title: Monitoring of inhibitors of enzymatic amplification in polymerase chain reaction and evaluation of efficacy of RNA extraction for the detection of hepatitis C virus using the internal control.
abstract: Inhibitors of enzymatic amplification in serum may cause false-negative results for direct detection of hepatitis C virus (HCV) by polymerase chain reaction (PCR). This study was undertaken to demonstrate the importance of the internal control in a PCR assay for detection of HCV-RNA to monitor false-negatives due to inhibitors. HCV-RNA was extracted using RNA extraction kit (SepaGene RV-R, Sanko Junyaku) and a prototype instrument for automated specific capture of HCV-RNA with probes and magnetic bead/fluid separation (Roche Molecular Systems). The extracted HCV-RNA and internal control were detected by an automated PCR machine (Cobas Amplicor, Roche Diagnostic Systems). Addition of hemoglobin (up to 4.5 g/l) to the sera followed by RNA extraction with SepaGene RV-R had no inhibitory effect on the detection of either HCV-RNA or the internal control. In contrast, addition of heparin to the sera showed an inhibitory effect with a dose-dependent manner on the detection of both HCV-RNA and the internal control, with a greater effect at lower copy number of HCV. When HCV-RNA was extracted by the automated system, the inhibitory effect of heparin was successfully eliminated. In the assays of 65 serum samples positive for anti-HCV antibodies, positivity for the internal control indicated efficient amplification and validated 14 negative and two equivocal results for detection of HCV-RNA. Detection of the internal control was negatively correlated with viral copy number in sera suggesting competitive inhibition of high viral copy number on amplification of the internal control. Extraction, co-amplification and detection of the internal control appears useful for estimating effects of inhibitors on amplification in each assay for the detection of HCV-RNA, and for evaluating efficacy of RNA extraction methods.
authors.affiliation: Department of Clinical Pathology, Tokai University School of Medicine, Isehara, Kanagawa, Japan. miyachi@is.icc.u-tokai.ac.jp
authors.forename: H
authors.initials: H
authors.lastname: Miyachi
authors.forename: A
authors.initials: A
authors.lastname: Masukawa
authors.forename: T
authors.initials: T
authors.lastname: Ohshima
authors.forename: H
authors.initials: H
authors.lastname: Fusegawa
authors.forename: T
authors.initials: T
authors.lastname: Hirose
authors.forename: C
authors.initials: C
authors.lastname: Impraim
authors.forename: Y
authors.initials: Y
authors.lastname: Ando
mesh_terms: D004791:Enzyme Inhibitors / Q000737:chemistry; D005188:False Negative Reactions; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D018937:Hepatitis C Antibodies / Q000097:blood; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004791:Enzyme Inhibitors; D018937:Hepatitis C Antibodies; D012367:RNA, Viral