Duplex — reverse transcription — polymerase chain reaction (D-RT-PCR)-a technique for the simultaneous detection of viruses causing sugarcane mosaic

Abstract

Sugarcane mosaic virus (SCMV) and Sugarcane streak mosaic virus (SCSMV) are the two major RNA viruses that are widely prevalent in most of the Asian countries including India and are the causative viruses of mosaic in sugarcane. We have established that these two viruses either alone or in combination cause mosaic in sugarcane through RT-PCR. Further studies were conducted to detect the associated viruses in a single reaction. To optimize simultaneous detection of these viruses, a new set of primers were designed from the coat protein region of the viruses to suit duplex reverse transcription polymerase chain reaction (D-RT-PCR) and the conditions were standardized to amplify the target viruses in this assay. Of the 29 sugarcane samples tested for the simultaneous occurrence of SCMV and SCSMV, 10 sugarcane varieties were found to be infected with both the viruses with positive amplifications of ∼860 bp and ∼690 bp respectively, for SCMV and SCSMV. In the remaining 19 varieties, SCSMV was detected alone as the causative virus of mosaic. None of the selected varieties were found infected with SCMV alone. Further, the D-RT-PCR was found equally reliable to uniplex RT-PCR performed with SCMV and SCSMV primers in separate reactions. The D-RT-PCR products were cloned, sequenced and the respective sequence information confirmed the authenticity of the viruses amplified in D-RT-PCR. The lengths of the amplified fragments from SCMV isolates were ranged between 812 and 877 bp and in SCSMV it ranged from 683 to 689 bp. This diagnostic approach will be useful to establish distribution map of the virus(es) associated with mosaic in India and in other sugarcane growing countries.