Towards an international standard for PCR-based detection of foodborne thermotolerant campylobacters:: interaction of enrichment media and pre-PCR treatment on carcass rinse samples

被引:33
作者
Josefsen, MH
Lübeck, PS
Hansen, F
Hoorfar, J
机构
[1] Danish Inst Food & Vet Res, DFVF, DK-1790 Copenhagen V, Denmark
[2] Danish Meat Res Inst, DK-4000 Roskilde, Denmark
关键词
Campylobacter; PCR; enrichment; carcass rinse; food;
D O I
10.1016/j.mimet.2004.03.001
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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). (C) 2004 Elsevier B.V. All rights reserved.
引用
收藏
页码:39 / 48
页数:10
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