A Simplified Isothermal Recombinase Polymerase Amplification Assay for Detection of Sugarcane bacilliform virus Infection

Sugarcane bacilliform virus (SCBV), a circular double stranded DNA genome, is considered as one of the economically important pathogens of sugarcane, limiting the sugarcane production worldwide. Establishment of highly sensitive, quick, simple and cost-effective nucleic acid detection method is essential for its timely detection and management. Present study reports an isothermal recombinase polymerase amplification (RPA) assay for detection of SCBV employing the primers targeting RT/RNase H region of viral genome. The developed RPA assay could detect the virus infection up to 10(-10) dilution of crude sap (original sap prepared by lysis of 100 mg plant tissues in 1 ml of extraction buffer) thus exhibiting sufficient sensitivity. The developed RPA could detect the SCBV infection in to 10 ag mu l(-1) DNA of SCBV infected plant and plasmid DNA containing viral gene inserts, which was equivalent to the limit of detection of PCR assay in respective templates. The study therefore reports a sensitive yet low-cost RPA based detection for SCBV diagnosis which can be used in laboratories of low resource settings. The developed RPA assay is versatile and can be applied for rapid lab-based detection of SCBV infection in sugarcane.