Carli K T, Unal C B, Caner V, Eyigor A
Department of Microbiology, Faculty of Veterinary Medicine, Uludag University, Gorukle Kampusu, 16059 Bursa, Turkey.
J Clin Microbiol. 2001 May;39(5):1871-6. doi: 10.1128/JCM.39.5.1871-1876.2001.
This report describes a rapid detection procedure for salmonellae from chicken feces by the combination of tetrathionate primary enrichment (preenrichment [PE])-bacterial lysis-capillary PCR and capillary gel electrophoresis. Pure Salmonella enterica serovar Enteritidis 64K was reisolated and detected by capillary PCR after buffered peptone water and nutrient broth, tetrathionate broth base Hajna (TTBH), and tetrathionate broth (TTB) preenrichments. When the same culture was mixed with intestinal homogenate, bacteriological reisolation and capillary PCR detection was achieved only by TTBH and TTB preenrichments. Capillary gel electrophoresis revealed that a Salmonella genus-specific 281-bp PCR product was detected when Salmonella strains but not non-Salmonella strains were tested. The detection limit of capillary PCR with whole-cell DNA extracted from pure Salmonella enterica serovars Enteritidis 64K, Typhimurium LT2-CIP60-62, and Gallinarum 64K was 3, 3, and 9 CFU ml(-1), respectively. The detection limit of capillary PCR from whole-cell DNA extracted from intestinal homogenate artificially contaminated with the same three strains was 3, 3, and 7 CFU ml(-1), respectively. We compared the results of the capillary PCR and bacteriological examination from the natural samples. Thirty-five of 53 naturally contaminated samples produced a specific PCR product. In 9 of the 35 PCR-positive samples, Salmonella could not be detected bacteriologically either by PE or a primary and delayed secondary enrichment (DSE) combination. In the 18 PCR-negative samples, 4 samples were found to harbor Salmonella by both PE and DSE and 14 samples were positive after DSE. Fifty-three additional intestinal homogenate samples, which were negative by their PE and DSE in bacteriological examination, were found to be also negative by their PCRs. The total time required to detect Salmonella with the capillary PCR method we used was approximately 20 h. If samples are from clinically diseased birds, the total time for PCR and detection is reduced to 2 h since the 18-h PE is not required. These results indicate that TTB enrichment, bacterial lysis, and genus-specific capillary PCR combined with capillary gel electrophoresis constitute a sensitive and selective procedure which has the potential to rapidly identify Salmonella-infected flocks.
本报告描述了一种通过四硫磺酸盐初次富集（预富集[PE]）-细菌裂解-毛细管PCR和毛细管凝胶电泳相结合的方法，从鸡粪便中快速检测沙门氏菌的程序。在缓冲蛋白胨水、营养肉汤、哈伊纳四硫磺酸盐肉汤基础培养基（TTBH）和四硫磺酸盐肉汤（TTB）预富集后，通过毛细管PCR重新分离并检测到纯肠炎沙门氏菌血清型肠炎亚种64K。当将相同培养物与肠道匀浆混合时，仅通过TTBH和TTB预富集实现了细菌重新分离和毛细管PCR检测。毛细管凝胶电泳显示，检测沙门氏菌菌株而非非沙门氏菌菌株时，可检测到沙门氏菌属特异性的281bp PCR产物。从纯肠炎沙门氏菌血清型肠炎亚种64K、鼠伤寒沙门氏菌LT2-CIP60-62和鸡沙门氏菌64K中提取全细胞DNA进行毛细管PCR的检测限分别为3、3和9 CFU ml(-1)。从人工污染相同三种菌株的肠道匀浆中提取全细胞DNA进行毛细管PCR的检测限分别为3、3和7 CFU ml(-1)。我们比较了天然样品的毛细管PCR结果和细菌学检查结果。53份天然污染样品中有35份产生了特异性PCR产物。在35份PCR阳性样品中的9份中，无论是通过PE还是初次和延迟二次富集（DSE）组合，均未通过细菌学方法检测到沙门氏菌。在18份PCR阴性样品中，有4份通过PE和DSE均检测到含有沙门氏菌，14份样品在DSE后呈阳性。另外53份肠道匀浆样品在细菌学检查中的PE和DSE均为阴性，其PCR检测也为阴性。我们使用的毛细管PCR方法检测沙门氏菌所需的总时间约为20小时。如果样品来自临床患病鸡，则由于不需要18小时的PE，PCR和检测的总时间可缩短至2小时。这些结果表明，TTB富集、细菌裂解以及属特异性毛细管PCR与毛细管凝胶电泳相结合构成了一种灵敏且具选择性的程序，有潜力快速识别感染沙门氏菌的鸡群。
