CPA恒温扩增技术检测炭疽芽胞杆菌方法的建立

CPA thermostatic expansion technique for detection of Bacillus anthracis

目的建立一种简便的新型交叉引物恒温核酸扩增结合层析试纸条方法,快速可视化检测炭疽芽胞杆菌核酸。方法通过序列比对挑选出炭疽芽胞杆菌质粒上的特异性基因pXO2,设计引物、探针并合成标记,建立核酸恒温扩增反应体系,用金标免疫层析试纸条快速检测扩增产物,并测试方法的灵敏度和特异性。结果建立的核酸恒温扩增结合金标条方法检测炭疽芽胞杆菌,采用阳性质粒DNA优化实验条件,灵敏度可达5.4pg/反应体系;建立的检测方法检测炭疽Sterne疫苗株的灵敏度为6×103CFU/ml;与枯草芽胞杆菌、苏云金芽胞杆菌、矮小芽胞杆菌、蜡样芽胞杆菌、蕈状芽胞杆菌等相似菌株无交叉反应。结论本研究建立的方法对炭疽芽胞杆菌检测具有很好的敏感性、特异性,为生物恐怖因子现场检测提供了新方法,在国境检疫及公共卫生安全领域有较好的应用前景。

Objective To establish a simple and novel method named cross priming amplification combine to nucleic acid test strip for rapid detection of Bacillus anthracis. Methods The Bacillus anthracis specific primers and probes were designed by sequence alignments, then isothermal nucleic acid amplification reactions were established and the amplified products were rapidly test by colloid gold immunochromatographic strip. The sensitivity and specificity of this method was evaluated. Results The method of cross priming amplification combine to nucleic acid test strip detected Bacillus anthracis was optimized by positive control standards plasmid with the sensitivity up to 10 pg /test, and sensitivity was 103CFU/ml for detection of Sterne strain, and no cross reaction was found to detect Bacillus subtilis, Bacillus thuringiensis, Bacillus pumilus, Bacillus cereus, Bacillus mycoides.Conclusion With high sensitivity and specificity, this method will be useful for detection of Bacillus anthracisin in the field of health quarantine and public health.