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 causing mosaic disease of sugarcane in India

Abstract

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 4°C 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.