Pentimalli Daniela, Pegels Nicolette, García Teresa, Martín Rosario, González Isabel
Dipartimento di Sanità Pubblica, Facoltà di Agraria, Università degli studi di Parma, Parma, Italy.
J Food Prot. 2009 Jul;72(7):1491-5. doi: 10.4315/0362-028x-72.7.1491.
An enrichment PCR assay using species-specific primers was developed for the detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in chicken meat. Primers for A. cryaerophilus, A. skirrowii, and A. cibarius were designed based on the gyrA gene to amplify nucleic acid fragments of 212, 257, and 145 bp, respectively. The A. butzleri-specific primers were designed flanking a 203-bp DNA fragment in the 16S rRNA gene. The specificity of the four primer pairs was assessed by PCR analysis of DNA from a panel of Arcobacter species, related Campylobacter, Helicobacter species, and other food bacteria. The applicability of the method was then validated by testing 42 fresh retail-purchased chicken samples in the PCR assay. An 18-h selective preenrichment step followed by PCR amplification with the four Arcobacter primer sets revealed the presence of Arcobacter spp. in 85.7% of the retail chicken samples analyzed. A. butzleri was the only species present in 50% of the samples, and 35.7% of the samples were positive for both A. butzleri and A. cryaerophilus. A. skirrowii and A. cibarius were not detected in any of the chicken samples analyzed. The enrichment PCR assay developed is a specific and rapid alternative for the survey of Arcobacter contamination in meat.
开发了一种使用种特异性引物的富集PCR检测方法，用于检测鸡肉中的布氏嗜冷栖热菌、嗜低温嗜冷栖热菌、斯氏嗜冷栖热菌和西巴氏嗜冷栖热菌。基于gyrA基因设计了嗜低温嗜冷栖热菌、斯氏嗜冷栖热菌和西巴氏嗜冷栖热菌的引物，分别扩增212、257和145bp的核酸片段。布氏嗜冷栖热菌特异性引物设计在16S rRNA基因中一个203bp DNA片段的侧翼。通过对一组嗜冷栖热菌属、相关弯曲菌属、幽门螺杆菌属物种以及其他食品细菌的DNA进行PCR分析，评估了这四对引物的特异性。然后通过在PCR检测中测试42份新鲜零售购买的鸡肉样品，验证了该方法的适用性。经过18小时的选择性预富集步骤，随后用四组嗜冷栖热菌引物进行PCR扩增，结果显示在所分析的零售鸡肉样品中，85.7%存在嗜冷栖热菌属。在50%的样品中仅存在布氏嗜冷栖热菌，35.7%的样品同时对布氏嗜冷栖热菌和嗜低温嗜冷栖热菌呈阳性。在所分析的任何鸡肉样品中均未检测到斯氏嗜冷栖热菌和西巴氏嗜冷栖热菌。所开发的富集PCR检测方法是一种用于肉类中嗜冷栖热菌污染调查的特异性快速替代方法。
