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Applied and Environmental Microbiology, July 2005, p. 3911-3916, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3911-3916.2005

Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

Mark G. Wise and Gregory R. Siragusa*

Poultry Microbiological Safety Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Russell Research Center, Athens, Georgia

Received 14 September 2004/ Accepted 26 January 2005

Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

* Corresponding author. Mailing address: ARS-USDA, Russell Research Center, 950 College Station Road, P.O. Box 5677, Athens, GA 30605. Phone: (706) 546-3596. Fax: (706) 546-3772. E-mail: siragusa{at}saa.ars.usda.gov.

Applied and Environmental Microbiology, July 2005, p. 3911-3916, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3911-3916.2005




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