Malorny Burkhard, Mäde Dietrich, Teufel Paul, Berghof-Jäger Cornelia, Huber Ingrid, Anderson Annette, Helmuth Reiner
Federal Institute for Risk Assessment, National Salmonella Reference Laboratory, Diedersdorfer Weg 1, D-12277 Berlin, Germany.
Int J Food Microbiol. 2007 Jun 30;117(2):211-8. doi: 10.1016/j.ijfoodmicro.2007.04.004. Epub 2007 Apr 25.
A collaborative study including 13 German laboratories was conducted to evaluate the performance of two non-patented real-time PCR methods for the detection of Salmonella in milk powder targeting the ttrC/ttrA- or the invA gene. The enrichment procedure and sample DNA preparation method prior to the real-time PCR was the same for both systems and the identical DNA extraction samples were analysed. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in each laboratory as the reference. The participants received twelve coded milk powder samples each of 25 g for the analysis. Four of them were Salmonella negative (level L0), four artificially contaminated with <3 MPN/g Salmonella Typhimurium (level L1) and four artificially contaminated with 3.6 MPN/g S. Typhimurium (level L2) to the beginning of the experiment. Of the 13 laboratories 12 used various models of real-time PCR blockcyclers conducting both real-time PCR assays and three laboratories the Light Cycler 2.0 system (Roche Bioscience) conducting the ttr-based real-time PCR assay only. The relative accuracy for both real-time PCR assays performed on blockcyclers was for level L0 97.5%. For level L1 the relative accuracy was 94.1% and for level L2 it was 100% for both assays. The relative accuracy on the Light Cycler 2.0 system was 100% for all levels applied to the ttr-real-time PCR.
一项由13个德国实验室参与的合作研究开展了，旨在评估两种非专利实时荧光定量PCR方法在检测奶粉中沙门氏菌的性能，这两种方法分别靶向ttrC/ttrA基因或invA基因。两种系统在实时荧光定量PCR之前的富集程序和样本DNA制备方法相同，且对相同的DNA提取样本进行了分析。每个实验室都采用了符合EN ISO 6579:2002标准的传统培养方法作为参考来检测食品中的沙门氏菌。参与者收到了12个编码的25克奶粉样本用于分析。在实验开始时，其中4个样本沙门氏菌呈阴性（L0水平），4个样本人工污染了每克低于3个MPN的鼠伤寒沙门氏菌（L1水平），4个样本人工污染了每克3.6个MPN的鼠伤寒沙门氏菌（L2水平）。13个实验室中有12个使用了各种型号的实时荧光定量PCR扩增仪进行两种实时荧光定量PCR检测，3个实验室仅使用Light Cycler 2.0系统（罗氏生物科学公司）进行基于ttr的实时荧光定量PCR检测。在扩增仪上进行的两种实时荧光定量PCR检测，L0水平的相对准确度为97.5%。对于L1水平，相对准确度为94.1%，对于L2水平，两种检测的相对准确度均为100%。在Light Cycler 2.0系统上，应用于ttr实时荧光定量PCR的所有水平的相对准确度均为100%。
