Published online by Cambridge University Press: 27 August 2009
There are several fundamentally different methods that have been developed to detect HCV RNA in blood. These include RT/PCR, branched chain signal amplification (bDNA), nucleic acid sequence-based amplification, ligase chain reaction, transcription-mediated amplification, and various postamplification hybrid capture systems (Hodinka, 1998; Allain, 2000). Northern blot analysis, in situ hybridization and RT/PCR have also been used to detect HCV RNA in tissue samples (Negro, 1998). However, RT/PCR has evolved as the most common method for viral RNA detection and quantification in clinical samples (Ch. 18). Briefly, total RNA is extracted and purified from clinical samples. The extraction procedure may involve pronase digestion in the presence of chaotrophic agents (e.g., guanidinium chloride) to release the RNA from the virion and with phenol extraction and ethanol precipitation to separate nucleic acids from protein. Alternatively, the clinical samples could be “lysed” by addition of detergent, and the released RNA purified and recovered from “mini-spin” columns, which bind nucleic acid and permit the removal of denatured proteins and potential inhibitors of PCR prior to RNA recovery. The HCV RNA in the resulting material is converted to DNA in a two-step procedure. Since HCV RNA is plus stranded, it is converted to minus strand DNA by addition of a complementary primer and by RT, which synthesizes the remaining minus strand. After minus strand synthesis, some protocols call for extraction and purification of the minus strand DNA followed by addition of plus and minus strand primers together with Taq polymerase and then finally by standard PCR.
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