title: Rapid identification and quantification of Campylobacter coli and Campylobacter jejuni by real-time PCR in pure cultures and in complex samples.
abstract: BACKGROUND
  Campylobacter spp., especially Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), are recognized as the leading human foodborne pathogens in developed countries. Livestock animals carrying Campylobacter pose an important risk for human contamination. Pigs are known to be frequently colonized with Campylobacter, especially C. coli, and to excrete high numbers of this pathogen in their faeces. Molecular tools, notably real-time PCR, provide an effective, rapid, and sensitive alternative to culture-based methods for the detection of C. coli and C. jejuni in various substrates. In order to serve as a diagnostic tool supporting Campylobacter epidemiology, we developed a quantitative real-time PCR method for species-specific detection and quantification of C. coli and C. jejuni directly in faecal, feed, and environmental samples.
  
  
  RESULTS
  With a sensitivity of 10 genome copies and a linear range of seven to eight orders of magnitude, the C. coli and C. jejuni real-time PCR assays allowed a precise quantification of purified DNA from C. coli and C. jejuni. The assays were highly specific and showed a 6-log-linear dynamic range of quantification with a quantitative detection limit of approximately 2.5 × 10² CFU/g of faeces, 1.3 × 10² CFU/g of feed, and 1.0 × 10³ CFU/m² for the environmental samples. Compared to the results obtained by culture, both C. coli and C. jejuni real-time PCR assays exhibited a specificity of 96.2% with a kappa of 0.94 and 0.89 respectively. For faecal samples of experimentally infected pigs, the coefficients of correlation between the C. coli or C. jejuni real-time PCR assay and culture enumeration were R² = 0.90 and R² = 0.93 respectively.
  
  
  CONCLUSIONS
  The C. coli and C. jejuni real-time quantitative PCR assays developed in this study provide a method capable of directly detecting and quantifying C. coli and C. jejuni in faeces, feed, and environmental samples. These assays represent a new diagnostic tool for studying the epidemiology of Campylobacter by, for instance, investigating the carriage and excretion of C. coli and C. jejuni by pigs from conventional herds.
authors: LUNAM Université, Oniris, UMR 1300 Biologie, Epidémiologie et Analyse des Risques, Nantes F-44307, France. leblanc.maridor@gmail.com
authors: Mily
authors: M
authors: Leblanc-Maridor
authors: François
authors: F
authors: Beaudeau
authors: Henri
authors: H
authors: Seegers
authors: Martine
authors: M
authors: Denis
authors: Catherine
authors: C
authors: Belloc
references: J Appl Microbiol. 2010 Mar;108(3):1041-1049
references: 19735321
references: Int J Food Microbiol. 1995 Jun;26(1):43-76
references: 7662519
references: Nature. 2000 Feb 10;403(6770):665-8
references: 10688204
references: J Food Prot. 2010 Jun;73(6):1057-63
references: 20537260
references: Zoonoses Public Health. 2008 Sep;55(7):352-60
references: 18667028
references: FEMS Microbiol Lett. 1998 Aug 15;165(2):341-6
references: 9742707
references: Appl Environ Microbiol. 2003 Dec;69(12):7409-13
references: 14660392
references: J Appl Microbiol. 2005;99(2):292-300
references: 16033460
references: Int J Food Microbiol. 1992 Jan-Feb;15(1-2):153-63
references: 1622752
references: J Immunol Methods. 2004 Apr;287(1-2):179-86
references: 15099766
references: Mol Cell Probes. 2009 Jun-Aug;23(3-4):201-3
references: 19269316
references: Trop Anim Health Prod. 2011 Jan;43(1):83-7
references: 20730490
references: Biotechniques. 1995 Aug;19(2):208-10
references: 8527139
references: Int J Food Microbiol. 1991 Jan;12(1):9-16
references: 2018709
references: J Food Prot. 2005 Apr;68(4):722-7
references: 15830662
references: Clin Chem Lab Med. 2000 Feb;38(2):87-91
references: 10834394
references: J Am Vet Med Assoc. 1999 Dec 1;215(11):1601-4
references: 14567422
references: Genome Biol Evol. 2010;2:646-55
references: 20688752
references: Appl Environ Microbiol. 2003 Jun;69(6):3435-47
references: 12788747
references: J Microbiol Methods. 2011 Apr;85(1):53-61
references: 21277341
references: FEMS Immunol Med Microbiol. 2003 Oct 15;38(3):265-71
references: 14522462
references: J Appl Microbiol. 2006 Apr;100(4):623-32
references: 16553716
references: Appl Environ Microbiol. 2010 Aug;76(15):5097-104
references: 20562292
references: Can J Microbiol. 2009 Jun;55(6):633-41
references: 19767832
references: Nucleic Acids Res. 1999 Feb 1;27(3):915-6
references: 9889293
references: J Clin Microbiol. 2003 Dec;41(12):5835
references: 14662997
references: J Appl Microbiol. 2010 May;108(5):1485-93
references: 19912432
references: Mol Cell Probes. 2006 Oct;20(5):269-79
references: 16644183
references: J Food Prot. 2007 Sep;70(9):2015-22
references: 17900077
references: Mol Cell Probes. 2006 Oct;20(5):311-6
references: 16704921
references: Res Vet Sci. 2000 Feb;68(1):75-8
references: 10684762
references: J Clin Microbiol. 2004 Nov;42(11):5125-32
references: 15528705
references: J Clin Microbiol. 2004 Jun;42(6):2445-9
references: 15184417
references: J Appl Microbiol. 2007 Oct;103(4):977-84
references: 17897201
references: Clin Microbiol Rev. 1996 Jul;9(3):405-22
references: 8809468
references: Appl Environ Microbiol. 1986 Sep;52(3):531-8
references: 3767358
references: Water Res. 2009 Nov;43(19):4918-28
references: 19631959
references: J Microbiol Methods. 2000 Oct;42(2):115-9
references: 11018267
references: Methods Mol Biol. 2003;216:109-21
references: 12512359
references: J Med Microbiol. 2005 Nov;54(Pt 11):1043-1047
references: 16192435
references: Vet Microbiol. 2008 Oct 15;131(3-4):309-17
references: 18539408
references: Appl Environ Microbiol. 2004 Apr;70(4):2296-306
references: 15066825
references: Res Microbiol. 2005 Jan-Feb;156(1):107-14
references: 15636755
references: Res Microbiol. 2008 Mar;159(2):88-93
references: 18272350
references: J Clin Microbiol. 1997 Apr;35(4):995-8
references: 9157172
references: J Clin Microbiol. 1997 Mar;35(3):759-63
references: 9041429
references: Mol Cell Probes. 2004 Aug;18(4):275-82
references: 15271389
references: Vet Microbiol. 2001 Sep 20;82(2):141-54
references: 11423205
references: Vet Microbiol. 2006 Aug 25;116(1-3):96-105
references: 16697127
references: Lett Appl Microbiol. 1999 Dec;29(6):406-10
references: 10664985
references: Appl Environ Microbiol. 2003 Aug;69(8):4343-51
references: 12902214
references: PLoS Biol. 2005 Jan;3(1):e15
references: 15660156
references: Nucleic Acids Res. 1997 Sep 15;25(18):3718-23
references: 9278496
references: FEMS Immunol Med Microbiol. 1997 Sep;19(1):47-56
references: 9322068
references: Appl Environ Microbiol. 2004 Feb;70(2):790-7
references: 14766556
references: Appl Environ Microbiol. 2000 Jan;66(1):1-9
references: 10618195
references: J Virol Methods. 1995 Nov;55(3):427-33
references: 8609207
references: Appl Environ Microbiol. 2003 Mar;69(3):1383-90
references: 12620820
references: Cytobios. 1995;82(329):73-9
references: 7587375
references: Vet Res. 2005 May-Jun;36(3):351-82
references: 15845230
references: J Appl Microbiol. 2001 Mar;90(3):470-5
references: 11298244