梨褪绿叶斑伴随病毒的RT-PCR和巢式RT-PCR检测技术建立

 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。

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

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, 3C and 5H 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 3C 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 5H 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.