The majority of transfusion-associated, non-A, non-B hepatitis cases are caused by hepatitis C virus (HCV), a positive-stranded
RNA virus. Although high titers of HCV in clinical specimens have been reported, in some cases extremely low titers of virus are not uncommon. Therefore, an extremely sensitive and reliable assay is required to determine viremia and replication of HCV accurately. We report here the systematic investigation of factors influencing the detection of HCV
RNA by a reverse transcription-polymerase chain reaction (RT-PCR) assay utilizing "drop in-drop out" heminested primers derived from the conserved 5' non-coding region of the viral
genome. A genetically engineered 5' noncoding region has been constructed and used as an inte
rnal control. Addition of the control
RNA to each test not only allowed semiquantitation of positive reactions but also validated the performance of reverse transcription and PCR for every specimen. The optimized heminested PCR (HN-PCR) protocol is capable of amplifying one molecule of cloned HCV DNA or 10 molecules of in vitro-transcribed HCV
RNA to levels detectable in ethidium bromide-stained agarose gels. We evaluated the improved method for the detection of HCV
RNA on a human plasma sample containing the pedigreed strain H of HCV with a chimpanzee infectious dose of 10(6)/ml. Utilizing the inte
rnal control
RNA, we calculated 2 x 10(7) virions in 1 ml of the original human plasma. The HN-PCR achieves the sensitivity and specificity of the double-nested PCR (DN-PCR) in a simplified format that avoids the false-positive results associated with DN-PCR.
Source - http://genome.cshlp.org/content/2/3/241.abstract?sid=40206306-7c2e-4b18-96b3-8319fa594a4b
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