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Journal of Clinical Microbiology, July 2001, p. 2477-2484, Vol. 39, No. 7
Microbial Food Safety Research Unit, U.S.
Department of Agriculture, Agricultural Research Service, Eastern
Regional Research Center, Wyndmoor, Pennsylvania
19038,1 and Department of Diagnostic
Medicine/Pathobiology2 and Food
Animal Health and Management Center,3 College of
Veterinary Medicine, Kansas State University, Manhattan, Kansas
66506
Received 23 January 2001/Returned for modification 4 March
2001/Accepted 29 April 2001
A total of 150 fecal and water samples from four swine farms were
tested for the presence of Salmonella enterica using
different enrichment techniques as follows: (i) 92 fecal samples from
nursery and farrowing barns at three swine farms were preenriched
overnight in tryptic soy broth (TSB) at 37°C followed by overnight
enrichment in Rappaport-Vassiliadis 10 broth (RV10) at 42°C; (ii) 24 water samples from the third farm were preenriched overnight in 3MC broth at 37°C followed by overnight enrichment in RV10 at 42°C; and
(iii) 34 fecal samples from a fourth farm, a finishing farm, were
enriched overnight in RV10 at 42°C with no additional enrichment. Following each of the enrichment techniques, samples were subcultured onto modified semisolid Rappaport-Vassiliadis (MSRV) agar prior to
transfer to Hektoen Enteric agar plates for the recovery of viable
Salmonella bacteria. Presumptive Salmonella
isolates were biochemically and serologically confirmed. For the PCR
detection of Salmonella, a 1-ml portion was removed from
each sample after the first overnight enrichment and the DNA was
extracted using a Sepharose CL-6B spin column. Amplicons (457 bp)
derived from primers to the invA and invE genes
were confirmed as Salmonella specific on ethidium
bromide-stained agarose gels by Southern hybridization with a 20-mer
oligonucleotide probe specific for the Salmonella invA
gene. Neither the standard microbiological method nor the molecular
method detected all of the 65 samples that tested positive by both
methods or either method alone. Salmonella bacteria were
detected by both cultivation and PCR-hybridization in 68% (17 of 25)
of the positive samples that were preenriched in TSB, in 73% (11 of
15) of the positive samples preenriched in 3MC broth, and in 24% (6 of
25) of the positive samples enriched in RV10. Agreement between
Salmonella detection using cultivation with preenrichment
and detection by PCR was 76% using the kappa statistic. However,
agreement between Salmonella detection using cultivation
without preenrichment and detection by PCR was about 6%; the PCR assay
detected 80% (20 of 25) of the 25 positive samples, while
Salmonella bacteria were recovered from only 44% (11 of 25) by cultivation. Our results indicate that the PCR-hybridization approach is equivalent to or better than cultivation for detecting Salmonella in swine feces or water samples from swine farms
when using the medium combinations evaluated in this study.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.7.2477-2484.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Comparison of Cultivation and PCR-Hybridization for Detection of
Salmonella in Porcine Fecal and Water Samples

*
Corresponding author. Present address: United States
Department of Agriculture, Agricultural Research Service, Eastern
Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038. Phone: (215) 233-6492. Fax: (215) 233-6581. E-mail:
ifeder{at}arserrc.gov.
Contribution 00-238-J from the Kansas Agricultural Experiment Station.
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