摘要
目的为了更加方便布鲁氏菌病(布病)检测,增加布病检测方法的多样性。方法根据布鲁氏菌特异基因的保守序列,设计引物与探针,利用Twist Amp? nfo试剂盒提供的聚合酶扩增体系对布鲁氏菌基因组进行快速等温扩增,扩增产物用PCRD试剂盒侧流试纸进行检测,进而判断结果。其实验过程为确定最佳反应条件,检测特异性和敏感性,并与QPCR同时检测样品,比较符合率。结果 38℃反应10 min为最佳条件,特异性结果良好,敏感性能达到10-5(8拷贝),与QPCR检测结果符合率高,以此建立本方法。结论该方法具有特异性强、敏感性好、操作过程简单、检测快速等优点,增加了布病检测方法的多样性,同时为野外田间布病的检测提供了一种可能。
In order to develop diversified and detection method for animal brucellosis, lateral flow test strips combined with FAM recombinase polymerase amplification (RPA) of Brucella was established, by designing primers and probes based on omp25 gene,Firstly,the Brucella genome was rapidly isothermally amplified using the polymerase amplification system by the Twist Amp^■nfo kit, then the amplified products were quickly detected by lateral flow test strip from PCRD kit. The results showed that the reaction at 38℃for 10 min was the best condition, and the sensitivity and specificity of it was also be proved. It can detect at least 8-copies DNA, and the coincidence rate is higher than that of QPCR.Therefore, this developed RPA has the advantages of high specificity, good sensitivity, simple operation process and rapid detection. It will provide strong support for detecting brucellosis in the field samples.
作者
葛志毅
周建华
尚佑军
李学瑞
刘永生
赵鹭
孙晶晶
曹小安
GE Zhi-yi;ZHOU Jian-hua;SHANG You-jun;Li Xue-rui;LIU Yong-sheng;ZHAO Lu;SUN Jing-jing;CAO Xiao-an(State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000,China)
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2019年第10期905-908,共4页
Chinese Journal of Zoonoses
基金
国家重点研发计划资助(No.2018YFD0500900)~~
关键词
布鲁氏菌
RPA
侧流试纸
田间检测
Brucella
RPA
lateral flow test strips
field detection
作者简介
通讯作者:曹小安,Email:caoxiaoan@caas.cn,ORCID:0000-0001-6064-1297.
