Malorny Burkhard, Bunge Cornelia, Helmuth Reiner
Federal Institute for Risk Assessment, Diedersdorfer Weg 1, D-12277 Berlin, Germany.
J Microbiol Methods. 2007 Aug;70(2):245-51. doi: 10.1016/j.mimet.2007.04.013. Epub 2007 Apr 29.
A robust duplex 5' nuclease (TaqMan) real-time PCR was developed and in-house validated for the specific detection of Salmonella enterica subspecies enterica serovar Enteritidis in whole chicken carcass rinses and consumption eggs. The assay uses specifically designed primers and a TaqMan probe to target the Prot6e gene located on the S. Enteritidis specific 60-kb virulence plasmid. As an internal amplification control to monitor Salmonella DNA in the sample, a second primer/TaqMan probe set detects simultaneously the Salmonella specific invA gene. The assay identified correctly 95% of the 79 Salmonella Enteritidis strains tested comprising 19 different phage types. None of the 119 non-Enteritidis strains comprising 54 serovars was positive for the Prot6e gene. The assay detection probability was for 10(2) or more genome equivalents 100% and for 10 equivalents 83%. A pre-PCR sample preparation protocol including a pre-enrichment step in buffered peptone water, followed by DNA extraction was applied on low levels of artificially contaminated whole chicken carcass rinses and eggs from hens as well as 25 potentially naturally contaminated chickens. The detection limit was less than three CFU per 50 ml carcass rinse or 10 ml egg. The sensitivity and specificity compared to the traditional culture-based detection method and serotyping were both 100%. Twenty-five potentially naturally contaminated chickens were compared by the real-time PCR and the traditional cultural isolation method resulting in four Salmonella positive samples of which two were positive for the Prot6e gene and serotyped as S. Enteritidis. We show also that Salmonella isolates which have a rough lipopolysaccharide structure could be assigned to the serovar Enteritidis by the real-time PCR. This methodology can contribute to meet the need of fast identification and detection methods for use in monitoring and control measures programmes.
开发了一种稳健的双链5'核酸酶（TaqMan）实时荧光定量PCR方法，并在内部进行验证，用于特异性检测全鸡胴体冲洗液和食用鸡蛋中的肠炎沙门氏菌肠炎亚种肠炎血清型。该检测方法使用专门设计的引物和TaqMan探针，靶向位于肠炎沙门氏菌特异性60 kb毒力质粒上的Prot6e基因。作为监测样品中沙门氏菌DNA的内部扩增对照，第二组引物/TaqMan探针同时检测沙门氏菌特异性invA基因。该检测方法正确鉴定了79株肠炎沙门氏菌中的95%，这些菌株包括19种不同的噬菌体类型。119株非肠炎血清型菌株（包括54个血清型）中，没有一株Prot6e基因呈阳性。该检测方法对于10²或更多基因组当量的检测概率为100%，对于10个当量的检测概率为83%。一种PCR前样品制备方案，包括在缓冲蛋白胨水中进行预富集步骤，然后进行DNA提取，应用于低水平人工污染的全鸡胴体冲洗液、母鸡产的鸡蛋以及25只可能自然污染的鸡。检测限为每50 ml胴体冲洗液或10 ml鸡蛋中少于3个CFU。与传统的基于培养的检测方法和血清分型相比，灵敏度和特异性均为100%。通过实时荧光定量PCR和传统培养分离方法对25只可能自然污染的鸡进行比较，结果有4份沙门氏菌阳性样品，其中2份Prot6e基因呈阳性，血清型鉴定为肠炎沙门氏菌。我们还表明，具有粗糙脂多糖结构的沙门氏菌分离株可通过实时荧光定量PCR鉴定为肠炎血清型。这种方法有助于满足监测和控制措施计划中快速鉴定和检测方法的需求。
