Alvarez Juan, Sota Mertxe, Vivanco Ana Belén, Perales Ildefonso, Cisterna Ramón, Rementeria Aitor, Garaizar Javier
Department of Immunology, Microbiology, and Parasitology, University of the Basque Country, Vitoria-Gasteiz, Spain.
J Clin Microbiol. 2004 Apr;42(4):1734-8. doi: 10.1128/JCM.42.4.1734-1738.2004.
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.
我们开发了一种用于沙门氏菌检测和流行病学分型的多重PCR检测方法。设计了六组引物来检测西班牙主要的沙门氏菌血清型和噬菌体类型。设计了一个内部扩增对照以检测PCR抑制情况。获得的不同扩增图谱使我们能够检测沙门氏菌，并区分临床上常见的肠炎沙门氏菌血清型肠炎亚种、鼠伤寒亚种和亚种I血清型4,5,12:i: -。使用这种方法，我们可以在鼠伤寒沙门氏菌血清型中检测到DT104和U302噬菌体类型的特异性条带。哈达尔沙门氏菌血清型和其他C2血清群菌株显示出两种特异性条带图谱。在验证阶段，除了一些C2血清群菌株外，该检测方法对所有研究的血清型都是可重复的。当该技术应用于临床粪便标本时，检测到常见的血清型肠炎亚种和鼠伤寒亚种，灵敏度为93%，特异性为100%，效率为98%。此外，PCR抑制率较低（8%）。使用科恩kappa指数，多重PCR与传统基于培养的技术在沙门氏菌分型方面的总体一致性为95%。
