干肉制品中霉菌PCR鉴定方法的建立

Establishment of a PCR Assay for Detection of Molds in Dried Meat Products

为了早期预防传统牛肉干发生霉变,建立了牛肉干中真菌PCR的鉴定方法,在特异性和敏感性方面对所建立的PCR方法进行了验证。其中特异性试验以霉变的鲜牛肉、酵母菌、乳酸球菌、乳酸杆菌、大肠杆菌为材料进行验证;敏感性试验以霉变的鲜牛肉为材料,提取其DNA并将其稀释成不同的浓度进行PCR验证。结果显示:特异性试验中仅霉菌样品在约300 bp处出现特异性条带,而其他样品在该处无特异性条带出现,表明具有良好的特异性;敏感性试验中,霉菌DNA原液、10倍稀释、100倍稀释、1 000倍稀释时均出现了约300 bp的特异性条带,即霉菌DNA模板稀释度为1 000倍(2 ng/μL)时仍对引物有敏感性。该试验为市场传统牛肉干真菌PCR检测工作提供了依据。

In order to prevent the mildewing of traditional beef jerky in the early stage of retention period, a PCR assay for detection of molds in beef jerky was established, and the specificity and sensibility of the PCR assay were verified. The mouldy fresh beef, yeast, Lactococcus lactis, Lactobacillus and E. coli were used as materials to verify the specificity of the established PCR assay, and a series of different concentration gradients of DNA solutions extracted from mouldy fresh beef were used to identify its sensibility. The results demonstrated that when the DNA extracted from yeast was used as a template, a specific 300 bp amplicon was obtained by the PCR assay; however, no amplicons were obtained using the DNA extracted from other samples as template, which indicated the good specificity of the established PCR assay. The specific bands were observed when the DNA stock solution extracted from mouldy fresh beef samples as well as the 10 times, 100 times and 1 000 times diluent of the stock solution were used as template in the PCR assay. The 2 ng/μL of the DNA template was still sensitive to the primers. The PCR assay developed in this study provided a new technique for the rapid detection of molds contamination of saled traditional beef jerky in market.