利用双重PCR-DHPLC技术检测水稻细菌性谷枯病菌的研究

Duplex PCR-DHPLC for detection of Burkholderia glumae

本研究建立了一种应用双重PCR结合变性高效液相色谱技术(polymerase chain reaction-denatured high performanceliquid chromatography,PCR-DHPLC)检测水稻细菌性谷枯病菌的方法。根据水稻细菌性谷枯病菌ITS序列(internal tran-scribed spacer)和gyrB基因序列,设计两对特异性PCR检测引物,对水稻细菌性谷枯病菌株和非水稻细菌性谷枯病菌株分别进行PCR-DHPLC及双重PCR-DHPLC检测,同时进行检测灵敏度及阳性菌株的同源性分析。结果显示,PCR-DHPLC检测的特异性强,灵敏度为菌浓度4×102cfu/mL,7株水稻细菌性谷枯病菌PCR产物同源性一致。该方法能简便、灵敏、高特异性地对水稻细菌性谷枯病菌进行高通量的自动化检测。

In this study,we developed a duplex PCR-DHPLC method for detecting Burkholderia glumae.It was established by using one pair of primers special to ITS（16 S-23 S rRNA interspacer region）sequence and the other pair of primers special to gyrB（β-subunit polyeptide of DNA gyrase）gene.With these two pairs of specific primers,PCR-DHPLC and duplex PCR-DHPLC methods for monitoring B.glumae could detect 4×10^2 cfu/mL bacterial cells.The positive PCR products of 7 strains of B.glumae showed the same sequence homology.The results provided a rapid,reliable and simple effective method for detection of B.glumae.