Sensitivity and specificity of nucleic acid probes for potato leafroll luteovirus detection
Robinson, D.J. | Romero, J.
Complementary DNA (cDNA) probes, prepared by nick-translation or by oligolabelling of a 520 bp fragment representing residues 4741 to 5261 in the potato leafroll luteovirus (PLRV) sequence, were equally sensitive, with a detection limit equivalent to sap from about 600 pg of infected potato or Nicotiana clevelandii leaf tissue and to RNA from about 120 micrograms tissue. Increasing the concentration of oligolabelled probe gave similar results with shorter autoradiographic exposures, but also resulted in positive signals with sap extracts from healthy plants. In contrast, a complementary RNA (cRNA) probe made by in vitro transcription of the cDNA insert could be used at higher concentration without giving rise to reactions with healthy plant extracts, and had a detection limit equivalent to 5 micrograms tissue/spot. Five oligolabelled probes representing different regions of the PLRV genome detected PLRV equally well. A probe that represented a portion of the particle protein gene also detected beet western yellows luteovirus (BWYV), with which it has 69% nucleotide sequence homology, and an English strain of the RPV form of barley yellow dwarf luteovirus, and reacted weakly with extracts from plants infected with groundnut rosette assistor luteovirus or carrot red leaf luteovirus. Probes for regions on either side of the particle protein gene also detected RPV, but not any of the other luteoviruses tested, in agreement with earlier suggestions that RPV is more closely related to PLRV than are BWYV or the other luteoviruses tested. An attempt to improve the detection of weak heterologous reactions by using a cRNA probe was unsuccessful, perhaps because tests using cRNA are more affected by mismatching than tests using cDNA probes.
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