Simplified reverse transcription polymerase chain reaction procedure with detection by microplate hybridization for routine screening of hepatitis A virus

Abstract

Reverse transcription polymerase chain reaction, using either nested or seminested primers, is used extensively for the detection of viruses in small quantities. However, existing methods are prone to false positive reactions. We report here an improved polymerase chain reaction technique based on the use of longer primers (39 nucleotides) with single-step amplification, applied to the detection of hepatitis A in low quantities. While the sensitivity of this technique (10 x the 50% tissue culture infective dose) is equivalent to that of existing methods, it is a simpler procedure, less time consuming, and less susceptible to contamination and therefore provides a more reliable tool for routine diagnosis. Finally, the development of a DNA enzyme immunoassay detection technique and the complete automation of the procedure allow a large number of samples to be processed in clinical laboratories.Key words: long primers, hepatitis A virus, reverse transcription polymerase chain reaction, seminested PCR, DNA enzyme immunoassay.