炭疽杆菌双毒力因子荧光PCR检测方法的建立及应用

Establishment and Application of a Fluorescent PCR Assay for Double Virulence Genes of Bacillus anthracis

由炭疽杆菌(Bacillus anthracis)引起的炭疽(anthrax)是严重危害人类和家畜健康的重大传染病。为了提高出入境口岸对不明粉未快件样品的查验效率,防范通过包裹夹带生化武器,本研究使用TaqMan探针技术,建立了针对炭疽杆菌两种毒力质粒pOX1和pOX2的双重荧光PCR检测方法,并对该方法的特异性、灵敏性和稳定性进行了试验。结果显示:本研究建立的炭疽杆菌双重荧光PCR检测方法具有较强的特异性,只特异性检出炭疽杆菌pOX1和pOX2,而与金黄色葡萄球菌等19种对照病原菌均无交叉反应;灵敏度与单重荧光PCR方法相近,最低检测限可达10 copies/μL;稳定性好,反应体系低温保存2、4、6、8个月后,检测阳性质粒的Ct值和峰值相差不大。应用该方法检测600份猪牛羊组织样本,均未检出炭疽杆菌阳性核酸。本研究建立的双重荧光PCR检测方法为炭疽杆菌的快速检测提供了技术支撑。

Anthrax caused by Bacillus anthracis was a major infectious disease that seriously endangers the health of human and livestock.In order to improve the performance of entry and exit ports for checking unidentified powder delivered by express,and to avoid any biochemical weapon carried within any package,a dual fluorescent PCR assay for two virulent plasmids pOX1 and pOX2 of Bacillus anthracis was established by using TaqMan probes,followed by the test of its specificity,sensitivity and stability.The results showed that the established method was with strong specificity,by which,only pOX1 and pOX2 were detected,but no cross reaction with 19 kinds of control pathogenic bacteria including Staphylococcus aureus was observed;its sensitivity was similar to that of single fluorescent PCR assay,and its minimum detection limit was up to 10 copies/μL;good stability was also observed,the Ct value and peak value of the positive plasmids were slightly different after the reaction system was stored at low temperature for 2,4,6 and 8 months.600 tissue samples collected from pigs,cattle and sheep were detected by the method,and no Bacillus anthracis positive nucleic acid was detected.Future rapid detection of Bacillus anthracis would be technically supported by the method established in the paper.