摘要
根据GenBank登录的肠炎沙门菌(SE)种特异性DNA序列,设计合成了1对能特异性扩增293 bp DNA片段的引物,首次建立了SE的种特异性聚合酶链式反应(PCR)诊断方法。该方法仅需10 h即可获得结果且只能特异性扩增SE,而对照菌株的扩增结果均为阴性;检测SE的最低DNA模板量为50 ng/L,最少检测到的细菌数为19CFU/mL。对10只SE人工感染死亡雏鸭的心、肝和粪便的检测结果与细菌分离的结果符合率为100%,而对脾、脑、法氏囊的检测阳性率极显著(P<0.01)高于细菌分离率;应用该技术对临床送检的228只疑似SE感染死亡鸭病例检测结果也显著高于细菌分离法。研究结果表明,建立的PCR方法检测SE具有较好的特异性和敏感性,比传统的细菌分离鉴定更加快捷、准确,可用于SE感染疑似病例的检测及其分子流行病学调查。
Based on the species specific DNA sequence of SE from GenBank, the specific primers which showed 293 bp fragment after PCR was designed and the species specific PCR method for SE detection was developed initially. This method required only less 10 hours until results were obtained. Applied this assay to several common Salmo- nellas and pathogenic bacterias of duck including S. choleraesuis, S. typhimurium, S. paratyphi, S. pullorunl, S. anatum, S. gallinarum, S. dublin ,Riemerella anatipesti fer , P. multocida, E. coli indicated that it was highly specific to SE,there was no amplification with no SE genera. Also, a series of sensitivity experiments were performed and proved that the detection limit of this method was 50 ng/L for genomic DNA, 19 CFU/mL for bacterial number. 10 artificial infected cases which were infected with SE were chosen to identify the accuracy and sensitivity of this PCR method after death. Results showed that the detection of heart,liver and faeces were identical between bacteria isolating and PCR. Used this assay to detect the spleen,, brain, bursa of fabricius indicated that the positive rates were significant difference（P〈0.01）, the PCR method was more sensitive. Meanwhile, suspected with SE infection of 228 dead-ducks were subjected to this PCR method showed that it was more sensitive than bacteria isolating method too. Thus,this PCR assay provides a more rapid and accurate method for identification of SE than traditional isolation methods. It will help to the isolation and identification of SE,detecting of SE infection suspected cases and investigation of molecular epidemiology.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2009年第2期153-157,共5页
Chinese Journal of Veterinary Science
基金
国家科技支撑计划资助项目(2007Z06-017)
教育部“长江学者和创新团队发展计划”资助项目(IRTO853)
教育部“新世纪优秀人才支持计划”项目(NCET-06-0818)
四川省基础研究重大项目(2008JO0003/2008JY0100/2008JY0102)
四川省重点建设学科项目(SZD0418)
