凤果花叶病毒的RT-PCR和核酸斑点杂交检测研究

Studies on the detection of pepino mosaic virus using RT-PCR and dot blot methods

0引言凤果花叶病毒(pepino mosaic virus,PepMV)属于线形病毒科(Flexiviridae)马铃薯X病毒属(Potexvirus),于1974年首次在秘鲁凤果植物内(Solanum muricatum)发现,后来于1999在荷兰发现该病毒还可侵染番茄[1-2]。被PepMV感染后,番茄生长迟缓,顶部叶片会表现皱缩、畸形和暗绿色不规则斑,下部叶片出现褐色坏死斑,有些叶片上出现亮黄色斑块及疱状突起;在一些大红果色番茄品种上,果实常会出现“大理石状”花纹病斑[3]。

Pepino mosaic virus(PepMV),belonging to genus Potexvirus of the Flexiviridae family,infects tomato(Solanum lycopersicum)and other solanaceae plants,resulting in severe yield losses in some of these infected plants,and has emerged as a severe problem of greenhouse tomato crops worldwide.It calls for effective detection methods to control the epidemics,but the detection methods remain limited.Here,the sap samples of PepMV-infected tobacco leaves were mechanically inoculated into tomato seedlings which were kept in green house.At 14 days post inoculation,the mosaic symptoms were developed on the seedlings inoculated with the PepMV-infected leaf saps,which were characterized by yellow blocks in irregular sizes and shapes on most of the leaves,similar to the symptoms that were previously described;no symptoms were observed on the control leaves.The inoculated seedlings together with the controls were subjected to detection of PepMV by RT-PCR,and the symptomatic seedlings instead of the controls were confirmed to be infected by PepMV.Moreover,the infection of PepMV in all these symptomatic leaves while not in the controls can detected by using a digoxin-labeled RNA riboprobe with a titer of 10-3.This study provides biological and molecular approaches for the detection of PepMV,and offers a technical support for controlling the important viral disease.