Multiplex RT-PCR Detection of Two Orchid Viruses with an Internal Control of Plant nad5 mRNA

以多對引子反轉錄聚合酶連鎖反應同時檢測兩種蘭花病毒和植物內在對照nad5 mRNA

李淑娟(Shu-Chuan Lee) ； 張雅君(Ya-Chun Chang)

《植物病理學會刊》 15卷3期 (2006/09) Pp. 187-196

https://doi.org/10.6649/PPB.200609_15(3).0005

摘要

針對感染蘭科植物的蕙蘭嵌紋病毒(Cymbidium mosaic virus，CymMV)及齒舌蘭輸斑病毒(Odontoglossum ringspot virus，ORSV)開發出一套利用多對引子反轉錄聚合酶連鎖反應(multiplex PT-PCR)爲基楚的檢测方法。由基因資料库(GenBank)中蒐集此二病毒的基因體序列，進行序列比對後，分别設計CymMV和ORSV之專一性引子對，以增幅個别病毒的鞘蛋白基因。除此之外，針對植物粒線體煙醯胺腺嘌呤二核酸去氫酶(NADH dehydrogenase，nad5)基因也設計另一組引子對，在進行反轉錄聚合酶連鎖反應時，可以專一性增幅出nad5的訊息RNA (mRNA)片段，以此作爲檢測時的反應内在對照組。利用nad5專一性引子對，不論在健康或是病毒感染的植物全RNA樣品中，皆可以稳定的增幅出nad5 mRNA片段。上述三組引子對不論是利用單對引子及對引子反轉錄聚合酶連鎖反應進行测試，結果證實皆具有高度專一性。當以多對引子反轉錄聚合酶連鎖反應测試方法的靈敏度時，不論樣品含有單一或兩種病毒，針對CymMV檢測靈敏度皆可逹1Pg，對ORSV的檢測靈敏度則爲10Pg。在複合感染的樣品中，此二病毒存在量的差異似乎不會影響檢測的結果。本檢測方法的研發可以應用於例行的病毒篩檢，同時可以降低檢測成本舆避免僞陰性反應，期望對於國内蘭花種苗病毒驗證作業有所助益。

關鍵字

蕙蘭嵌紋病毒 ； 齒舌蘭輸斑病毒 ； 多封引子反轉錄聚合酶連鎖反應 ； nad5 ； 反應内在對照組

並列摘要

A multiplex RT-PCR method was developed for the detection of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) in orchids. Specific primers were designed based on the available sequences of CymMV and ORSV from the GenBank and were used to RT-PCR amplify the respective viral coat protein genes. In addition, one primer pair derived from the plant mitochondrial NADH dehydrogenase gene (nad5) was tested for the amplification of nad5 mRNA as internal control in multiplex RT-PCR. The fragment of internal control mRNA was constantly amplified from total RNA of healthy and infected plants. The specificity of three designed primer pairs for CymMV, ORSV and nad5 mRNA was confirmed by means of simplex and multiplex RTPCR assays. The detection sensitivity of multiplex RT-PCR was 1 pg for CymMV and 10pg for ORSV no mater one or two kinds of viral RNAs existing in samples. The quantity difference of viral RNA seemed to have no influence on the result of multiplex RT-PCR. Application of multiplex RTPCR could greatly reduce the cost and false negative results for routine detection; therefore, it may have great help to the certification program of orchids.

並列關鍵字

Cymbidium mosaic virus ； Odontoglossum ringspot virus ； multiplex RT-PCR ； nad5 ； internal control