梨褪绿叶斑伴随病毒的RT-PCR和巢式RT-PCR检测技术建立

RT-PCR and nested RT-PCR detection techniques for pear chlorotic leaf spot-associated virus

梨褪绿叶斑伴随病毒(Pear chlorotic leaf spot-associated virus,PCLSaV)是新近发现的为害梨树的欧洲花楸环斑病毒属(Emaravirus)病毒,该病毒基因组由5条负义单链RNA组成。本研究比较分析了反转录引物pd(N)6、3C和5H及基于该病毒基因组RNA3和RNA5链序列设计的4对引物用于RT-PCR检测梨样品中PCLSaV的效果,结果显示,采用与该病毒基因组RNA链3′末端互补的引物3C用于cDNA合成及基于该病毒RNA5链序列的引物5-F/R用于PCR扩增时,检测PCLSaV的灵敏度相较采用引物pd(N)6和5H合成cDNA为模板时高10-100倍;不同部位和不同发病状况的梨树组织中PCLSaV检测结果差异明显。进一步建立了具有高灵敏度的巢式RT-PCR技术,采用外侧引物5-F/R和内巢引物5-IF/IR结合可用于梨不同组织样品中PCLSaV的检测。本研究为系统分析PCLSaV在我国栽培梨树上的危害状况及无病毒梨种质培育奠定了技术基础。

Pear chlorotic leaf spot-associated virus(PCLSaV) is a newly identified pear-infecting virus in the genus Emaravirus. The genome of PCLSaV is composed of at least five negative-sense single-stranded RNA(-ssRNA) segments. In this study, RT-PCR analyses of PCLSaV were carried out by using primers pd(N)6, 3 C and 5 H for cDNA synthesis combined with four sets of PCR primers designed based on the viral RNA3 and RNA5 sequences. Results showed that RT-PCR using primer 3 C for cDNA synthesis and primer set 5-F/5-R specific for the viral RNA5 sequences for the virus detection had 10-100 times higher sensitivity than that with pri-mers pd(N)6 and 5 H for cDNA synthesis. Our results showed that RT-PCR efficiency for the PCLSaV detection varied depending on pear tissues and disease degrees. Furthermore, a highly sensitive nested RT-PCR assay was developed for the detection of PCLSaV in pear leaf samples by using 5-F/R as an outside primer set and 5-IF/IR as a nested primer set. The presented results provide a useful tool for investigation of the virus in pear trees and certification of pear virus-free nursery stocks.