ONE-STEP RNA-POLYMERASE CHAIN-REACTION FOR DETECTION OF HEPATITIS-C VIRUS-RNA

Although detection of hepatitis C virus RNA with polymerase chain reac tion has become the standard for diagnosis, extensive application has been thwarted by polymerase chain reaction's labor intensiveness, risk of false-positive results through contamination and time required for individual assays. To minimize these limitations, we developed and va lidated a one-step hepatitis C virus RNA polymerase chain reaction ass ay. The one-step method was compared with traditional hepatitis C viru s RNA polymerase chain reaction using primers from the highly conserve d 5' untranslated region of the hepatitis C virus genome. Variables st udied in the one-step method included the source and quantity of rever se transcriptase (RTase), the concentration of MgCl2 and the duration of reverse transcription and complementary DNA amplification cycles. O ptimal conditions for the one-step method were obtained with 25 U of r everse transcriptase and 2 mmol/L MgCl2. The one-step method substanti ally reduced the time required for analysis. The sensitivity of the on e-step method was comparable to that of traditional hepatitis C virus RNA polymerase chain reaction using serially diluted RNA extracted fro m the serum of a hepatitis C virus-infected patient. The specificity o f the one-step method was confirmed on Southern-blot hybridization. Th e results exhibited 100% concordance with results of traditional hepat itis C virus RNA polymerase chain reaction in 50 serum samples, includ ing those of positive and negative controls. In addition, 100% concord ance was observed between the two methods' results when sera containin g low levels of hepatitis C virus RNA were used. In serum samples cont aining positive- and negative-stranded hepatitis C virus RNA, the one- step method produced stronger polymerase chain reaction product signal s than did traditional polymerase chain reaction performed with only a n antisense primer for reverse transcription. These results indicated that both strands were reverse-transcribed in the one-step technique. The one-step method is a sensitive and specific alternative to traditi onal hepatitis C virus RNA polymerase chain reaction. Substantial redu ctions in labor intensiveness, risks of contamination and time require d for analysis make it suitable for testing multiple clinical samples.