Application of Bi-Directional PCR to Citrus Tristeza Virus: Detection and Strain Differentiation

B Cevik, S Pappu, H Pappu, D Benscher, M Irey, R Lee, C Niblett
The monoclonal antibody MCA13 reacts predominantly with severe strains of citrus tristeza virus (CTV) occurring in many regions of the world. Its specificity is based on a single nucleotide (AIT) difference a t position 371 of the capsid protein gene (CPG). Based on this single nucleotide difference, we designed two internal primers, one specific for mild strains and the other specific for severe strains of CTV. These primers, along with two terminal primers for the ends of the CPG, were used
more » ... he CPG, were used to develop a bi-directional, reverse transcription/polymerase chain reaction (BDPCR) to differentiate mild and severe strains of CTV. Under our standard PCR conditions, a full length CPG (-700 bp) and a 400 bp DNA fragment were produced with mild strains, and a 700 and a 300 bp DNA fragment were produced with severe strains. With a mixture of mild and severe strains, the 700,400 and 300 bp DNA fragments were all produced. Reducing both the terminal primer concentrations and extension times eliminated the 700 bp fragment. Thus, this method can be manipulated for different purposes. Tests of BDPCR with samples from naturally-infected trees indicated that BDPCR were more sensitive than ELISA, because BDPCR amplified a 300 bp fragment from some samples which were MCA13 negative by ELISA tests.