Joint Sino-US Food Safety Research Center & Bor Luh Food Safety Center, School of Agriculture & Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
Int J Food Microbiol. 2010 Feb 28;137(2-3):168-74. doi: 10.1016/j.ijfoodmicro.2009.12.004. Epub 2010 Jan 7.
A 5'-nuclease real-time PCR assay using a minor groove binding probe was developed for the detection of Salmonella enterica from artificially contaminated foods. S. enterica-specific sequences were identified by a comparative genomic approach. Several species-specific target sequences were evaluated for specificity. A real-time PCR assay was developed targeting a nucleotide sequence within the putative type III secretion ATP synthase gene (ssaN). An internal amplification control (IAC) probe was designed by randomly shuffling the target probe sequence and a single-stranded oligonucleotide was synthesized to serve as an IAC. The assay demonstrated 100% inclusivity for the 40 Salmonella strains tested and 100% exclusivity for 24 non-Salmonella strains. The detection limit of the real-time PCR assay was 41.2 fg/PCR with Salmonella Typhimurium genomic DNA and 18.6 fg/PCR using Salmonella Enteritidis genomic DNA; 8 and 4 genome equivalents, respectively. In the presence of a natural background flora derived from chicken meat enrichment cultures, the sample preparation and PCR method were capable of detecting as few as 130 Salmonella cfu/mL. Using the developed real-time PCR method to detect Salmonella in artificially contaminated chicken, liquid egg and peanut butter samples, as few as 1 cfu/10 g of sample was detectable after a brief (6h) non-selective culture enrichment.
建立了一种使用小沟结合探针的 5'-核酸酶实时 PCR 检测方法，用于检测人工污染食品中的沙门氏菌。通过比较基因组方法鉴定了沙门氏菌特异性序列。评估了几种种特异性靶序列的特异性。针对假定的 III 型分泌 ATP 合酶基因（ssaN）内的核苷酸序列开发了实时 PCR 检测方法。通过随机打乱目标探针序列设计了内部扩增对照（IAC）探针，并合成了单链寡核苷酸作为 IAC。该检测方法对 40 株沙门氏菌测试菌株的包容性为 100%，对 24 株非沙门氏菌的排他性为 100%。实时 PCR 检测方法的检测限为 41.2 fg/PCR，使用鼠伤寒沙门氏菌基因组 DNA，18.6 fg/PCR 使用肠炎沙门氏菌基因组 DNA；分别为 8 和 4 个基因组当量。在来自鸡肉富集培养物的天然背景菌群存在的情况下，样品制备和 PCR 方法能够检测到低至 130 cfu/mL 的沙门氏菌。使用开发的实时 PCR 方法检测人工污染鸡肉、液态蛋和花生酱样品中的沙门氏菌，在经过简短（6 小时）非选择性培养富集后，可检测到低至 1 cfu/10 g 的样品。
