番茄褪绿病毒RT-PCR检测技术的优化及河南分离物的鉴定

Technique optimization of RT-PCR for detecting Tomato chlorosis virus and molecular identification of isolates in Henan Province

为获得高效、灵敏和稳定的番茄褪绿病毒(Tomato chlorosis virus,ToCV)RT-PCR检测体系,选取文献报道的和重新设计的共9套ToCV的RT-PCR引物,经过一系列测试和比较,筛选出了灵敏度、特异性均较高的引物组合,并对反应条件、参数进行了优化,确定了最优反应条件。应用优化的ToCV RT-PCR检测体系对从河南设施蔬菜生产区采集的疑似感病番茄样品进行检测,扩增产物测序后经BLAST比对发现,河南分离物的CP和HSP70基因序列与GenBank上注册的ToCV典型分离物序列的相似性均达94%以上,通过对ToCV病毒粒子的电镜观察而进一步确定ToCV已在河南侵染设施番茄。

To obtain stable, sensitive and specific RT-PCR detection system for Tomato chlorosis virus （ToCV）, 9 primer pairs redesigned or selected from those published research papers were compared for their productivity, sensitivity and specificity in the RT-PCR detection system, a series of tests were conducted and the primer pairs that most sensitive and specific among them were selected and confirmed, and the RT-PCR reaction conditions were optimized, The optimized RT-PCR detection system was used to detect the ToCV-suspected tomato samples from greenhouse vegetable-growing area in Henan Province. Sequence analysis revealed that the CP and HSPTO homologue gene of Henan isolates shared 97% nucleotide sequence identity with that of ToCV isolates registered in the NCBI, indicating the tomato plants were infected by ToCV in Henan. This results were also confirmed by electron microscopy observation of the virus particles extracted from the samples.