辣椒轻斑驳病毒的分子杂交检测

Molecular hybridization detection for pepper mild mottle virus

为探索辣椒轻斑驳病毒(pepper mild mottle virus,PMMo V)的分子杂交技术检测方法,以感病辣椒叶片为试材,用改良CTAB法从辣椒叶片中提取总核酸,运用RT–PCR技术扩增出PMMo V特异片段。以带有PMMo V特异片段的质粒DNA为模板,以用PCR技术制备的地高辛标记PMMo V c DNA探针检测PMMo V。结果表明:1)干燥叶片、新鲜叶片中提取的总核酸稀释80、640倍(相当于12.5、1.60μg叶片)后仍可检测到其中的PMMo V;干燥叶片利于保存及长距离转运,利于对大批量样品进行集中检测;2)采用快速提取法提取叶片总核酸,可从稀释80倍(约12.5μg叶片)的总核酸样品中检测到PMMo V。该技术体系可基本满足常规检测试验的需要。

To develop a diagnosis system for detection pepper mild mottle virus （PMMoV） via molecular hybridization, total nucleic acid was extracted with modified CTAB method from pepper leaves infected by PMMoV, and the specific fragment was amplified using RT-PCR technology. PMMoV was detected by adopted the plasmid DNA from specific fragment of PMMoV as template, and the PMMoV-cDNA labeled with digoxigenin prepared via PCR as probe. The results indicated that： 1） PMMoV could be even detected from the total nucleic both diluted to 80-fold （amount to 12.5 μg leaves） for dry leaves and 640-fold （amount to 1.6 μg leaves） for fresh leaves. Easy conservation and long distance transport for dry leaves were advantageous to perform centralized detection for collected samples in bulk; 2） nucleic acid extracted using the rapid method from pepper leaves could be diluted to 80-fold （amount to 12.5 μg leaves） for PMMoV detection. The diagnostic system could meet the need of routine detection of PMMoV.