基于核酸横流生物传感器的沙门氏菌快速、可视化检测方法  

Rapid and Visual Detection Method of Salmonella Based on Lateral Flow Nucleic Acid Biosensor

作　　者：王月[1] 吴海燕[2] 孙秀伟 付兵 王奎宇 常洪标 万家余[3] 陈志宝[1] 武志敏 Wang Yue;Wu Haiyan;Sun Xiuwei;Fu Bing;Wang Kuiyu;Chang Hongbiao;Wan Jiayu;Chen Zhibao;Wu Zhimin(College of Life Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319;College of Animal Science and Technology,Heilongjiang Bayi Agricultural University;Institute of Military Veterinary,Academy of Military Medical Sciences;Bureau of Animal Husban,Monggol nationality Autonomous County)

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,大庆163319 [2]黑龙江八一农垦大学动物科技学院 [3]长春军事医学科学院军事兽医研究所 [4]杜尔伯特蒙古族自治县畜牧局

出　　处：《黑龙江八一农垦大学学报》2018年第6期63-68,81,共7页journal of heilongjiang bayi agricultural university

基　　金：农垦总局课题"提高母猪年生产力关键技术集成与示范"(HNK135-04-06-02)

摘　　要：为了建立沙门氏菌的快速、可视化检测方法,根据核酸横流生物传感器(lateral flow nucleic acid biosensor,LFNAB)的检测原理,对沙门氏菌核酸扩增产物进行改良。以沙门氏菌DNA为模板,通过PCR技术和DNA酶的介导,将生物素-三磷酸碱基脱氧核苷酸(Biotin-dNTP)引入DNA链中,使高拷贝的DNA双链携带一定含量的生物素分子。在DNA链上,由于上游引物修饰的异硫氰酸荧光素抗原(FAM)和dNTP上携带的生物素分子(Biotin),导致新产生的PCR产物可采用LFNAB进行检测。实验证明,Biotin-dUTP最佳的使用量是脱氧胞嘧啶核苷三磷酸(dTTP)和Biotin-dUTP混合物的总量的5%,且LFNAB的样品检验效果清晰直观、肉眼可见,能够检测出101 CFU·mL-1浓度的沙门氏菌。To establish a rapid and visual detection method for Salmonella,the natural dsDNA of Salmonella was improved based on the principle of lateral flow nucleic acid biosensor(LFNAB).The experiment used Salmonella DNA as a template to introduce biotindNTPs into DNA strands by PCR and DNase mediation,so that high copy dsDNA could carry a certain amount of biotin molecule.On DNA strands,newly generated PCR products could be detected using LFAAB due to forward primer-modified fluorescein isothiocyanate antigen(FAM)and biotin molecules carried on dNTPs.Experiments showed that the optimal amount of Biotin-dUTP was5%of the total amount of dTTP and Biotin-dUTP mixture,and the test results of the LFNAB were clear and intuitive,visible to the naked eye,and could detect101CFU·mL^-1concentrations of Salmonella.

关 键 词：核酸横流生物传感器 沙门氏菌 生物素化双链DNA 灵敏性比较 可视化检测 

分 类 号：Q7[生物学—分子生物学]