Multimeric non-radioactive cRNA probes improve detection of potato spindle tuber viroid (PSTVd)

Abstract

Experimental data showed that multimeric, complementary RNA (cRNA) probes, labelled with non-radioactive digoxigenin (DIG), improved sensitivity of detection of the potato spindle tuber viroid (PSTVd) RNA by 2- to 30-fold as compared with corresponding multimeric cDNA probes. The degree of PSTVd detectability improvement depended upon the type of alkaline phosphatase substrate (colorimetric vs. chemiluminescent) used. Use of hexameric DIG-labelled cRNA probes in combination with chemiluminescent (Lumi-Phos 530) substrate resulted in detection of 0.48 pg of PSTVd RNA. The size of the synthesized cRNA probes corresponded to the size of the respective PSTVd cDNA templates. Interestingly, there was no relationship between the size of the synthesized, DIG-labelled DNA probes and that of the PSTVd cDNA template. This type of anomaly was not observed with other plant viral cDNA templates. Monomeric or multimeric cDNA probes detected both a mild and a severe PSTVd strain in viroid-infected potato leaf extracts diluted 1024 to 2048 times. In comparison, cRNA probes exhibited a much greater dilution end point; PSTVd RNA was detectable in viroid-infected potato leaf tissue diluted up to 16 384 times. Comparable levels of PSTVd sensitivity of detection were obtained with viroid-infected potato tuber tissue.