Towards an international standard for PCR-based detection of foodborne thermotolerant campylobacters: interaction of enrichment media and pre-PCR treatment on carcass rinse samples.
As part of a large EU project for standardisation of polymerase chain reaction (PCR), a systematic evaluation of the interaction of enrichment media, type of DNA polymerase and pre-PCR sample treatment for a PCR detecting thermotolerant campylobacters was carried out. The growth-supporting capacity and PCR compatibility of enrichment in Preston, Mueller-Hinton and Bolton broth (blood-containing and blood-free) were evaluated. The effect of resin-based DNA extraction and DNA extraction by boiling on the final PCR assay was investigated. The time-course studies indicated that a 20-h sample enrichment in blood-containing Bolton broth, followed by a simple resin-based extraction of DNA and a PCR amplification using Tth polymerase, resulted in strong and clear PCR amplicons for target (287 bp) and internal amplification control (IAC, 124 bp). The enrichment PCR-based method, tested on 68 presumably naturally contaminated poultry-rinse samples, showed a diagnostic sensitivity of 97.5% (39 PCR-positive/40 total positive samples) and a diagnostic specificity of 100% (28 PCR-negative/28 total negative samples; P=0.32) when compared to a standard bacteriological method (ISO 10272).
Josefsen
M H
MH
Danish Institute for Food and Veterinary Research (DFVF), 27 Bülowsvej, DK-1790 Copenhagen V, Denmark.
Lübeck
P S
PS
Hansen
F
F
Hoorfar
J
J
D000818:Animals; D002167:Campylobacter / Q000235:genetics / Q000254:growth & development*; D002169:Campylobacter Infections / Q000517:prevention & control; D002645:Chickens / Q000382:microbiology; D003470:Culture Media / Q000592:standards; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000592:standards; D004372:Ducks / Q000382:microbiology; D005516:Food Microbiology; D008460:Meat / Q000382:microbiology* / Q000592:standards; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012680:Sensitivity and Specificity
D003470:Culture Media; D004269:DNA, Bacterial; C068508:Tth polymerase; D004259:DNA-Directed DNA Polymerase