CAJ | 학술논문

应用环介导等温扩增（LAMP）技术建立了布鲁氏菌可视化快速检测方法。针对布鲁氏菌外膜蛋白OMP25基因保守区设计6条特异引物，反应前加入染料羟基萘芬蓝（HNB）作为LAMP扩增的指示剂，63℃恒温反应60min，根据HNB的颜色变化进行结果判定。分别评价所建立LAMP方法的特异性和灵敏性，并对60份牛布鲁氏菌病虎红平板凝集试验（RBT）阳性血清样本，经LAMP和B4但5-PCR方法进行平行检测。结果显示，本方法最低检出限约为17垃布鲁氏菌基因组DNA。本方法特异性良好，布鲁氏菌反应管均出现特异性LAMP扩增反应，而猪大肠杆菌K99、巴氏杆菌C48—1、猪链球菌ST171、绿脓杆菌等对照组均未出现扩增。针对60份RBT阳性血清的平行检测结果显示，LAMP和B4／B5．PCR这两种方法间的结果符合率为85．0％。B4／B5-PCR检测为阳性的43份样品，经本方法检测全部为阳性；B4／B5-PCR检测为阴性的17份样品，经本方法检测，9份为阳性，8份为阴性。LAMP的敏感性高于B4／B5-PCR方法。实验表明，本文所建立的基于颜色判定的布鲁氏菌LAMP检测方法具有特异、灵敏、设备要求简单等特点，适用于基层兽医部门进行布鲁氏菌的快速检测。
응용배개도등온확증（LAMP）기술건립료포로씨균가시화쾌속검측방법。침대포로씨균외막단백OMP25기인보수구설계6조특이인물，반응전가입염료간기내분람（HNB）작위LAMP확증적지시제，63℃항온반응60min，근거HNB적안색변화진행결과판정。분별평개소건립LAMP방법적특이성화령민성，병대60빈우포로씨균병호홍평판응집시험（RBT）양성혈청양본，경LAMP화B4단5-PCR방법진행평행검측。결과현시，본방법최저검출한약위17랄포로씨균기인조DNA。본방법특이성량호，포로씨균반응관균출현특이성LAMP확증반응，이저대장간균K99、파씨간균C48—1、저련구균ST171、록농간균등대조조균미출현확증。침대60빈RBT양성혈청적평행검측결과현시，LAMP화B4／B5．PCR저량충방법간적결과부합솔위85．0％。B4／B5-PCR검측위양성적43빈양품，경본방법검측전부위양성；B4／B5-PCR검측위음성적17빈양품，경본방법검측，9빈위양성，8빈위음성。LAMP적민감성고우B4／B5-PCR방법。실험표명，본문소건립적기우안색판정적포로씨균LAMP검측방법구유특이、령민、설비요구간단등특점，괄용우기층수의부문진행포로씨균적쾌속검측。
A loop-mediated isothermal amplification （LAMP） assay was developed for rapid visual detection of Brucella. A set of six specific primers specific to eight regions of OMP25 gene were designed. The reaction was performed in a single tube at 63℃ with the addition ofhydroxynaphthol blue （HNB） dye prior to amplification and the result was determined by the color change of HNB. The sensi- tivity and specificty of LAMP method were evaluated with the optimized reaction system and conditions. Meanwhile, sixty bovine bru- cellosis positive serum samples diagnosed by rose bengal precipitation test （RBT） were detected by LAMP and B4/B5-PCR. The results showed the detection limit of the LAMP assay was about 17 fg genomic DNA, about 100 times higher than conventional PCR method. Also, the specificity of the LAMP assay showed there was no cross reactivity with other related bacteria including Escherichia coli K99, Pasteurella multocida C48-1, Streptococcus ST171, Pseudomonas aeruginosa. Furthermore, the LAMP assay was evaluated by 60 seropositive samples from brucellosis epidemic areas, and the results showed 53 positive samples including all 43 B4/B5-PCR positive samples. The coincidence rate of two methods was 85.0%. These results suggested that the LAMP assay with HNB dye provided a useful tool for the rapid detection of Brucella.