title: Rapid identification of Campylobacter jejuni from poultry carcasses and slaughtering environment samples by real-time PCR.
abstract: The objective of this study was to develop a real-time PCR assay for rapid identification of Campylobacter jejuni and to apply the method in analyzing samples from poultry processing. A C. jejuni-specific primer set targeting a portion of the C. jejuni hippuricase gene was developed. The specificity of the newly designed primer pair was verified using 5 C. jejuni strains and 20 other bacterial strains. Sensitivity was determined to be as low as 1 genome copy per reaction. A total of 73 samples were collected at different sites along the processing line during 2 visits to a poultry slaughterhouse and were examined by direct plating onto modified charcoal cefoperazone deoxycholate agar or after enrichment in Bolton broth followed by plating on modified charcoal cefoperazone deoxycholate agar. The newly developed real-time PCR assay was used to identify the presumptive colonies as belonging to C. jejuni. A real-time PCR assay targeting 16S ribosomal RNA was also applied to determine Campylobacter spp. prevalence. Results from the real-time PCR analysis indicated considerable variability in Campylobacter contamination, with incidence rates of 72.7 and 27.6% for sampling days A and B, respectively. Campylobacter was isolated from 100% of prescalded and preeviscerated carcasses on sampling day A. In contrast, on sampling day B, the highest number of Campylobacter-positive carcasses was recovered after evisceration (60%). The chilling process significantly reduced (P < 0.05) Campylobacter population, but the percentage of positive samples on sampling day A increased to 80%. All samples collected from the processing environment, except scalding tank 3 and the prechiller and chiller tanks, were 100% positive on day A, whereas no campylobacters were isolated from machinery on sampling day B. Our results revealed the widespread of C. jejuni in poultry processing and proved that the newly developed real-time PCR assay is a simple, specific, and inexpensive method for rapid C. jejuni identification. The newly developed PCR method can be easily used in laboratories for reliable and unambiguous identification of C. jejuni in poultry samples.
authors.affiliation: Department of Biotechnology, University of Food Technologies, 26 Maritza Blvd, 4002 Plovdiv, Bulgaria.
authors.forename: Mirena
authors.initials: M
authors.lastname: Ivanova
authors.affiliation: School of Public Health and Zoonoses, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, India PB-141004.
authors.forename: Randhir
authors.initials: R
authors.lastname: Singh
authors.affiliation: Department of Food, Nutrition, and Packaging Sciences, Clemson University, Clemson, SC 29634.
authors.forename: Muthu
authors.initials: M
authors.lastname: Dharmasena
authors.affiliation: Department of Food, Nutrition, and Packaging Sciences, Clemson University, Clemson, SC 29634.
authors.forename: Chao
authors.initials: C
authors.lastname: Gong
authors.affiliation: Department of Biotechnology, University of Food Technologies, 26 Maritza Blvd, 4002 Plovdiv, Bulgaria.
authors.forename: Albert
authors.initials: A
authors.lastname: Krastanov
authors.affiliation: Department of Food, Nutrition, and Packaging Sciences, Clemson University, Clemson, SC 29634 xiuping@clemson.edu.
authors.forename: Xiuping
authors.initials: X
authors.lastname: Jiang
mesh_terms: D000003:Abattoirs; D000581:Amidohydrolases / Q000235:genetics*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D002169:Campylobacter Infections / Q000453:epidemiology / Q000382:microbiology / Q000662:veterinary*; D016123:Campylobacter jejuni / Q000235:genetics* / Q000302:isolation & purification; D002645:Chickens; D005511:Food Handling; D005516:Food Microbiology / Q000379:methods; D015994:Incidence; D008460:Meat / Q000382:microbiology; D011201:Poultry Diseases / Q000453:epidemiology* / Q000382:microbiology; D015995:Prevalence; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; D012336:RNA, Ribosomal, 16S; D000581:Amidohydrolases; C021356:hippurate hydrolase
keywords: Campylobacter jejuni; hippuricase gene; poultry carcass; real-time PCR; slaughtering environment