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

Abstract

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 MgCl2 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 mmol/L MgCl2. 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 virus-infected 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatitis C virus F protein: A double-edged sword in the potential contribution of chronic inflammation to carcinogenesis

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Immortalized intrahepatic mouse biliary epithelial cells: Immunologic characterization and immunogenicity

“…37 The oligonucleotide primers were: sense: 5Ј-GACAACATGGAACA-CATACCTACG-3Ј and antisense: 3Ј-GCAAGCAAAGTGTCAGCCACTC-5Ј. Total RNA was isolated from the IBEC lines using TRI ZOL reagent (GIBCO BRL).…”

Direct detection of hepatitis C virus (HCV) RNA from whole blood, and comparison with HCV RNA in plasma and peripheral blood mononuclear cells

“…Reaction Assays For most assays, the one-step HCV RNA, RT-PCR method of Hu et al [1993] was performed, with modifications to allow nesting of primers. After denaturation of RNA, various volumes of the RNA (.02-5.0 pl) were added to a 50 pl reaction mixture containing 10 mM Tris-HCL (pH 8.31, 50 mM KCL, 2 mM MgCl,, 1.0 micromole of each outside sense and antisense primer, 0.01% BSA, and 250 pM each of dNTP, 20 U RNasin, and 25 U MMLV reverse transcriptase.…”