Braun Sascha D, Methner Ulrich
Friedrich-Loeffler-Institute, Institute of Bacterial Infections and Zoonoses, Jena, Germany.
Berl Munch Tierarztl Wochenschr. 2011 May-Jun;124(5-6):177-85.
There is a strong interest to reduce the expenditure for the detection of Salmonella spp. from animal faeces and environmental samples from primary production according to ISO 6579:2002 Annex D by including a rapid and effective method to detect Salmonella spp. already after pre-enrichment in BPW. It has been shown that real-time PCR methods are very effective to detect Salmonella organisms after pre-enrichment of foods. However, materials from primary animal production compose of much higher amounts of substances which might inhibit the sensitivity of real-time PCR. Different techniques of DNA isolation after pre-enrichment of artificially inoculated bovine faecal material were used to compare their detection limit and detection probability using an invA 5' nuclease real-time PCR approach. A detection probability of 100% was shown at 10(5) cfu/ml using the QIAamp DNA Stool Mini Kit (Qiagen, Germany), at 10(4) cfu/ml using the High Pure PCR Template Preparation Kit (Roche, Germany) and at 10(3) cfu/ml using thermal cell lysis or an in-house lab protocol, respectively. In comparison DNA isolation by thermal cell lysis revealed a very good detection limit, low costs and almost no risks of contamination. Furthermore, caecal contents from pigs were analysed by ISO 6579:2002 Annex D and the invA real-time PCR using thermal cell lysis for DNA extraction. As a result neither false positive nor false negative findings were obtained. Inclusion of the real-time PCR after pre-enrichment of samples in BPW followed by bacterial detection of Salmonella only with samples positive with real-time PCR might be a valuable tool to fulfil the international standard of ISO 6579:2002 Annex D but also to diminish the expenditures. However, it must be stated that the modification of an international standard method and its use in routine diagnostic requires the validation and registration of national and/or international competent authorities.
人们强烈希望降低按照ISO 6579:2002附录D从动物粪便和初级生产环境样本中检测沙门氏菌属的支出，方法是纳入一种快速有效的方法，以便在BPW中预富集后即可检测沙门氏菌属。已经表明，实时PCR方法在预富集食品后检测沙门氏菌非常有效。然而，初级动物生产的材料含有大量可能会抑制实时PCR灵敏度的物质。使用不同的DNA分离技术对人工接种的牛粪便材料进行预富集后，采用invA 5'核酸酶实时PCR方法比较它们的检测限和检测概率。使用QIAamp DNA Stool Mini试剂盒（德国Qiagen公司）时，在10(5) cfu/ml时检测概率为100%，使用高纯PCR模板制备试剂盒（德国Roche公司）时在10(4) cfu/ml时检测概率为100%，分别使用热细胞裂解或内部实验室方案时在10(3) cfu/ml时检测概率为100%。相比之下，热细胞裂解进行DNA分离显示出非常好的检测限、低成本且几乎没有污染风险。此外，按照ISO 6579:2002附录D对猪盲肠内容物进行分析，并使用热细胞裂解提取DNA进行invA实时PCR。结果未获得假阳性或假阴性结果。在样本于BPW中预富集后进行实时PCR，然后仅对实时PCR呈阳性的样本进行沙门氏菌细菌检测，这可能是满足ISO 6579:2002附录D国际标准以及减少支出的有价值工具。然而，必须指出的是，修改国际标准方法并将其用于常规诊断需要国家和/或国际主管当局的验证和注册。
