Development of reverse transcription duplex PCR (RT-d-PCR) for simultaneous detection of the Citrus Tristeza Virus and Indian Citrus Ringspot virus

Citrus tristeza virus (CTV) is an aphid transmitted Closterovirus having an RNA genome of ~ 19.3 kb has destroyed millions of productive citrus fruit trees worldwide. CTV, singly or as mixed infection with Indian citrus ringspot virus (ICRSV), having ~7.5 kb RNA genome under the genus Mandarivirus, plays a significant role in causing citrus decline particularly in Kinnow mandarin in India. Since citrus is predominantly a vegetatively propagated crop, development of rapid, reliable and sensitive detection techniques are important to prevent the spread of these two viruses by infected planting material in the nursery. Sero-diagnosis of these two pathogens are not preferred in many laboratories due to difficulties in virus purification, production of polyclonal antibodies, uneven distribution of the virus in infected citrus trees, low titer of the virus in plant tissues, contamination of host proteins in purified preparation and necessity to produce separate antibodies against individual target viruses etc. As an alternative, different PCR-based diagnostic tools have become popular worldwide. In the present study, a rapid and reliable duplex RT-PCR protocol was standardized for the simultaneous detection of both CTV and ICRSV in infected citrus trees. A set of primers designed based on respective pathogen isolate sequence data available in GenBank were synthesized and used in the present experiment to obtain anticipated products of calculated size. Using RT-d-PCR, two different fragments of ~ 627 bp specific to RNA binding protein of CTV (p23) and ~ 309 bp specific to partial coat protein (CP) gene of ICRSV were simultaneously amplified. The consistent result of duplex RT-PCR was compared with simplex RT-PCR for detection of individual pathogen. The duplex RT-PCR method developed in the present investigation has proven to be a highly sensitive, cost-effective and reliable method for the simultaneous detection of CTV and ICRSV in infected citrus plants. Therefore, the rapid diagnostic technology developed can be used for disease survey and eradication of pathogen infection from the existing citrus orchards, varietal improvement programme as well as in implementing citrus budwood certification programme.