该研究建立了检测多种生物胺产生菌的多重聚合酶链反应（PCR）方法，首先检测了已报道的针对酪胺、腐胺产生菌和革兰氏阴性组胺产生菌的多对引物的特异性，针对革兰氏阴性菌的组氨酸脱羧酶基因的引物特异性差，重新设计了该引物，在此基础上，建立了可同时检测酪胺、腐胺和组胺产生菌的多重PCR方法，并优化了多重PCR反应条件为退火温度50 ℃，Mg2+浓度1.0 mmol/L，引物浓度配比为Tdc-F/R 0.2 μmol/L，Hdc1/2 0.4 μmol/L和Odc3/4 0.4 μmol/L。该方法特异性强，对酪胺、腐胺和组胺产生菌的灵敏度分别为1.2 ng/μL、1.5 ng/μL和1.5 ng/μL，可用于黄酒酒曲中生物胺产生菌的快速检测。
Abstract
A multiplex polymerase chain reaction (mPCR) method for the detection of biogenic amines-producing bacteria established in this research. Firstly, the specificity of some reported primers against tyramine, putrescine and gram-negative histamine-producing bacteria was detected, and the poor primer-specificity against gram-negative histamine-producing bacteria was observed. To solve this problem, the primers for gram-negative histamine producing bacteria were redesigned. A mPCR method for simultaneous detection of tyramine, putrescine and histamine-producing bacteria was established. After that, the annealing temperature was 50 ℃, the concentration of Mg2+ was 1.0 mmol/L and the concentration ratio of the Tdc-F/R, Hdc1/2, Odc3/4 primers were 0.2 μmol/L, 0.4 μmol/L, and 0.4 μmol/L, respectively. This method was specific for amplification of the biogenic amine-producing bacteria. The detect sensitivity of tyramine, putrescine and histamine were 1.2 ng/μl, 1.5 ng/μl, and 1.5 ng/μl, respectively. The method can be applied in the rapid detection biogenic amines-producing bacteria in Chinese rice wine Jiuqu.
关键词
多重聚合酶链反应 /
检测 /
黄酒酒曲 /
生物胺产生菌
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Key words
multiplex polymerase chain reaction /
detection /
Chinese rice wine Jiuqu /
biogenic amines-producing bacteria
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