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%) 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.
Similar content being viewed by others
References
Altekruse SF, Stern NJ, Fields PI, Swerdlow DL (1999) Campylobacter jejuni—an emerging foodborne pathogen. Emerg Infect Dis 5:28
Asakura M, Samosornsuk W, Taguchi M, Kobayashi K, Misawa N, Kusumoto M, Nishimura K, Matsuhisa A, Yamasaki S (2007) Comparative analysis of cytolethal distending toxin (cdt) genes among Campylobacter jejuni, C. coli and C. fetus strains. Microb Pathog 42:174
Bhaduri S, Cottrell B (2004) Survival of cold-stressed Campylobacter jejuni on ground chicken and chicken skin during frozen storage. Appl Environ Microbiol 70:7103
Birk T, Ingmer H, Andersen MT, Jorgensen K, Brondsted L (2004) Chicken juice, a food-based model system suitable to study survival of Campylobacter jejuni. Lett Appl Microbiol 38:66
Bonjoch X, Calvó L, Soler M, Ruiz-Rueda O, Garcia-Gil LJ (2010) A new multiplexed real-time PCR assay to detect Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis. Food Anal Methods 3:40
Callicott KA, Hargardottir H, Georgsson F, Reiersen J, Frigriksdottir V, Gunnarsson E, Michel P, Bisaillon JR, Kristinsson KG, Briem H, Hiett KL, Needleman DS, Stern NJ (2008) Broiler Campylobacter contamination and human campylobacteriosis in Iceland. Appl Environ Microbiol 74:6483
CDC (2007) Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food—10 states, 2006. MMWR Morb Mortal Wkly Rep 56:336
Champion OL, Best EL, Frost JA (2002) Comparison of pulsed-field gel electrophoresis and amplified fragment length polymorphism techniques for investigating outbreaks of enteritis due to Campylobacters. J Clin Microbiol 40:2263
Chan KF, Le Tran H, Kanenaka RY, Kathariou S (2001) Survival of clinical and poultry-derived isolates of Campylobacter jejuni at a low temperature (4 degrees C). Appl Environ Microbiol 67:4186
Che Y, Li Y, Slavik M (2001) Detection of Campylobacter jejuni in poultry samples using an enzyme-linked immunoassay coupled with an enzyme electrode. Biosens Bioelectron 16:791
Chen CY, Nace GW, Irwin PL (2003) A 6 × 6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli. J Microbiol Methods 55:475
Dingle KE, Colles FM, Wareing DR, Ure R, Fox AJ, Bolton FE, Bootsma HJ, Willems RJ, Urwin R, Maiden MC (2001) Multilocus sequence typing system for Campylobacter jejuni. J Clin Microbiol 39:14
Frost JA, Gillespie IA, O'Brien SJ (2002) Public health implications of Campylobacter outbreaks in England and Wales, 1995–9: epidemiological and microbiological investigations. Epidemiol Infect 128:111
Ge B, White DG, McDermott PF, Girard W, Zhao S, Hubert S, Meng J (2003) Antimicrobial-resistant Campylobacter species from retail raw meats. Appl Environ Microbiol 69:3005
Gillespie IA, O'Brien SJ, Frost JA, Adak GK, Horby P, Swan AV, Painter MJ, Neal KR (2002) A case–case comparison of Campylobacter coli and Campylobacter jejuni infection: a tool for generating hypotheses. Emerg Infect Dis 8:937
Gilpin B, Cornelius A, Robson B, Boxall N, Ferguson A, Nicol C, Henderson T (2006) Application of pulsed-field gel electrophoresis to identify potential outbreaks of campylobacteriosis in New Zealand. J Clin Microbiol 44:406
Hannis JC, Manalili SM, Hall TA, Ranken R, White N, Sampath R, Blyn LB, Ecker DJ, Mandrell RE, Fagerquist CK, Bates AH, Miller WG, Hofstadler SA (2008) High-resolution genotyping of Campylobacter species by use of PCR and high-throughput mass spectrometry. J Clin Microbiol 46:1220
Hindiyeh M, Jense S, Hohmann S, Benett H, Edwards C, Aldeen W, Croft A, Daly J, Mottice S, Carroll KC (2000) Rapid detection of Campylobacter jejuni in stool specimens by an enzyme immunoassay and surveillance for Campylobacter upsaliensis in the greater Salt Lake City area. J Clin Microbiol 38:3076
Hong J, Jung WK, Kim JM, Kim SH, Koo HC, Ser J, Park YH (2007) Quantification and differentiation of Campylobacter jejuni and Campylobacter coli in raw chicken meats using a real-time PCR method. J Food Prot 70:2015
Keener KM, Bashor MP, Curtis PA, Sheldon BW, Kathariou S (2004) Comprehensive review of Campylobacter and poultry processing. Comp Rev Food Sci Food Safety 3:105
Kemp R, Leatherbarrow AJ, Williams NJ, Hart CA, Clough HE, Turner J, Wright EJ, French NP (2005) Prevalence and genetic diversity of Campylobacter spp. in environmental water samples from a 100-square-kilometer predominantly dairy farming area. Appl Environ Microbiol 71:1876
Klena JD, Parker CT, Knibb K, Ibbitt JC, Devane PM, Horn ST, Miller WG, Konkel ME (2004) Differentiation of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis by a multiplex PCR developed from the nucleotide sequence of the lipid A gene lpxA. J Clin Microbiol 42:5549
Kramer JM, Frost JA, Bolton FJ, Wareing DR (2000) Campylobacter contamination of raw meat and poultry at retail sale: identification of multiple types and comparison with isolates from human infection. J Food Prot 63:1654
LaGier MJ, Joseph LA, Passaretti TV, Musser KA, Cirino NM (2004) A real-time multiplexed PCR assay for rapid detection and differentiation of Campylobacter jejuni and Campylobacter coli. Mol Cell Probes 18:275
Mandrell RE, Harden LA, Bates A, Miller WG, Haddon WF, Fagerquist CK (2005) Speciation of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Appl Environ Microbiol 71:6292
Miller WG, On SL, Wang G, Fontanoz S, Lastovica AJ, Mandrell RE (2005) Extended multilocus sequence typing system for Campylobacter coli, C. lari, C. upsaliensis, and C. helveticus. J Clin Microbiol 43:2315
Miller WG, Wang G, Binnewies TT, Parker CT (2008) The complete genome sequence and analysis of the human pathogen Campylobacter lari. Foodborne Pathog Dis 5:371
Nachamkin I, Allos BM, Ho T (1998) Campylobacter species and Guillain–Barré syndrome. Clin Microbiol Rev 11:555
Nachamkin I, Szymanski CM, Blaser MJ (2008) Campylobacter, 3rd edn. ASM, Washington, D.C
Newell DG, Fearnley C (2003) Sources of Campylobacter colonization in broiler chickens. Appl Environ Microbiol 69:4343
Oliveira TC, Barbut S, Griffiths MW (2005) Detection of Campylobacter jejuni in naturally contaminated chicken skin by melting peak analysis of amplicons in real-time PCR. Int J Food Microbiol 104:105
Park SF (2002) The physiology of Campylobacter species and its relevance to their role as foodborne pathogens. Int J Food Microbiol 74:177
Parkhill J, Wren BW, Mungall K, Ketley JM, Churcher C, Basham D, Chillingworth T, Davies RM, Feltwell T, Holroyd S, Jagels K, Karlyshev AV, Moule S, Pallen MJ, Penn CW, Quail MA, Rajandream MA, Rutherford KM, van Vliet AH, Whitehead S, Barrell BG (2000) The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403:665
Persson S, Olsen KE (2005) Multiplex PCR for identification of Campylobacter coli and Campylobacter jejuni from pure cultures and directly on stool samples. J Med Microbiol 54:1043
Quinones B, Parker CT, Janda JM Jr, Miller WG, Mandrell RE (2007) Detection and genotyping of Arcobacter and Campylobacter isolates from retail chicken samples by use of DNA oligonucleotide arrays. Appl Environ Microbiol 73:3645
Richardson JF, Frost JA, Kramer JM, Thwaites RT, Bolton FJ, Wareing DR, Gordon JA (2001) Coinfection with Campylobacter species: an epidemiological problem? J Appl Microbiol 91:206
Ridley AM, Allen VM, Sharma M, Harris JA, Newell DG (2008) Real-time PCR approach for detection of environmental sources of Campylobacter strains colonizing broiler flocks. Appl Environ Microbiol 74:2492
Rudi K, Hoidal HK, Katla T, Johansen BK, Nordal J, Jakobsen KS (2004) Direct real-time PCR quantification of Campylobacter jejuni in chicken fecal and cecal samples by integrated cell concentration and DNA purification. Appl Environ Microbiol 70:790
Sails AD, Fox AJ, Bolton FJ, Wareing DR, Greenway DL (2003) A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture. Appl Environ Microbiol 69:1383
Suo B, He Y, Tu S, Shi X (2010) A multiplex real-time polymerase chain reaction for simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes in meat products. Foodborne Pathog Dis. doi:10.1089=fpd.2009.0430
USDA, Food Safety and Inspection Service, Microbiology Laboratory Guidebook, 3rd Edition (1998) US Government Printing Office, Washington, DC 20250.
Volokhov D, Chizhikov V, Chumakov K, Rasooly A (2003) Microarray-based identification of thermophilic Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis. J Clin Microbiol 41:4071
Werno AM, Klena JD, Shaw GM, Murdoch DR (2002) Fatal case of Campylobacter lari prosthetic joint infection and bacteremia in an immunocompetent patient. J Clin Microbiol 40:1053
Zhao C, Ge B, De Villena J, Sudler R, Yeh E, Zhao S, White DG, Wagner D, Meng J (2001) Prevalence of Campylobacter spp., Escherichia coli, and Salmonella serovars in retail chicken, turkey, pork, and beef from the Greater Washington, D.C., area. Appl Environ Microbiol 67:5431
Acknowledgments
We thank Dr. George Paoli and Sue Reed in the Microbial Biophysics and Residue Chemistry Research Unit at the USDA–ARS–ERRC for kindly providing the non-Campylobacter strains and critically reviewing the manuscript.
Author information
Authors and Affiliations
Corresponding author
Additional information
Yiping He and Xiaomin Yao contributed equally to this work.
Rights and permissions
About this article
Cite this article
He, Y., Yao, X., Gunther, N.W. et al. Simultaneous Detection and Differentiation of Campylobacter jejuni, C. coli, and C. lari in Chickens Using a Multiplex Real-Time PCR Assay. Food Anal. Methods 3, 321–329 (2010). https://doi.org/10.1007/s12161-010-9136-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12161-010-9136-6