摘要
目的采用xMAP液相悬浮芯片检测方法,建立一管同时检测金黄色葡萄球菌、沙门菌、副溶血弧菌、单增李斯特菌、大肠杆菌O157∶H7、创伤弧菌和空肠弯曲菌的快速筛查方法。方法根据GenBank公布的金黄色葡萄球菌肠毒素nuc基因、沙门菌侵袭性基因ssaR、副溶血弧菌通用基因toxR、单增李斯特菌的hlyA基因、大肠杆菌O157∶H7的rfbE基因、创伤弧菌的vvh基因和空肠弯曲菌的gyrA基因,设计特异性引物和探针,利用上游引物和生物素标记的下游引物对7个靶序列进行不对称实时PCR扩增,7重实时PCR扩增产物和制备的7种偶联探针的微球在同一体系中进行杂交,最后利用藻红蛋白和生物素的亲和作用报告荧光,从而建立7种食源性致病菌的xMAP液相悬浮芯片检测法。结果 xMAP液相悬浮芯片体系检测140株菌株,均出现特异性荧光中值信号,7种食源性致病菌检测互不干扰。DNA和菌液检出限分别为1～100ng和105～108cfu/ml对56份各类食品标本进行检测,检出金黄色葡萄球菌2份、沙门菌4份、副溶血弧菌7份、单增李斯特菌1份、创伤弧菌3份,未检出大肠杆菌O157∶H7和空肠弯曲菌,此结果与传统方法分离结果相一致。结论xMAP液相悬浮芯片技术从样品处理到检测结果需3.5h左右,该方法可应用于食品安全风险监测中食源性致病菌的快速筛查和食源性致病菌的分型鉴定。
Objective To develop an assay for the simultaneous detection of 7 common foodborne pathogens with xMAP liquid chip in a single-tube reaction.Methods Seven specific primers and probes were designed and synthesized based on the target gene sequences from GenBank.Target bacterial sequences were amplified by asymmetric PCR.The biotinylated products were hybridized to seven probe beads in a multiplex reaction and analyzed by using streptavidin conjugated to a fluorescent reporter molecule.The developed liquid chip xMAP assay was used to test 140 strains of bacteria and then 56 food samples.Results No cross-reaction and false signals were observed.The detection limit was 1-100 pg and 105-106 cfu/ml.The results tested by xMAP were in accordance with the traditional culture method.Conclusion The processing of xMAP liquid chip assay,including DNA preparation and sample detection,could be finished within 3.5 hours and could be applied to the classification and identification of foodborne pathogens in the food safety monitoring.
出处
《卫生研究》
CAS
CSCD
北大核心
2012年第1期96-101,共6页
Journal of Hygiene Research
基金
深圳市食品安全委员会项目(No.SZSAW2010002)
