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发明公开
CN108048585A 饲料中沙门氏菌的微滴数字PCR检测方法 无效 - 撤回

饲料中沙门氏菌的微滴数字PCR检测方法
摘要：
本发明公开了鉴定沙门氏菌的PCR扩增引物及探针引物，包括：沙门氏菌引物正向序列：5＇‑GCGGCGTTGGAGAGTGATA‑3＇，反向序列；5＇‑AGCAATGGAAAAAGCAGGATG‑3＇；探针序列：5＇‑FAM‑CATTTCTTAAACGGCGGTGTCTTTCCCT‑TAMRA‑3＇；一种饲料中沙门氏菌的微滴数字PCR检测方法，包括以下步骤：称取样品至盛有225mL无菌培养液的均质袋中，用拍击式均质器拍打，稀释为1:10的样品匀液，进行增菌培养，得到增菌液；提取待测样品、阳性对照、阴性对照的DNA；获得鉴定沙门氏菌的PCR扩增引物及探针引物；将所得DNA进行微滴数字PCR扩增，获得反应液；对反应液进行检测与分析。结果表明该引物对沙门氏菌的扩增具有特异性；同时微滴数字PCR对其扩增稳定，具有重复性；取浓度为108‑103的基因序列进行检测，灵敏度良好。
摘要（英）：

The invention discloses a PCR amplification primer and probe primer for authenticating salmonella. A salmonella primer has the forward sequence of 5'-GCGGCGTTGGAGAGTGATA-3' and the backward sequence of 5'-AGCAATGGAAAAAGCAGGATG-3'; a probe has the sequence of 5'-FAM-CATTTCTTAAACGGCGGTGTCTTTCCCT-TAMRA-3'. A droplet digital PCR detecting method of salmonella in feed includes the following steps of weighing a sample into a homogeneous bag containing 225 mL of sterile culture solution, conducting batting through a batting type homogenizer, conducting diluting to obtain a sample homogeneous solutionwith the ratio of 1:10, conducting bacteria increasing culturing to obtain a bacterial increasing solution, extracting DNA of a to-be-detected sample, a positive contrast and a negative contrast, obtaining the PCR amplification primer and probe primer for authenticating salmonella, conducting droplet digital PCR amplification on the obtained DNA to obtain a reaction solution, and detecting and analyzing the reaction solution. It is shown through the result that the primers have specificity for the amplification of salmonella; meanwhile, the droplet digital PCR amplification is stable, and repeatability is achieved; detection conducted through a gene sequence with the concentration of 103-108 shows that sensitivity is high.

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