Direct Detection of eae-Positive Bacteria in Human and Veterinary Colorectal Specimens by PCR

A PCR test based on the amplification of an eae-specific sequence was designed and evaluated for its ability to directly detect homologous sequences in enteropathogenic Escherichia coli and Citrobacter spp. (amplification of eae open reading frame, 178 bp) in sections of the intestines of humans and animals with colonic lesions. Positive PCR results were observed with eae-positive reference strains of E. coli and Citrobacter rodentium (Citrobacter freundii biotype 4280). Known eae-negative reference strains of E. coli and other laboratory strains of enteric bacteria were negative by the amplification test. The sensitivity of the PCR for detection of eae-positive E. coli and C. rodentium was between 1 and 2 CFU. To detect these sequences directly from sections of fixed colon from human and veterinary sources, PCR conditions were modified by the addition of 0.1 mM 8-methoxypsoralen to eliminate extraneous bacterial DNA from the PCR amplification cocktail without added template. Sections of colon from three pigs experimentally affected with colon lesions due to enteropathogenic (attaching and effacing) E. coli were PCR positive for bacterial eae genome. Sections from control animals were negative. Sections of colon from one of 18 biopsies from confirmed AIDS patients and from 22 of 35 colorectal cancer patients were PCR positive for bacterial eae genome. The PCR test was a simple and quick method of detecting bacterial eae genome in human and veterinary clinical specimens. This method may remove the need for initial culture and detection of the gene by DNA probing from potential associated lesions. The clear relationship of bacteria containing the eae gene with colonic lesions in the pigs and mice indicates that a similar relationship is possible for human patients having similar lesions.

本研究设计并验证了一种基于eae特异性序列扩增的聚合酶链式反应（Polymerase Chain Reaction，PCR）检测方法，用于直接在伴结肠病变的人类及动物肠道组织切片中，检测肠致病性大肠杆菌（enteropathogenic Escherichia coli）与枸橼酸杆菌属（Citrobacter spp.）的同源序列，扩增靶标为eae开放阅读框，扩增片段长度为178 bp。针对eae阳性的大肠杆菌参考菌株及啮鼠枸橼酸杆菌（Citrobacter rodentium，即弗劳地枸橼酸杆菌生物型4280），该PCR检测均呈阳性结果。已知的eae阴性大肠杆菌参考菌株及其他肠道细菌实验室菌株经该扩增检测均呈阴性。该PCR方法对eae阳性大肠杆菌及啮鼠枸橼酸杆菌的检测灵敏度可达1~2个菌落形成单位（Colony Forming Unit，CFU）。为直接从人类及兽医来源的固定结肠组织切片中检测上述序列，本研究对PCR反应体系进行优化：添加0.1 mM 8-甲氧基补骨脂素，以消除未添加模板时PCR扩增混合液中存在的外源细菌DNA干扰。3头经实验诱导感染肠致病性（黏附脱落型）大肠杆菌并引发结肠病变的猪的结肠组织切片，其细菌eae基因组PCR检测均呈阳性。对照组动物的结肠组织切片检测结果均为阴性。在18份确诊艾滋病患者的结肠活检组织切片中，仅1份呈细菌eae基因组PCR阳性；35份结直肠癌患者的结肠活检组织切片中，有22份呈阳性。该PCR检测方法操作简便、快速高效，可直接在人类及兽医临床样本中检测细菌eae基因组。该方法可省去传统检测流程中需先进行菌株培养，再通过DNA探针技术从潜在病变组织中检测目标基因的步骤。猪及小鼠体内携带eae基因的细菌与结肠病变间存在明确关联，这提示携带eae基因的细菌或与人类出现的类似结肠病变存在潜在关联。