以18S rRNA为内参照的多重RT-PCR检测3种百合病毒

Optimization and application of a multiplex RT-PCR system for simultaneous detection of three lily viruses using 18S rRNA as internal control

本研究对多重PCR体系各成分和循环参数进行了摸索和优化,建立了以18S rRNA为内参照的同时检测3种百合病毒的多重RT-PCR体系,所检测的3种病毒是黄瓜花叶病毒(CMV)、百合斑驳病毒(LMoV)和百合无症病毒(LSV)。它们为侵染我国百合的主要病毒。在RT反应体系中加入3种病毒和18S rRNA的特异性反向引物的混合物,使反应体系中各反向引物终浓度均为0.5μmol/L,反转录酶(AMV)反转录合成各病毒和18S rRNA的互补第一链cDNAs。多重RT-PCR条件实验显示:将标准RT-PCR体系中的TaqHS DNA聚合酶量改为0.100 U/μL,Mg2+浓度改为4.0 mmol/L,各引物浓度选择0.2μmol/L,那么25个循环反应以上就能在一个反应管中以18S rRNA为内参照同时检测百合中的3种常见病毒。用该体系检测了10个百合样品,同时与32P同位素标记膜杂交检测作对照,结果显示,该检测体系检测灵敏度更高。

Reaction components and reaction cycling parameters were optimized for a multiplex RT-PCR protocol to simultaneous detection for three lily viruses with 18S ribosomal RNA used as an internal control. The first strand eDNA was synthesized using reverse transcriptase and primer mixture of three virus-specific reverse primers （0.5 μmol/L each） and 18S rRNA reverse primer （0.5 μmol/L）. A series of conditional assays resuited in modification of the standard reaction components. Utilization of a DNA polymerase concentration of 0. 100 U/μL, Mg^2＋ of 4.0 mmol/L, and four primer pairs of 0.2 μmol/L each was set for simultaneously amplificating the cDNAs of Lily mottle virus （LMoV）, Cucumber mosaic virus （CMV）, Lily symptomless virus （LSV） and 18S rRNA with more than 25 cycles. The above multiplex system was able to detect different virus combinations. The feasibility of multiplex RT-PCR was investigated by analyzing 10 leaf tissue samples. Compared to ^32p labeling nucleic acid hybridization, this optimized multiplex RT-PCR system was of higher sensitivity and specificity for detection of the above viruses infecting lily plants.