The Application of hydroxy naphthol blue in loop-mediated isothermal amplification of pseudorabies virus | 羟基萘酚蓝在伪狂犬病病毒环介导等温扩增检测方法中的应用

Chinese. 伪狂犬病毒(Pseudorabies virus, PRV)传统诊断方法具有程序繁琐、周期长、成本高、特异性差等缺点，本实验建立了敏感、特异的PRV的环介导等温扩增(loop-mediated isothermal amplification, LAMP)检测方法，并在此基础上应用金属离子指示剂羟基萘酚蓝(hydroxy naphthol blue, HNB)进行反应结果判定。实验选取保守性较高的gB基因(glycoprotein B, 糖蛋白gB)筛选引物，确定反应体系后，可在45 min内完成检测。LAMP方法特异性与PCR方法一致，敏感性是PCR方法的100倍，最低能够检测10个拷贝的目的基因。在HNB的应用试验中，发现LAMP反应体系中，HNB浓度为150 μmol/L时，阴阳性的颜色对比明显且反应的敏感性最高，可用于LAMP扩增产物的判断。临床样本的检测结果表明，PCR方法和LAMP方法的检出率一致。因此，本研究中所建立的LAMP方法是一种高度特异敏感的，可替代PCR方法的检测技术。

English. The traditional diagnostic method for Pseudorabies virus (PRV) has the defect of cumbersome procedures, long period, high cost and poor specificity. Thus we built a sensitive and specific loop-mediated isothermal amplification (LAMP) method for the detection of PRV, and used hydroxy naphthol blue (HNB) as indicator to judge the reaction result. The highly conserved gB (glycoprotein B) gene was selected to design primers. The detection could be finished within 45 min after the reaction system was established. Both LAMP and PCR were highly specific to PRV，but he LAMP-based assay, whose detection limit was 102 copies of the target sequence，was 10 times more sensitive than that of the PCR assay. In HNB test, when the ultimate concentration of HNB was 150 μmol/L, the color contrast of the LAMP products between positive samples and negative control was obvious and the sensisity of LAMP reaction was the best.The detection of clinical samples showed that the LAMP reaction had the similar detection rate with the PCR assay. In this experiment, the LAMP method is a highly specific and sensitive detection technology, which can be used instead of traditional PCR method.