Department of Biotechnology and Food Science, University of Burgos, Pza. Misael Bañuelos s/n, 09001, Burgos, Spain.
Department of Advanced Materials, Nuclear Technology and Applied Nano/Biotechnology, University of Burgos, Parque Científico, Edificio I+D+I, Plaza Misael Bañuelos s/n, 09001, Burgos, Spain.
Int J Food Microbiol. 2015 Dec 23;215:16-24. doi: 10.1016/j.ijfoodmicro.2015.08.002. Epub 2015 Aug 16.
Weissella viridescens has been identified as one of the lactic acid bacteria (LAB) responsible for the spoilage of "morcilla de Burgos". In order to identify and quantify this bacterium in "morcilla de Burgos", a new specific PCR procedure has been developed. The primers and Taqman probe were designed on the basis of a sequence from the gene recN. To confirm the specificity of the primers, 77 strains from the genera Carnobacterium, Enterococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Vagococcus and Weissella were tested by conventional PCR. The specificity of the primers and the correct functioning of the probe was confirmed by performing real-time PCR (qPCR) with 21 W. viridescens strains and 27 strains from other LAB genera. The levels of detection and quantification for the qPCR procedure proposed herein were determined for a pure culture of W. viridescens CECT 283(T) and for "morcilla de Burgos" artificially inoculated with this species. The primers were specific for W. viridescens, with only one product of 91 bp being observed for this species. Similarly, the qPCR reactions were found to be specific, amplifying at a mean CT of 15.0±0.4 only for W. viridescens strains. The limit of detection (LOD) and quantification (LOQ) for this procedure was established in 0.082 pg for genomic DNA from W. viridescens. With regard to the artificially inoculated "morcilla", the limit of quantification was established in 80 CFU/reaction and the limit of detection in 8 CFU/reaction. Consequently, the qPCR developed herein can be considered to be a good, fast, simple and accurate tool for the specific detection and quantification of W. viridescens in meat samples.
青霉菌已被鉴定为导致“布尔戈斯血肠”变质的乳酸菌（LAB）之一。为了在“布尔戈斯血肠”中鉴定和定量这种细菌，开发了一种新的特定 PCR 程序。引物和 Taqman 探针是基于 recN 基因的序列设计的。为了确认引物的特异性，通过常规 PCR 测试了来自 Carnobacterium、Enterococcus、Lactobacillus、Leuconostoc、Pediococcus、Streptococcus、Vagococcus 和 Weissella 属的 77 株菌株。通过用 21 株青霉菌和 27 株其他 LAB 属菌株进行实时 PCR（qPCR），确认了引物的特异性和探针的正确功能。提出的 qPCR 程序的检测和定量水平是通过对纯培养的青霉菌 CECT 283(T) 和用该物种人工接种的“布尔戈斯血肠”进行确定的。引物对青霉菌具有特异性，仅观察到该物种的 91bp 产物。同样，qPCR 反应被发现是特异性的，仅对青霉菌菌株的平均 CT 值为 15.0±0.4 进行扩增。该程序的检测限（LOD）和定量限（LOQ）在青霉菌基因组 DNA 中设定为 0.082pg。对于人工接种的“布尔戈斯血肠”，定量限设定为 80CFU/反应，检测限设定为 8CFU/反应。因此，本文开发的 qPCR 可以被认为是一种用于检测和定量肉类样品中青霉菌的快速、简单、准确的方法。
