Simultaneous Detection and Differentiation of Campylobacter jejuni , C. coli , and C. lari in Chickens Using a Multiplex
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Simultaneous Detection and Differentiation of Campylobacter jejuni, C. coli, and C. lari in Chickens Using a Multiplex Real-Time PCR Assay Yiping He & Xiaomin Yao & Nereus W. Gunther IV & Yanping Xie & Shu-I Tu & Xianming Shi
Received: 25 January 2010 / Accepted: 18 March 2010 / Published online: 21 April 2010 # US Government 2010
Abstract A multiplex real-time PCR (qPCR) assay was developed for simultaneous detection and differentiation of the three most important Campylobacter species in chickens. Three novel sets of PCR primers and TaqMan probes were designed to amplify the unique DNA sequences within the hipO, cdtA, and pepT genes which are specific to Campylobacter jejuni, Campylobacter coli, and Campylobacter lari, respectively. To avoid competition in the multiple target amplifications, the concentrations of primers and probes were optimized. By using the optimized qPCR conditions together with a minor-groove binding probe of pepT, amplification efficiency greater than 92% and detection sensitivity of 38 genome copies/reaction have been achieved for all three targets. The assay was highly specific for C. jejuni, C. coli, and C. lari with testing of 33 Campylobacter strains and 20 non-Campylobacter strains. In chicken samples spiked with known quantities of Campylobacter cells, the assay was able to detect 1 CFU/ g after a 24-h enrichment. Application of the assay in food was further evaluated using 21 fresh chicken samples obtained from local supermarkets. The results revealed that, after a 24-h or 48-h enrichment, 14 samples (66.7%) Yiping He and Xiaomin Yao contributed equally to this work. Y. He (*) : N. W. Gunther IV : S.-I. Tu Microbial Biophysics and Residue Chemistry Research Unit, United States Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center (USDA–ARS–ERRC), 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA e-mail: [email protected] X. Yao : Y. Xie : X. Shi Department of Food Science and Technology & Bor Luh Food Safety Center, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China
were positive for C. jejuni, five samples (23.8%) were positive for C. coli, and none of the samples was contaminated by C. lari. Taken together, the multiplex qPCR assay combined with an enrichment step is a sensitive, species-specific, and non-labor-intensive method suitable for rapid detection of C. jejuni, C. coli, and C. lari in chicken samples. Keywords Multiplex Real-Time PCR . Campylobacter Species . Detection . Chicken Samples
Introduction Campylobacter is an important foodborne pathogen and a common cause of bacterial gastroenteritis in humans throughout the world (Altekruse et al. 1999; Frost et al. 2002). In the USA, an estimated 2.4 million cases of bacterial gastroenteritis are caused by Campylobacter each year, which is higher than the number of Salmonella infection (CDC 2007). Campylobacter infection can cause fever and diarrhea, which is often bloody in humans. In a few complicated cases, Campylobacter jejuni infection can lead to Guillain–Bar
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