Development of a Multiplex RT-PCR Assay for Detection of the Causal Agents of Citrus Tristeza and Cachexia Diseases with Coamplification of Plant mRNA as an Internal Control

Plant certification programs need reliable, fast, cheap and sensitive methods for detection of systemic pathogens with special interest in virus and viroid detection. Reverse transcriptase-polymerase chain reaction (RT-PCR) has been documented as an alternative assay for certification of plant propagating materials. The main object of the present study was the optimization of a multiplex RT-PCR assay for simultaneous detection of Citrus tristeza virus (CTV) and Hop stunt viroid (HSVd), the casual agents of citrus tristeza and cachexia, together with the plant mRNA as an internal control for citrus certification in the country. Total RNA was extracted from healthy; CTV- and HSVd- infected citrus tissues and subjected to cDNA synthesis by M-MuLV H- reverse transcriptase, followed by optimization of simplex, diplex and multiplex RT-PCR (s-, d-, and mRT-PCR). Amplified fragments were further sequenced for evaluation of the accuracy of the assays. CTV, HSVd and the plant internal control (Nad 5 gene) were successfully amplified in all assays and the sequence information revealed the accuracy of all assays in citrus certification programs. Results from the developed s-, d-, and mRT-PCR assays revealed these detection methods as excellent candidates for citrus certification.