Duplex-immunocapture-RT-PCR for detection and discrimination of two distinct potyviruses naturally infecting sugarcane (Saccharum spp. hybrid).

ABSTRACT

A sensitive duplex-immunocapture-RT-PCR (D-IC-RT-PCR) technique was developed for detection and discrimination of taxonomically distinct Sugarcane streak mosaic virus (SCSMV) and Sugarcane mosaic virus (SCMV) that naturally infect sugarcane. D-IC-RT-PCR was performed using polyclonal antisera for capture of virions. Oligo 5′-d(T)18(AGC)-3′ as a common reverse primer for both viruses and virus specific forward primers, 5′-aagtggttaaacgcctgtgg-3′ and 5′-ATGTC(GA)AAGAA(GA)ATGCGCTTGC-3′ were used for amplifying ~1400 and ~900 bp fragments of SCSMV and SCMV genomes, respectively from their 3′ termini. To assess the applicability of the developed technique, 67 mosaic affected sugarcane samples were initially screened by direct antigen coating-enzyme-linked immunosorbent assay (DAC-ELISA) followed by D-IC-RT-PCR. In DAC-ELISA, ~69 % of tested samples were shown to be positive for presence of SCSMV, ~28 % for SCMV and ~10 % for both viruses. In D-IC-RT-PCR both viruses were detected up to the dilution of 10-4. In D-IC-RT-PCR, ~76 % of tested samples were found to be positive for SCSMV, ~37 % for SCMV and ~16 % for both viruses. The sequence analyses of D-IC-RT-PCR amplicons of 3 isolates of each virus revealed that the designed primers were virus-specific. The developed technique had potential application for sensitive parallel detection of two viruses in sugarcane.