Development and Evaluation of a Multiplex Real-Time Polymerase Chain Reaction Procedure to Clinically Type Prevalent Salmonella enterica Serovars
Development and Evaluation of a Multiplex Real-Time Polymerase Chain Reaction Procedure to Clinically Type Prevalent Salmonella enterica Serovars
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DOI：
10.2353/jmoldx.2010.090036
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发表时间：
2010-03-01
影响因子：
4.1
通讯作者：
Cardona-Castro, Nora
中科院分区：
文献类型：
--
作者：
Munoz, Nelida;Diaz-Osorio, Miguel;Cardona-Castro, Nora
建立了一种多重实时荧光聚合酶链式反应方法，用于鉴定肠炎沙门氏菌亚种临床分离株。肠系膜。利用编码0、H和Vi抗原的rib、flc、fljB和viab组基因设计了15对针对rfbJ、wzx、flc、fljB、wcdb、SDF-I序列和invA基因的TaqMan探针，并将其作为内部扩增对照。将引物和探针以不同的方式组合成六组。第一轮反应使用其中的两组来检测沙门氏菌0：4、0：9、0：7、0：8和0：3、10个血清组。一旦确定了血清组，使用鞭毛抗原1基因1，2；e，h；g，m；d；e，n，x和z(10)以及Vi基因的引物和探针的第二轮反应的结果被用来识别单个血清型。该程序使用18个沙门氏菌标准菌株和其他肠道细菌进行标准化。对随机抽取的267株血清分型沙门氏菌临床分离株进行了可靠性和敏感性的评估。该方法的敏感性为95.5%，特异性为100%。因此，我们的技术是一种快速、敏感、可靠和特异的鉴定肠杆菌血清型的方法，并可与传统的血清学分型结合使用。可以使用其他的引物和探针组合来增加可识别的血清型的数量。(J摩尔诊断2010年，12：220225doi：10.2353/jmoldx.2010.090036)
A multiplex real-time polymerase chain reaction procedure was developed to identify the most prevalent clinical isolates of Salmonella enterica subsp. enterica. Genes from the rib, fliC, fljB, and viaB groups that encode the 0, H, and Vi antigens were used to design 15 primer pairs and TaqMan probes specific for the genes rfbJ, wzx, fliC, fljB, wcdB, the sdf-I sequence, and invA, which was used as an internal amplification control. The primers and probes were variously combined into six sets. The first round of reactions used two of these sets to detect Salmonella 0:4, 0:9, 0:7, 0:8, and 0:3,10 serogroups. Once the serogroups were identified, the results of a second round of reactions that used primers and probes for the flagellar antigen 1 genes, 1,2; e,h; g,m; d; e,n,x; and z(10), and the Vi gene were used to identify individual serovars. The procedure was standardized using 18 Salmonella reference strains and other enterobacteria. The procedure's reliability and sensitivity was evaluated using 267 randomly chosen serotyped Salmonella clinical isolates. The procedure had a sensitivity of 95.5% and was 100% specific. Thus, our technique is a quick, sensitive, reliable, and specific means of identifying S. enterica serovars and can be used in conjunction with traditional serotyping. Other primer and probe combinations could be used to increase the number of identifiable serovars. (J Mol Diagn 2010, 12:220-225: DOI: 10.2353/jmoldx.2010.090036)