Multiplex RT-PCR detection of two orchid viruses with an internal control of plant nad5 mRNA.

Abstract

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.