摘要
为建立同时快速检测沙门氏菌和大肠杆菌078的方法，本研究根据沙门氏菌invA基因和大肠杆菌O78O-抗原特异基因序列设计引物，并在细菌16SrDNA区设计内参引物，通过各项参数调整优化，建立了能够直接从样品中同时检测沙门氏菌和大肠杆菌078的多重PCR检测方法。结果显示：该方法可以同时扩增出0781113bp和沙门氏菌821bp的特异片段以及细菌16SrDNA527bp的通用片段，而对于其他13种肠道致病菌只能扩增出527bp的细菌同源片段，具有较强的特异性。该体系检测沙门氏菌和大肠杆菌O78的最低检出限分别为103cfu／mL和102 cfia／mL。增菌5h，鸡粪模拟污染菌样品中沙门氏菌和大肠杆菌078的检测灵敏度分别为10 3cfu／mL和10 2cfu／mL。本研究建立的检测方法能够在8h内完成鸡粪样品中两种靶标菌的快速检测，对于沙门氏菌和大肠杆菌078的鉴别诊断和快速检测具有重大意义。
To establish protocols for simultaneous detection of Salmonella sp. and Escherichh coli 078, a multiplex PCR assay was developed with primers designed according to invA gene of Salmonella sp., O-antigen gene of E.coli 078 and 16S rDNA as housekeeping gene of bacteria. The assay was highly specific to amplify the DNA fragments from Salmonella sp. and E.coli 078 with size of 821 bp and 1,113 bp, respectively, but not from other 13 related bacteria. Universal fragment sized 527 bp of 16S rDNA was amplified for all tested bacteria to avoid false-negative. In addition, the sensitivity of the multiplex PCR assay was l0t cfu/mL for Salmonella sp. and 102 cfu/mL for E.coli 078, respectively. Moreover, the multiplex PCR assay was able to detect Salmonella sp. and E.coli 078 in fresh chicken manure samples as low as 103 cfu/mL for Salmonella sp. and 102 cfu/mL for E.coli 078 within 8 hrs including 5 hrs for bacteria enrichment culture. Therefore, this multiplex PCR assay has a potential application in rapid detection and differential diagnosis of Salmonella sp. and E.coli 078 mixed infections.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2016年第1期58-62,共5页
Chinese Journal of Preventive Veterinary Medicine
