Direct RNA polymerase chain reaction for TMV detection in crude cell extracts

Abstract

A part of the 30,000 bp transport protein gene of tobacco mosaic virus (TMV) RNA was amplified via direct RNA PCR and via traditional reverse transcription followed by cDNA PCR. Both amplified cDNA products were restricted with NcoI or HaeIII endonucleases and identical restriction fragments were produced. Two efficient methods of viral RNA concentration from an infected tobacco leaf extract were used; both 3–3.5 M sodium acetate alone and 3 M LiCl with 4 M urea quantitatively precipitated TMV RNA from the extracts. TMV RNA thus obtained could be readily amplified by direct RNA PCR. These results demonstrate that direct RNA PCR can be applied for the detection or/and analysis of high molecular weight RNA and for diagnosis of viral infections.