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.
作者
王月
吴海燕
孙秀伟
付兵
王奎宇
常洪标
万家余
陈志宝
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)
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)
