Moreno Y, Herńandez M, Ferrús M A, Alonso J L, Botella S, Montes R, Hernández J
Departamento de Biotecnología, Universidad Politécnica, Valencia, Spain.
Res Microbiol. 2001 Jul-Aug;152(6):577-82. doi: 10.1016/s0923-2508(01)01232-3.
We have evaluated the use of PCR and fluorescent in situ hybridization (FISH) techniques for the detection of thermotolerant campylobacters in naturally contaminated chicken products. 16S rRNA sequence data was used to design two specific primers and an oligonucleotide probe for PCR and FISH analyses, respectively. The PCR protocol amplified a 439-bp fragment corresponding to a portion of specific 16S RNA gene from thermotolerant campylobacters. The detection range of the PCR assay varied between 10 cells (after enrichment) to 10(2) cells per mL (without enrichment). FISH probes were able to identify thermotolerant Campylobacter species in 'spiked' and 'unspiked' naturally contaminated samples. PCR and FISH were performed on naturally contaminated samples and compared with the isolation of cells on selective media. The in situ hybridization technique was less sensitive than PCR, although its sensitivity of detection was increased considerably after 22 h of enrichment. These results confirm the usefulness of 16S rRNA-based techniques for the direct detection of campylobacters in food samples.
我们评估了聚合酶链反应（PCR）和荧光原位杂交（FISH）技术在检测天然污染鸡肉产品中耐热弯曲杆菌方面的应用。分别利用16S rRNA序列数据设计了用于PCR和FISH分析的两种特异性引物和一种寡核苷酸探针。PCR方案扩增出一个439碱基对的片段，对应于耐热弯曲杆菌特异性16S RNA基因的一部分。PCR检测的范围在每毫升10个细胞（富集后）至10²个细胞（未富集）之间。FISH探针能够在“加标”和“未加标”的天然污染样品中识别耐热弯曲杆菌种类。对天然污染样品进行了PCR和FISH检测，并与在选择性培养基上分离细胞的结果进行了比较。原位杂交技术的灵敏度低于PCR，不过在富集22小时后其检测灵敏度有了显著提高。这些结果证实了基于16S rRNA的技术在直接检测食品样品中弯曲杆菌方面的实用性。
