Multiplex RT-PCR for parallel detection of three RNA viruses associated with stripe and mosaic diseases of sorghum in India

摘要

Stripe of sorghum caused by Maize stripe virus (MSpV-Sorg) and mosaic of sorghum caused by Sugarcane streak mosaic virus (SStMV-Sorg) and a newly identified rhabdovirus (tentatively named Indian sorghum mosaic virus-ISMV) are the two prevalent and important viral diseases of sorghum in India. In this study, a multiplex-reverse transcription-polymerase chain reaction (M-RT-PCR) was developed for the simultaneous detection and discrimination of these three taxonomically distinct RNA viruses. Three sets of primers were successfully evaluated for their sensitivity and specificity by both uniplex- and M-RT-PCR by optimizing the conditions for simultaneous detection. The designed primers amplified 962, 749 and 497 bp fragments of MSpV, SStMV and ISMV, respectively. The specificities of multiplex primers were confirmed by sequencing the M-RT-PCR products and the generated sequences of the respective virus isolates were deposited in the GenBank. Using the developed M-RT-PCR, MSpV-Sorg up to 1:1000 dilution while SStMV-Sorg and ISMV up to 1:5000 dilutions of total RNA extracts from respective virus infected sorghum leaf samples were clearly detected. Further, the developed technique was successfully evaluated for detection of above viruses in field-collected sorghum samples with stripe and mosaic symptoms.