Reischl Udo, Youssef Mohammad T, Wolf Hans, Hyytia-Trees Eija, Strockbine Nancy A
Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany.
J Clin Microbiol. 2004 Sep;42(9):4092-100. doi: 10.1128/JCM.42.9.4092-4100.2004.
To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.
为便于诊断人类肠产毒性大肠杆菌（ETEC）感染，我们针对罗氏LightCycler（LC）开发并评估了针对与人类疾病相关菌株中常见肠毒素基因的实时荧光PCR检测方法。使用LC杂交探针形式，针对I型不耐热肠毒素（LT I）和I型耐热肠毒素（ST I）基因设计了具有相同循环条件的单独LC-PCR检测方法。为检测ST I的主要核苷酸序列变体ST Ia和ST Ib，需要一种针对ST I的双重检测方法，该方法包含两组扩增引物和三种杂交探针。将161株人源大肠杆菌分离株（138株ETEC和23株非ETEC）的LC-PCR检测结果与通过块循环PCR分析获得的结果进行比较。LC-PCR检测方法对LT I和ST I基因的敏感性和特异性均为100%。还比较了LC-PCR和块循环PCR检测方法在不同样品制备方法的加标粪便标本中检测LT I和ST I基因的能力。这些实验结果表明，LC-PCR检测方法的检测限与块循环PCR检测方法相同或显著更低。对扩增的LT I和ST I基因进行熔解曲线分析，揭示了每个基因内的序列变异，对于ST I基因，这种变异与ST Ia和ST Ib的存在相关。LC-PCR检测方法的快速性、敏感性和特异性使其成为用于ETEC检测和鉴定的块循环PCR检测方法的有吸引力的替代方法。
