title: Evaluation of a new PCR assay with competitive internal control sequence for blood donor screening.
abstract: BACKGROUND
  High-throughput nucleic acid testing for transfusion-relevant viruses by PCR requires contamination-proof methods with high sensitivity and validity. A new PCR reagent kit (TaqMan, PE BioSystems) reduces the risk of carry-over contamination by eliminating post-PCR processing.
  
  
  METHODS
  Oligonucleotide design was done with software specialized for designing the assays' (TaqMan) primers and probes. A template-derived competitive internal control sequence designed through site-directed mutagenesis was used to reveal failures in amplification. Assay sensitivity was determined for single-donor and single-patient testing and by spiking sample mini-pools. Three seroconversion panels were tested.
  
  
  RESULTS
  Sensitivity is high, reaching 300 HBV genomes per mL of single-patient material on direct testing. A detection limit of 1000 HBV genome equivalents per mL of donor plasma is achieved for 96 pooled samples. The window period for HBV infection was reduced by 17, 10, and 63 days from that for HBsAg screening in three seroconverting donors.
  
  
  CONCLUSIONS
  The assay provides sufficient sensitivity to be superior to HBsAg screening in transfusion medicine and will be useful in clinical laboratories because of its ease of handling.
authors.affiliation: Institute of Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service of Hesse, Frankfurt am Main, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Drosten
authors.forename: M
authors.initials: M
authors.lastname: Weber
authors.forename: E
authors.initials: E
authors.lastname: Seifried
authors.forename: W K
authors.initials: WK
authors.lastname: Roth
mesh_terms: D001782:Blood Donors; D004279:DNA, Viral / Q000097:blood*; D005069:Evaluation Studies as Topic; D016679:Genome, Viral; D006509:Hepatitis B / Q000097:blood / Q000175:diagnosis* / Q000821:virology; D006510:Hepatitis B Antibodies / Q000096:biosynthesis / Q000097:blood; D006515:Hepatitis B virus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D008403:Mass Screening / Q000379:methods*; D016133:Polymerase Chain Reaction; D012015:Reference Standards; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013997:Time Factors; D014766:Viremia / Q000175:diagnosis*
chemical_list: D004279:DNA, Viral; D006510:Hepatitis B Antibodies