Evaluation of four viroid RNA extraction methods for the molecular diagnosis of CEVd in Citrus lemon using RT-PCR, Dot blot and Northern blot

An effi cient method for RNA extraction that leads to RNA high yield and purity is a technical issue relevant for development and optimization of molecular diagnostic methods aimed to detect viroid infections in citrus varieties. Residual contaminants may affect RNA detection depending on the molecular diagnosis approaches. This condition can be evaluated through RNA absorption spectrum analysis. Functionally, it is assessed through observation of RTPCR amplifi cation products and Northern blot and Dot-blot signal intensities, displaying levels of analytical response/ sensitivity. Four RNA extraction methods were evaluated to determine their effects on the capacity to detect viroid CEVd presence/absence in Citrus limon through four molecular diagnostic approaches: 1) conventional viroid extraction (CVE); 2) phenol/guanidine thiocyanate (PGT), 3) SDS/potassium acetate (SPA); and 4) formaldehyde/ SSC (FS). Phloem tissue quantifi cations showed values between 7500 ng/μL and 1200 ng/μL and ranged 1.3-2.0 OD260/280. Evaluations through RT-PCR showed the expected amplifi cations of the entire CEVd genome, but erratic scenarios still remained. Non-radioactive probe hybridization techniques revealed high intensity signals (132 RU) for infected tissue, by using the CVE method, and a positivity cut-off for the presence of infection was established (78 RU). Nevertheless, molecular hybridization tools can jeopardize the diagnosis due to the thoroughness of the protocol and the RNA template conditions. The diagnostic ability of the association of Northern blot with CVE viroid extraction analyses as starting point was evidenced for successful detection, among the molecular methods tested.