A highly sensitive heminested RT-PCR assay for the detection of citrus psorosis virus targeted to a conserved region of the genome

Psorosis is a widespread and damaging disease of citrus in many parts of th e world. The causal agent is a multipartite virus with RNA genome present i n very low concentration in infected citrus tissue. Diagnosis is made by bi ological indexing on indicator citrus seedlings, but it is a slow and costl y procedure and therefore it is not used generally. No sensitive wide-spect rum assay for Citrus Psorosis virus (CPsV) has been reported based on RT-PC R. A highly sensitive heminested RT-PCR assay is described for the detectio n of CPsV. Fragments of 313 bp amplified from RNA 1 of different isolates w ere cloned and sequenced. Very high homology was found among six isolates f rom the citrus producing region of Argentina: 96.6-100% in nucleotide seque nce. The consensus sequence obtained was used for the design of the primers for heminested PCR assay. It has been tested on different Argentine isolat es, employing various methods for RNA extraction from infected tissue. This test is able to detect CPsV in dilutions of 10(10) of the original sample. (C) 2000 Elsevier Science B.V. All rights reserved.