基于环介导等温扩增技术检测橡树疫霉菌

Rapid diagnostic methods for Phytophthora ramorum using LAMP

应用环介导等温核酸扩增技术(loop-mediated isothermal amplification,LAMP)建立了基于颜色判定的简单、快速和灵敏的橡树疫霉菌(Phytophthora ramorum)检测方法。以ITS为靶序列对橡树疫霉菌的分子检测存在无法区分近似种的问题,笔者在橡树疫霉菌的基因组研究中,发现了PrA3aPro类转座子序列。生物信息学分析表明,该序列非常适合作为大豆疫霉分子检测的靶标。利用LAMP技术,以PrA3aPro为靶序列,设计LAMP特异性引物,建立LAMP反应体系与条件,并进行灵敏度和特异性试验。结果表明:整个检测过程仅需1 h,即可通过肉眼直接目测试验结果。在等温条件下(64℃)进行核酸扩增反应1 h,然后经3种方法验证扩增结果:1)浊度仪验证浊度变化;2)琼脂糖凝胶电泳验证;3)在扩增前加入染料HNB(羟基萘酚蓝)作为反应指示剂,根据HNB的颜色变化判定。特异性试验中,在橡树疫霉菌株中能够产生浊度曲线,扩增到梯形条带,同时HNB显色观察到天蓝色的阳性反应,而在其他疫霉、腐霉和真菌供试菌株中均没有观察到这些现象。在灵敏度试验中,PrA3aPro-LAMP技术最低检测限为1 ng.μL-1。该方法的建立为橡树疫霉菌的检疫及其所致病害的快速诊断提供了新的技术。

This study reports the development of a loop-mediated isothermal amplification （LAMP） assay targeting the PrA3aPro element for visual detection of Phytophthora rarnorum, rDNA sequences from closely related species are highly conserved,limiting the development of species-specific detection primers. The PrA3aPro-LAMP assay efficiently amplified the target element in less than 60 min at 64℃and was evaluated for specificity and sensitivity. The specificity was evaluated against P. ramorum,Phytophthora spp. , Pythium spp. , and true fungi isolates. Magnesium pyrophosphate resulting from the LAMP of P. ramorum could be detected by real- time measurement of turbidity. Meanwhile, the P. ramorum DNA products were visualised as a ladder-like banding pattern on 2% gel electrophoresis. A positive colour（ sky blue）was only observed in the presence of P. ramorum by addition of hydroxynaphthol blue （HNB） prior to amplification, whereas none of other isolates showed a eolour change. The detection limit of the PrA3aPro-specific LAMP assay for P. ramorum was 1 ng. μL-1 of genomic DNA per reaction. Establishment of LAMP provides a new alternative method for the rapid detection of P. ramorum.