O'Regan Edel, McCabe Evonne, Burgess Catherine, McGuinness Sheila, Barry Thomas, Duffy Geraldine, Whyte Paul, Fanning Séamus
Centres for Food Safety and Food-borne Zoonomics, UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland.
BMC Microbiol. 2008 Sep 21;8:156. doi: 10.1186/1471-2180-8-156.
A real-time multiplex PCR assay was developed for the detection of multiple Salmonella serotypes in chicken samples. Poultry-associated serotypes detected in the assay include Enteritidis, Gallinarum, Typhimurium, Kentucky and Dublin. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in parallel. The real-time PCR based method comprised a pre-enrichment step in Buffered Peptone Water (BPW) overnight, followed by a shortened selective enrichment in Rappaport Vasilliadis Soya Broth (RVS) for 6 hours and subsequent DNA extraction.
The real-time multiplex PCR assay and traditional cultural method showed 100% inclusivity and 100% exclusivity on all strains tested. The real-time multiplex PCR assay was as sensitive as the traditional cultural method in detecting Salmonella in artificially contaminated chicken samples and correctly identified the serotype. Artificially contaminated chicken samples resulted in a detection limit of between 1 and 10 CFU per 25 g sample for both methods. A total of sixty-three naturally contaminated chicken samples were investigated by both methods and relative accuracy, relative sensitivity and relative specificity of the real-time PCR method were determined to be 89, 94 and 87%, respectively. Thirty cultures blind tested were correctly identified by the real-time multiplex PCR method.
Real-time PCR methodology can contribute to meet the need for rapid identification and detection methods in food testing laboratories.
开发了一种实时多重PCR检测方法，用于检测鸡肉样本中的多种沙门氏菌血清型。该检测方法中检测到的与家禽相关的血清型包括肠炎沙门氏菌、鸡沙门氏菌、鼠伤寒沙门氏菌、肯塔基沙门氏菌和都柏林沙门氏菌。同时采用了符合EN ISO 6579:2002标准的传统培养方法来检测食品中的沙门氏菌。基于实时PCR的方法包括在缓冲蛋白胨水（BPW）中过夜预富集步骤，随后在Rappaport Vasilliadis大豆肉汤（RVS）中进行6小时的缩短选择性富集，以及后续的DNA提取。
实时多重PCR检测方法和传统培养方法在所有测试菌株上均显示出100%的包容性和100%的排他性。在检测人工污染鸡肉样本中的沙门氏菌时，实时多重PCR检测方法与传统培养方法一样灵敏，并且能够正确鉴定血清型。对于两种方法，人工污染的鸡肉样本每25克样本的检测限在1至10 CFU之间。两种方法共检测了63份自然污染的鸡肉样本，实时PCR方法的相对准确度、相对灵敏度和相对特异性分别确定为89%、94%和87%。实时多重PCR方法正确鉴定了30份盲测培养物。
实时PCR方法有助于满足食品检测实验室对快速鉴定和检测方法的需求。
