G-四链体与As-PCR联用可视化检测沙门氏菌  

Visual Detection of Salmonella by Combined Application of G-quadruplex and As-PCR Methods

作　　者：刘健慧 张先舟[1] 张蕴哲[1] 王红静 李雪瑶 耿凤珍[3] 檀建新[1] Liu Jianhui;Zhang Xianzhou;Zhang Yunzhe;Wang Hongjing;Li Xueyao;Geng Fengzhen;Tan Jianxin(College of Food Science and Technology,Hebei Agricultural University,Hebei Bioinformatic Utilization and Technological Innovation Center for Agricultural Microbes,Baoding 071000,Hebei;School of Biology and Food Science,Hebei Normal University for Nationalities,Chengde 067000,Hebei;Affiliated Hospital of Hebei University,Baoding 071000,Hebei)

机构地区：[1]河北农业大学食品科技学院,河北省农业微生物生物信息利用技术创新中心,河北保定071000 [2]河北民族师范学院生物与食品科学学院,河北承德067000 [3]河北大学附属医院,河北保定071000

出　　处：《中国食品学报》2023年第5期371-380,共10页Journal of Chinese Institute Of Food Science and Technology

基　　金：河北省自然科学基金重大项目(C2019204342)。

摘　　要：沙门氏菌是食源性病原菌,常在食品媒介中传播,因此快速检测是控制该病原菌的关键。本研究用沙门氏菌基因组DNA作模板,通过对不对称PCR(As-PCR)扩增条件的优化,证明当非限制性引物HF终浓度0.4μmol/L,与限制性引物GR终浓度比10∶1、退火温度58℃,扩增40个循环时可获得最大浓度的单链DNA(ssDNA)产物。带有G-四链体序列的ssDNA与40μmol/L氯化血红素作用60 min时,G-四链体显示出最高的过氧化物酶活性。在此基础上,将As-PCR和G-四链体联用,实现了可视化检测沙门氏菌invH基因,在2 pg/μL~258 ng/μL的质量浓度区间,质量浓度对数与吸光值A421nm呈线性关系(y=0.0691x+0.3085,R2=0.9729)。对污染的牛奶样品检测,检出灵敏度高,为35 CFU/mL,且操作便捷,为病原微生物检测提供了新技术支撑。Salmonella(SALM)is a foodborne pathogen that often transmit via food as media,so rapid detection is the key to controlling the pathogen.In this study,Salmonella genomic DNA was used as templates,the amplification conditions of asymmetric PCR(As-PCR)were optimized.The results demonstrated that the maximum concentration of single-strand DNA(ssDNA)could be obtained when the final concentration of the excess primer(HF)was 0.4μmol/L,the ratio of the final concentration of HF to GR was 10∶1,the annealing temperature was 58℃and the amplification cycle was 40 cycles.When ssDNA with G-quadruplex sequence interacted with 40μmol/L hemins for 60 min,the peroxidase activity of G-quadruplex was the highest.On this basis,the visual detection of the invH gene of Salmonella was realized by combining As-PCR with G-quadruplex technology.The method could detect Salmonella genomic DNA in a range of 2 pg/μL-258 ng/μL,and the logarithm of its concentration had a good linear relationship with the absorbance value at 421 nm(y=0.0691x+0.3085,R2=0.9729).The detection limit of Salmonella for contaminated milk samples was 35 CFU/mL,showing that this method had the advantages of simple operation with a high sensitivity and providing new technical support for the detection of these pathogenic microorganisms.

关 键 词：不对称PCR 可视化检测 沙门氏菌 G-四链体 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]