title: Two-round rapid-cycle RT-PCR in single closed capillaries increases the sensitivity of HCV RNA detection and avoids amplicon carry-over.
abstract: BACKGROUND
  For the detection of hepatitis C virus (HCV) specific nucleic acids the polymerase chain reaction (PCR) is widely used. Rapid-cycle PCR is performed in glass capillaries with the LightCycler instrument and allows PCR including product analysis to be performed within a closed system in about 1 h. Thus, rapid-cycle PCR appears especially suitable for routine diagnostic applications. However, the volume of the PCR vessel is restricted to about 20 microl, which may limit the sensitivity of the PCR. To increase its sensitivity two-round or nested primer PCR protocols have been developed. In rapid-cycle PCR first-round PCR products are usually collected from the capillaries by centrifugation, a procedure prone to cross-contamination.
  
  
  OBJECTIVE
  Development of a two-round rapid-cycle reverse transcription-polymerase chain reaction (RT-PCR) in single closed LightCycler capillaries for the sensitive detection of HCV RNA in serum or plasma.
  
  
  METHODS
  A set of two pairs of nested primers was selected. The first-round RT-PCR reaction mixture was separated from the second-round PCR mixture by silicone oil. Reverse transcription followed by the first-round PCR was performed. Then, the second-round mixture was combined with first-round products by a centrifugation step followed by second round PCR during which fluorescence intensities were recorded and used for quantification.
  
  
  RESULTS
  To establish the sensitivity of this novel assay a serial dilution of HCV reference standard was used. In plasma samples about 100 IU/ml HCV were consistently detected using the high pure viral RNA kit for nucleic acid purification. This detection limit was found to be about 20 fold increased compared with single-round RT-PCR and corresponded to 3.4 IU of HCV per capillary. Using a panel of HCV genotype standards the novel assay exhibited similar sensitivity for all HCV genotypes. The applicability for clinical routine testing was demonstrated by examining 156 clinical samples.
  
  
  CONCLUSIONS
  Two-round RT-PCR with the LightCycler instrument using a single closed capillary throughout the procedure was found ideally suited for rapid (100 min), accurate and sensitive molecular diagnosis of active HCV infections. Since the capillaries remained closed during the procedure carry-over contamination was precluded.
authors.affiliation: Institute of Clinical Pathology, Robert Bosch Hospital, Auerbachstrasse 110, D-70376 Stuttgart, Germany. dieter.ratge@rbk.de
authors.forename: D
authors.initials: D
authors.lastname: Ratge
authors.forename: B
authors.initials: B
authors.lastname: Scheiblhuber
authors.forename: O
authors.initials: O
authors.lastname: Landt
authors.forename: J
authors.initials: J
authors.lastname: Berg
authors.forename: C
authors.initials: C
authors.lastname: Knabbe
mesh_terms: D001483:Base Sequence; D004279:DNA, Viral; D016174:Hepacivirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D006526:Hepatitis C / Q000097:blood / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000097:blood*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic