炭疽芽胞杆菌染色体特异序列的筛选及实时定量检测

筛选117条炭疽芽胞杆菌(Bacillusanthracis)特异序列,经双重特异性验证后得到19条理想的特异序列(genomicsignatures),其中6条符合设计TaqMan探针建立实时定量PCR的要求,根据常规PCR检测结果选择其中C04片段与炭疽芽胞杆菌毒性质粒pX01、pX02上的pagA、capB基因建立实时定量PCR检测体系。经试验证实这一体系检测灵敏度达到每PCR反应10~100个拷贝。利用12种相关菌株评价后获得100%特异性,对10份模拟污染标本和20份对照标本检测,所有污染标本均被检出,所有对照标本均为阴性。此方法特异、灵敏、高效,在炭疽芽胞杆菌感染的诊断和环境污染的检测等领域有潜在的应用前景。

Screening and real-time quantitative detection for genomic signatures of Bacillus anthracis

A total amount of 117 candidate chromosomal sequences were screened for the genomic signatures of Bacillus anthracis by a 2_step approach.Out of them, 19 genomic signatures sequences were selected,among which, 6 genomic signatures were competent as the target sequence of TaqMan real_time PCR method. With the most significant alignment and specificity, fragment C04, together with pagA gene and capB gene from virulence plasmids pX01 and pX02, was selected to establish a TaqMan real_time PCR assay. For each target sequence, as little as 10 to 100 copies per reaction could be detected. Twelve bacterial species including 7 from Bacillus cereus group which were closely related to Bacillus anthracis were used to test the specificity of this assay. Data revealed that the assay gained a 100% specificity. Further performance of the assay was assessed with 10 simulative contaminated environmental samples and 20 negative control environmental samples; all of the Bacillus anthracis contaminated samples gave strong positive signals while the control samples were negative. This specific and sensitive real_time PCR assay could be used in rapid confirmation or exclusion of potential bio_attacks and the diagnosis of anthrax.