One-step RNA polymerase chain reaction for detection of hepatitis C virus RNA

✍ Scribed by Ke-Qin Hu; Chang-Hong Yu; John M. Vierling

• Publisher: John Wiley and Sons
• Year: 1993
• Tongue: English
• Weight: 583 KB
• Volume: 18
• Category: Article
• ISSN: 0270-9139
• DOI: 10.1002/hep.1840180208

✦ Synopsis

Although detection of hepatitis C virus RNA with polymerase chain reaction 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 validated a one-step hepatitis C virus RNA polymerase chain reaction assay. The one-step method was compared with traditional hepatitis C virus RNA polymerase chain reaction using primers from the highly conserved 5' untranslated region of the hepatitis C virus genome. Variables studied in the one-step method included the source and quantity of reverse transcriptase (RTase), the concentration of MgCl, and the duration of reverse transcription and complementary DNA amplification cycles. Optimal conditions for the one-step method were obtained with 25 U of reverse transcriptase and 2 mmo1L MgCl,. The one-step method substantially reduced the time required for analysis. The sensitivity of the one-step method was comparable to that of traditional hepatitis C virus RNA polymerase chain reaction using serially diluted RNA extracted from the serum of a hepatitis C virusinfected patient. The specificity of the one-step method was confirmed on Southern-blot hybridization. The results exhibited 100% concordance with results of traditional hepatitis C virus RNA polymerase chain reaction in 50 serum samples, including those of positive and negative controls. In addition, 100% concordance was observed between the two methods' results when sera containing low levels of hepatitis C virus RNA were used. In serum samples containing positive-and negative-stranded hepatitis C virus RNA, the one-step method produced stronger polymerase chain reaction product signals than did traditional polymerase chain reaction performed with only an antisense primer for reverse transcription. These re-