Comparison of direct binding polymerase chain reaction with recombinant coat protein antibody based dot-blot immunobinding assay and immunocapture-reverse transcription-polymerase chain reaction for the detection of sugarcane streak mosaic virus caus

摘要

Direct-binding polymerase chain reaction (DB-PCR) test was developed for the detection of sugarcane streak mosaic virus (SCSMV-AP), a member of the new, undescribed genus in the family Potyviridae. Its sensitivity levels were compared to recombinant coat protein antibody-based tests like dot-blot immunobinding assay (DBIA) and immunocapture-reverse transcription-PCR (IC-RT-PCR) to detect virus in purified preparations and in sugarcane leaf extracts. In DBIA, DB-PCR and IC-RT-PCR, the virus was detected up to 5 ng, 0.01 ng and 0.001 ng in purified virus preparations and 10(-2), 10(-3) and 10(-4) dilutions in infected sugarcane leaf extracts, respectively. When compared to DBIA, IC-RT-PCR appears to be 5000-fold and 100-fold more sensitive with respect to purified virus and infected leaf samples respectively. DB-PCR is 10-fold less sensitive when compared to IC-RT-PCR. Further, shelf-life of recombinant antibody-coated tubes stored at 4degreesC was checked; it was found that they can be used for PCR up to one month. These PCR-based tests could be useful to screen sugarcane germplasm and in breeding and quarantine programmes.