Detection of salmonellae in chicken feces by a combination of tetrathionate broth enrichment, capillary PCR, and capillary gel electrophoresis

This report describes a rapid detection procedure for salmonellae from chic ken feces by the combination of tetrathionate primary enrichment (preenrich ment [PE])-bacterial lysis capillary PCR and capillary gel electrophoresis. Pure Salmonella enterica serovar Enteritidis 64K was reisolated and detect ed by capillary PCR after buffered peptone water and nutrient broth, tetrat hionate broth base Hajna (TTBH), and tetrathionate broth (TTB) preenrichmen ts. When the same culture was mixed with intestinal homogenate, bacteriolog ical reisolation and capillary PCR detection was achieved only by TTBK and TTB preenrichments. Capillary gel electrophoresis revealed that a Salmonell a genus-specific 281-bp PCR product aas detected when Salmonella strains bu t not non-Salmonella strains were tested. The detection limit of capillary PCR with whole-cell DNA extracted from pure Salmonella enterica serovars En teritidis 64K, Typhimurium LT2-CIP60-62, and Gallinarum 64K was 3, 3, and 9 CPU ml(-1), respectively. The detection limit of capillary PCR from whole cell DNA extracted from intestinal homogenate artificially contaminated wit h the same three strains was 3, 3, and 7 CFU ml(-1), respectively We compar ed the results of the capillary PCR and bacteriological examination from th e natural samples. Thirty-five of 9 naturally contaminated samples produced a specific PCR product. In 9 of the 35 PCR-positive samples, Salmonella co uld 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 w ere positive after DSE. Fifty-three additional intestinal homogenate sample s, 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 de tect Salmonella with the capillary PCR method we used was approximately 20 h, If samples are from clinically diseased birds, the total time for PCR an d detection is reduced to 2 h since the 18-h PE is not required. These resu lts indicate that TTB enrichment, bacterial lysis, and genus-specific capil lary PCR combined,vith capillary gel electrophoresis constitute a sensitive and selective procedure which has the potential to rapidly identify Salmon ella-infected flocks.