary electrophoresis.RESULTS The predicted specific DNA amplication bands were demonstrated at sequences of 580 bp,423 bp,257 bp,245 bp and 194 bp.Assay on sensitivity of this multiplex PCR revealed that it could be detected as little as 101-2 cfu/ml from salmonella,101 cfu/ml from shigella,102 cfu/ml from vibrio parahaemolyticus,102 cfu/ml from Staphylococcus aureus and 101 cfu/ml from Escherichia coli O157 in simulation experiments.CONCLUSION The multiplex PCR method is a method with the aspects of rapid,specific and sensitive as well as high distinguishability,which could be useful for the rapid detection of food-borne bacterial pathogens.

关 键 词：多重PCR 振荡培养 毛细管电泳 食品检验 

分 类 号：R378.22[医药卫生—病原生物学]