Macroarray Detection of Plant RNA Viruses Using Randomly Primed and Amplified Complementary DNAs from Infected Plants

Abstract

ABSTRACT Membrane-based macroarrays provide a relatively inexpensive technology with the potential to detect hundreds of pathogens in a single assay. For the simultaneous detection of a large number of pathogens, it is necessary to obtain sufficient nucleic acids for labeling, and any amplification reactions need to be performed using unbiased, pathogen-non-specific primers. A nonradioactive macroarray system is described to test for plant RNA viruses using 70-mer oligonucleotide probes immobilized on nylon membranes. Starting with a total plant RNA extract, complementary DNA (cDNA) and second-strand syntheses were carried out using an anchor primer sequence with random pentamers coupled at the 3' end. Subsequent synthesis by polymerase chain reaction using the anchor primer alone resulted in a relatively unbiased amplification of plant and viral RNAs. These cDNAs were chemically labeled and the product used as a target in hybridization analyses. The system was validated using RNA extracts from plants infected with Cucumber mosaic virus, Potato virus Y, and Potato leaf roll virus (PLRV). Despite the relative excess of host-derived nonviral sequences, viral RNAs were amplified between 100- and 1,000-fold and were detected in single and mixed infections. The macroarray sensitivity was comparable to that of double-antibody sandwich enzyme-linked immunosorbent assay, with PLRV being detected in sap dilutions of 1:100. The potential for the development of a relatively inexpensive multipathogen detection system is discussed.