CAJ | 학술논문

目的建立快速、高通量的基因悬浮芯片检测方法,用于生物恐怖病原体的快速筛检.方法选择保守的细菌16 S rDNA序列,并针对炭疽芽孢杆菌(Bacillus anthracis,B.a)、鼠疫耶尔森菌(Yersinia pestis,Y.p)、布鲁菌属(Brucella spp.,Bru)、土拉弗朗西斯菌(Francisella tularensis,F.t)和类鼻疽伯克霍尔德菌(Burkholderia pseudomallei,B.p)等5种生物恐怖细菌设计种(属)特异性探针,经16 S rDNA通用引物341A、519B扩增基因组DNA,用基因悬浮芯片方法进行检测,并对方法的灵敏度、特异度、重复性、检测能力进行验证.结果经16 S rDNA通用引物扩增,特异性探针杂交检测,能将样本细菌鉴定到属水平,同属菌出现交叉反应;检出限分别为类鼻疽伯克霍尔德菌1.5 pg/μl,布鲁菌属20 ps/μl,炭疽芽孢杆菌7 pg/μl,土拉弗朗西斯菌0.1 pg/μl,鼠疫耶尔森菌1.1 pg/μl;15次重复检测各探针变异系数(CV)为5.18%～17.88%,具有较好的重复性;方法可正确检出模拟白色粉末样本中炭疽芽孢杆菌和鼠疫耶尔森菌.结论建立了通用型基因悬浮芯片检测体系快速高通量筛查生物恐怖细菌的方法.
목적 건립쾌속、고통량적기인현부심편검측방법,용우생물공포병원체적쾌속사검.방법 선택보수적세균16 S rDNA서렬,병침대탄저아포간균(Bacillus anthracis,B.a)、서역야이삼균(Yersinia pestis,Y.p)、포로균속(Brucella spp.,Bru)、토랍불랑서사균(Francisella tularensis,F.t)화류비저백극곽이덕균(Burkholderia pseudomallei,B.p)등5충생물공포세균설계충(속)특이성탐침,경16 S rDNA통용인물341A、519B확증기인조DNA,용기인현부심편방법진행검측,병대방법적령민도、특이도、중복성、검측능력진행험증.결과 경16 S rDNA통용인물확증,특이성탐침잡교검측,능장양본세균감정도속수평,동속균출현교차반응;검출한분별위류비저백극곽이덕균1.5 pg/μl,포로균속20 ps/μl,탄저아포간균7 pg/μl,토랍불랑서사균0.1 pg/μl,서역야이삼균1.1 pg/μl;15차중복검측각탐침변이계수(CV)위5.18%～17.88%,구유교호적중복성;방법가정학검출모의백색분말양본중탄저아포간균화서역야이삼균.결론 건립료통용형기인현부심편검측체계쾌속고통량사사생물공포세균적방법.
Objective To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria. Methods 16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp. and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B,the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed. Results After PCR amplification by 16 S rDNA primers and specific probe hybridization,the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/μl (Burkholderia pseudomallei),20 pg/μl (Brucella spp.), 7 pg/μl (Bacillus anthracis), 0.1 pg/μ1 (Francisella tularensis), and 1.1 pg/μl (Yersinia pestis),respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%,it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples. Conclusion The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.