食品用菌种粘液乳杆菌属主要包括发酵粘液乳杆菌和罗伊氏粘液乳杆菌。基于可食用菌种快速、准确鉴定的需求,该文开发了粘液乳杆菌属菌种的实时荧光PCR(qPCR)检验方法。基于发酵粘液乳杆菌dnaJ基因和罗伊氏粘液乳杆菌groL基因,设计特异性引物和探针。同时收集了33株乳酸菌、芽孢菌及其他菌种作为交叉反应的阴性菌株,实验结果表明该文设计的引物和探针对其他菌种没有交叉反应。发酵粘液乳杆菌和罗伊氏粘液乳杆菌qPCR方法的扩增效率(E)分别为87.9%和95.6%,LOD95%值分别为1.91×102、5.33×102 pg/μL。为了验证方法的适用性,采用qPCR方法从食品样品中分离鉴定发酵粘液乳杆菌和罗伊氏粘液乳杆菌,同时采用16S rRNA基因测序,验证了2种方法鉴定菌种的一致性。针对食品样品,该文基于SYBR Green qPCR方法对5株发酵粘液乳杆菌分离株和4株罗伊氏粘液乳杆菌分离株的5种抗生素耐药基因进行了检测,发现四环素类tetA耐药基因检出率较高,提示存在食品安全隐患。实验结果表明,该文建立的qPCR方法适用于粘液乳杆菌属菌种鉴定,特异性好,扩增效率和灵敏度高。
Food strains of Limosilactobacillus mainly include Limosilactobacillus fermentum and Limosilactobacillus reuteri.Based on the needs of rapid and accurate identification of edible strains, a real-time fluorescent PCR (qPCR) test for Limosilactobacillus strains was developed.Specific primers and probes were designed based on the L. fermentum dnaJ gene and the L.reuteri groL gene.At the same time, 33 strains of Lactobacillus, Bacillus, and other strains were collected as negative strains for cross-reactivity, and the experimental results showed that the primers and probes designed in this paper had no cross-reactivity to other strains.The amplification efficiencies (E) of the qPCR methods for L.fermentum and L.reuteri were 87.9% and 95.6%, respectively, and the LOD values were 1.91×102 and 5.33×102 pg/μL.To verify the applicability of the methods, the qPCR methods were used to isolate and characterize L.fermentum and L.reuteri from food samples, while the 16S rRNA gene sequencing was used to verify the consistency of the two methods to identify the strains.For food samples, this study used the SYBR Green qPCR method to detect five antibiotic resistance genes in five strains of L.fermentum and four strains of L.reuteri.It was found that the detection rate of tetracycline tetA resistance genes was high, indicating the existence of food safety hazards.The experimental results showed that the qPCR method established was suitable for the identification of Limosilactobacillus species with good specificity, high amplification efficiency and sensitivity.
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