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Journal of Clinical Microbiology, May 2001, p. 1871-1876, Vol. 39, No. 5
Department of
Microbiology1 and Department of Food
Hygiene & Technology,3 Faculty of Veterinary
Medicine, Uludag University, Gorukle Kampusu, 16059 Bursa, and
Iontek Biotechnology Research and Development Co., Mavi
Cadde, 8. Sok. No 1, Bursa,2 Turkey
Received 18 October 2000/Returned for modification 24 January
2001/Accepted 8 March 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
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.1871-1876.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Detection of Salmonellae in Chicken Feces by a
Combination of Tetrathionate Broth Enrichment, Capillary PCR, and
Capillary Gel Electrophoresis
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.
*
Corresponding author. Mailing address: Department of
Microbiology, Faculty of Veterinary Medicine, Uludag University,
Gorukle Kampusu, 16059 Bursa, Turkey. Phone: 90 (224) 442 9200, ext.
137. Fax: 90 (224) 442 8025. E-mail:
tayfun{at}uludag.edu.tr.
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