Determination of HCV RNA concentration by direct quantitation of the products from a single RT-PCR

Hepatitis C virus (HCV) infection is one of the world's major health problems, and the identification of efficient HCV inhibitors is a major goal. Here we report the isolation of efficient anti-HCV internal ribosome entry site (IRES) RNA molecules identified by a new in vitro selection method. The newly developed procedure consists of two sequential steps that use distinct criteria for selection: selection for binding and selection for cleaving. The selection protocol was applied to a population of more than 10(15) variants of an anti-hepatitis C virus ribozyme covalently linked to an aptamer motif. The ribozyme was directed against positions 357 to 369 of the HCV IRES, and the cleavage substrate was a 691-nucleotide-long RNA fragment that comprises the entire HCV IRES domain. After six selection cycles, seven groups of RNA variants were identified. A representative of each group was tested for its capacity to inhibit IRES activity using in vitro translation assays. All selected RNAs promoted significant inhibition, some by as much as 95%.

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Manganese-independent Reverse Transcriptase Activity of Tth DNA Polymerase with Two Amino Acid Substitutions

Luo

Xue

Chen

et al. 2023

PPL

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Background: The DNA polymerase of Thermus thermophilus (Tth pol) presents reverse transcriptase activity with Mn2+, and can be used for one-step RT-qPCR. However, Mn2+ would reduce amplification fidelity and cause nonspecific products. Objective: Eliminating the Mn2+ dependence of the reverse transcriptase activity of Tth pol by point mutations. Methods: We constructed three variants I640F, I709K, and I640F/I709K, and measured their DNA polymerase and reverse transcriptase activities without Mn2+. Their enzymatic characteristics and PCR inhibitor resistance were also tested. Finally, these variants were applied in one-step RT-qPCR. Results: All three variants presented reverse transcriptase activity with Mg2+ only and increased DNA polymerase activity. The variants, except I709K, showed no significant difference in thermostability, optimal pH, optimal NaCl concentration, storage stability and PCR inhibitor resistance compared to the wild type. Variant I640F/I709K had good performance in one-step RT-qPCR with Mg2+ only, whereas both variants with single substitution exhibited nonspecific amplification. Conclusion: We successfully constructed three Tth pol variants possessing Mn2+ independent reverse transcriptase activity. The variant I640F/I709K was suitable for one-step RT-qPCR because of its good performance.

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Determination of HCV RNA concentration by direct quantitation of the products from a single RT-PCR

Cited by 3 publications

References 36 publications

Microchip and capillary electrophoresis for quantitative analysis of hepatitis C virus based on RT-competitive PCR

Microchip and capillary electrophoresis for quantitative analysis of hepatitis C virus based on RT-competitive PCR

Interfering with hepatitis C virus IRES activity using RNA molecules identified by a novel in vitro selection method

Manganese-independent Reverse Transcriptase Activity of Tth DNA Polymerase with Two Amino Acid Substitutions

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