布鲁氏菌环介导等温扩增（LAMP）可视化检测方法的建立

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/B5-PCR方法进行平行检测。结果显示,本方法最低检出限约为17fg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。

Visual Detection of Brucella by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye

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.