RT-fqPCR检测腐乳耐药基因方法的建立及应用  

Establishment and application of RT-fqPCR method for detection of drug resistance gene in sufu

作　　者：张娜娜 刘洋[1] 赵磊 赵燕[1] 俞漪[1] ZHANG Nana;LIU Yang;ZHAO Lei;ZHAO Yan;YU Yi(Shanghai Institute of Quality Inspection and Technical Research,National Institute ofQuality Inspection and Research on Product in Shanghai,Shanghai 200233,China)

机构地区：[1]国家级上海产品质量监督检验研究院上海市质量监督检验技术研究院,上海200233

出　　处：《中国酿造》2024年第5期237-241,共5页China Brewing

基　　金：国家市场监督管理总局科技计划项目(2017QK167)。

摘　　要：该研究根据腐乳中微生物全基因组测序结果中耐药基因的丰度筛选出3种耐药基因(Baca、Emra、Imrp)设计引物,建立一种非培养方式—实时荧光定量聚合酶链式反应(RT-fqPCR)方法检测不同腐乳样品中微生物可能存在的耐药基因,并对该方法的灵敏度、抗干扰能力及重复性进行检测,最后应用于市售腐乳样品中耐药基因的检测。结果表明,建立的RT-fqPCR方法对3种耐药基因检测的灵敏度较高,均达到5.4 pg;添加黄豆基因对检测结果影响不显著,抗干扰能力较好;重复性试验结果的相对标准偏差(RSD)均<1.8%,重复性较好。采用该方法对市售的47批次腐乳样品进行检测发现,3种耐药基因Baca、Emra、Imrp在腐乳中的检出率较高,分别为96.5%、92.2%和97.2%;青方腐乳、白方腐乳、红方腐乳3类腐乳含有的耐药基因分别为84.4%、83.3%和89.6%。综上,建立的RT-fqPCR方法可快速检测不同腐乳中的3种耐药基因。In this study,three kinds of drug resistance genes(Baca,Emra,Imrp)were screened to design primers according to the abundance of drug resistance genes in the sequencing results of the whole genome of microorganisms in sufu.A non culture method—real-time fluorescent quantitative polymerase chain reaction(RT-fqPCR)was established to detect the possible resistance genes of microorganisms in different sufu samples,the sensitivity,anti-interference ability and repeatability of the method were tested,and finally applied to the detection of drug resistance genes in sufu samples on the market.The results showed that the established RT-fqPCR method had high sensitivity for detecting three drug resistance genes,all reaching 5.4 pg.The addition of soybean genes had no significant impact on the detection results,but had good anti-interference ability.The relative standard deviation(RSD)of the repeatability test results were all less than 1.8%,and the repeatability was good.This method was used to detect 47 batches of commercial sufu samples,the results showed that the detection rates of Baca,Emra and Imrp in sufu were 96.5%,92.2%and 97.2%,respectively,and the resistance genes of grey sufu,white sufu and red sufu were 84.4%,83.3%and 89.6%,respectively.In conclusion,the established RT-fqPCR method could rapidly detect three kinds of drug resistance genes in different sufu.

关 键 词：腐乳 微生物 实时荧光定量聚合酶链式反应 耐药基因 检测 

分 类 号：Q936[生物学—微生物学]