Development of a multiplex PCR technique for detection and epidemiological typing of Salmonella in human clinical samples
Development of a multiplex PCR technique for detection and epidemiological typing of Salmonella in human clinical samples
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DOI：
10.1128/jcm.42.4.1734-1738.2004
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发表时间：
2004-04-01
影响因子：
9.4
通讯作者：
Garaizar, J
中科院分区：
文献类型：
--
作者：
Alvarez, J;Sota, M;Garaizar, J
我们建立了一种用于沙门氏菌检测和流行病学分型的多重PCR方法。设计了6套检测西班牙主要沙门氏菌血清型和噬菌体类型的引物。设计了一种内部扩增对照，用于检测PCR的抑制作用。获得的不同扩增图谱使我们能够检测到沙门氏菌，并区分临床流行的肠炎沙门氏菌、鼠伤寒沙门氏菌和亚种I型4、5、12：I：-。用该方法可以检测到鼠伤寒沙门氏菌的DT104和U302噬菌体类型的特异性条带。肠沙门氏菌血清型Hadar和其他C2血清型菌株表现出两种特殊的带型。在验证阶段，除了一些C2血清型菌株外，该方法对所有研究的血清型都是可重复性的。将该技术应用于临床粪便标本，检测肠炎和鼠伤寒流行血清型的敏感性为93%，特异性为100%，有效率为98%。同时获得了较低的聚合酶链式反应抑制率(8%)。用Cohen‘s kappa指数进行沙门氏菌分型，多重聚合酶链式反应与传统培养技术的总体符合率为95%。
We have developed a multiplex PCR assay for Salmonella detection and epidemiological typing. Six sets of primers were designed to detect the major Salmonella serotypes and phage types in Spain. An internal amplification control was designed in order to detect PCR inhibition. The different amplification profiles obtained allowed us to detect Salmonella bacteria and to distinguish the clinically prevalent Salmonella enterica serotypes Enteritidis, Typhimurium and subspecies I serotype 4,5,12:i:-. Using this method, we could detect a specific band for DT104 and U302 phage types in Salmonella serotype Typhimurium. Salmonella enterica serotype Hadar and other C2 serogroup strains showed two specific band profiles. In the validation stage, the assay was reproducible for all serotypes studied, apart from some C2 serogroup strains. When the technique was applied to clinical stool specimens, the prevalent serotypes Enteritidis and Typhimurium were detected with a sensitivity of 93%, specificity of 100%, and efficiency of 98%. Also, a low PCR inhibition rate (8%) was obtained. The overall agreement of the multiplex PCR with conventional culture-based techniques was 95% for Salmonella typing using Cohen's kappa index.