A Simplified Multiplex PCR Assay for Simultaneous Detection of Six Viruses Infecting Diverse Chilli Species in India and Its Application in Field Diagnosis

Chilli is infected by at least 65 viruses globally, with a mixed infection of multiple viruses leading to severe losses being a common occurrence. A simple diagnostic procedure that can identify multiple viruses at once is required to track their spread, initiate management measures and manage them using virus-free planting supplies. The present study, for the first time, reports a simplified and robust multiplex PCR (mPCR) assay for the simultaneous detection of five RNA viruses, capsicum chlorosis orthotospovirus (CaCV), chilli veinal mottle virus (ChiVMV), large cardamom chirke virus (LCCV), cucumber mosaic virus (CMV), and pepper mild mottle virus (PMMoV), and a DNA virus, chilli leaf curl virus (ChiLCV) infecting chilli. The developed mPCR employed six pairs of primer from the conserved coat protein (CP) region of the respective viruses. Different parameters viz., primer concentration (150–450 nM) and annealing temperature (50 °C), were optimized in order to achieve specific and sensitive amplification of the target viruses in a single reaction tube. The detection limit of the mPCR assay was 5.00 pg/µL to simultaneously detect all the target viruses in a single reaction, indicating a sufficient sensitivity of the developed assay. The developed assay showed high specificity and showed no cross-amplification. The multiplex PCR assay was validated using field samples collected across Northeast India. Interestingly, out of 61 samples collected across the northeastern states, only 22 samples (36%) were positive for single virus infection while 33 samples (54%) were positive for three or more viruses tested in mPCR, showing the widespread occurrence of mixed infection under field conditions. To the best of our knowledge, this is the first report on the development and field validation of the mPCR assay for six chilli viruses and will have application in routine virus indexing and virus management.