Real-time PCR method for Salmonella spp. targeting the stn gene
Real-time PCR method for Salmonella spp. targeting the stn gene
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DOI：
10.1111/j.1365-2672.2006.03079.x
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发表时间：
2007-02-01
影响因子：
4
通讯作者：
Feist, M. D.
中科院分区：
文献类型：
--
作者：
Moore, M. M.;Feist, M. D.
建立沙门氏菌的实时荧光PCR检测方法。通过用肠道沙门氏菌（Salmonella enterica）cDNA序列的BLAST发现Bongori沙门氏菌（Salmonella bongori）基因组中存在cDNA。人工比对的cDNA序列表明，Salm. bongori与Salm的同源性为88%。肠设计了两条引物（stnL-433和stnR-561）和一条探针（stnR-452），以靶向DNA中的保守区域，并满足5'-核酸酶测定的要求。对353个分离株（包括255个Salm）进行了引物和探针的检测。肠杆菌代表158个血清型，沙门氏菌14个。bongori代表12种血清型，84种非沙门氏菌代表31属56种。所有分离株均得到正确鉴定，但3株柠檬酸杆菌分离株除外，其为假阳性。该方法对沙门氏菌的检出限为3CFU/反应，对沙门氏菌的包容性为100%，特异性为96.4%，检出限为3CFU/反应。bongori，是两种沙门氏菌的有效靶标。沙门氏菌属实时荧光定量PCR是一种有效的沙门氏菌属鉴定方法。
To develop a real-time PCR assay for Salmonella spp. targeting the stn gene.The presence of stn in the Salmonella bongori genome was found by a BLAST with Salmonella enterica stn sequence. Manual alignment of stn sequences showed that Salm. bongori had 88% sequence identity with Salm. enterica. Two primers (stnL-433 and stnR-561) and a probe (stnP-452) were designed to target conserved regions in stn and meet the requirements of a 5'-nuclease assay. The primers and probe were evaluated against 353 isolates, including 255 Salm. enterica representing 158 serotypes, 14 Salm. bongori representing 12 serotypes and 84 non-Salmonella representing 56 species from 31 genera. All isolates were correctly identified, with the exception of three isolates of Citrobacter amalonaticus, which gave false positives. The limit of detection with cultured Salmonella was 3 CFU per reaction.The stn real-time PCR method had 100% inclusivity, 96.4% exclusivity and a level of detection of 3 CFU per reaction for cultured Salmonella spp.The study showed that stn is present in Salm. bongori and is a valid target for both species of Salmonella. The Salmonella s tn real-time PCR is a useful method for identifying Salmonella spp.