Bischoff C, Lüthy J, Altwegg M, Baggi F
Department of Medical Microbiology, University of Zurich, Gloriastrasse 30/32, 8028 Zurich, Switzerland.
J Microbiol Methods. 2005 Jun;61(3):335-41. doi: 10.1016/j.mimet.2004.12.007. Epub 2005 Jan 11.
Enterovirulent Escherichia coli are among the most important causes of acute diarrhea in developing as well as in developed countries. We have adapted classical PCR to detect these organisms in stool specimens to real-time PCR using the LightCycler (LC) SYBR Green format followed by melting curve analysis. With only two different cycling protocols we could detect enteropathogenic E. coli (EPEC) and verocytotoxin-producing E. coli (VTEC) (duplex assay for both Verotoxin 1 (VT1) and Verotoxin 2 (VT2)) in one run and enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) (duplex assay detecting both heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT)) in another run. Using serial dilutions of control strains, the LC proved to be clearly more sensitive than conventional PCR for five out of seven investigated targets: VTEC (VT1 and VT2), ETEC (ST and LT) and EIEC. For EPEC and EAEC, LC and conventional PCR had identical sensitivities. With stool samples, we found an optimal agreement between LC-PCR and the conventional PCR when samples were tested in a 1:10 dilution. Only one specimen was discrepant, being repetitively positive for VT by LightCycler but not by conventional PCR. Given the significantly higher sensitivity of the LC-PCR for the VT target (up to a 10(-4) dilution factor by melting curve analysis and up to a 10(-6) dilution factor following gel electrophoresis), this is probably a false negative result by conventional PCR. We conclude that LightCycler PCR is more rapid, easier than and at least as sensitive as our conventional PCR for the detection of enterovirulent E. coli in stool specimens after culture on MacConkey.
肠毒性大肠杆菌是发展中国家和发达国家急性腹泻的最重要病因之一。我们已将用于在粪便标本中检测这些微生物的经典PCR方法，改进为使用LightCycler（LC）SYBR Green技术的实时PCR方法，并进行熔解曲线分析。仅通过两种不同的循环方案，我们就能在一次运行中检测出肠致病性大肠杆菌（EPEC）和产志贺毒素大肠杆菌（VTEC）（针对志贺毒素1（VT1）和志贺毒素2（VT2）的双重检测），并在另一次运行中检测出肠集聚性大肠杆菌（EAEC）、肠侵袭性大肠杆菌（EIEC）和产肠毒素大肠杆菌（ETEC）（针对耐热肠毒素（ST）和不耐热肠毒素（LT）的双重检测）。通过对对照菌株进行系列稀释，结果表明，在七个被研究的目标中，有五个目标的LC检测明显比传统PCR更灵敏：VTEC（VT1和VT2）、ETEC（ST和LT）以及EIEC。对于EPEC和EAEC，LC和传统PCR具有相同的灵敏度。对于粪便样本，当样本以1:10的稀释度进行检测时，我们发现LC-PCR与传统PCR之间具有最佳的一致性。只有一个样本存在差异，即通过LightCycler检测VT呈重复性阳性，但传统PCR检测为阴性。鉴于LC-PCR对VT目标的灵敏度显著更高（通过熔解曲线分析可达10^(-4)稀释倍数，凝胶电泳后可达10^(-6)稀释倍数），这可能是传统PCR的假阴性结果。我们得出结论，在MacConkey培养基上培养后，对于粪便标本中肠毒性大肠杆菌的检测，LightCycler PCR比我们的传统PCR更快速、更简便，且至少具有相同的灵敏度。
