PMID- 10405896
OWN - NLM
STAT- MEDLINE
DCOM- 19990902
LR  - 20191103
IS  - 1386-6532 (Print)
IS  - 1386-6532 (Linking)
VI  - 13
IP  - 1-2
DP  - 1999 Jun
TI  - High-throughput extraction, amplification, and detection (HEAD) of HCV-RNA in 
      individual blood donations.
PG  - 95-103
AB  - BACKGROUND: High-throughput nucleic acid amplification techniques (NATs) are 
      required for the detection of viral genomes in individual blood donations and 
      might be helpful in any virological laboratory. OBJECTIVE: To develop and 
      automate a method for the detection of hepatitis C virus RNA in individual blood 
      donations, compatible with the time schedule of routine blood bank screening an 
      product release. STUDY DESIGN: The viral RNA was isolated with the use of target 
      specific capture oligonucleotides and magnetic beads. This extraction method was 
      combined with reverse transcription/amplification (RT/PCR) and fluorescence 
      detection. We adapted our method on a pipetting robot and pipetted all steps in a 
      single room. When the pipetting was completed, microtiter plates were heat-sealed 
      with foils and placed into a thermocycler. Positive reactions were detected with 
      a fluorescent dye in a second room. Aerosols were avoided with programmed slow 
      pipetting steps and with a special device constructed for the removal of the used 
      disposable tips. During a 7 month period, we used this method in routine testing 
      of individual donations prior to the release of all blood components. RESULTS: 
      The total number of 11,700 individual donations including platelet concentrates 
      were analysed. We tested up to 192 specimens in one run within 7 h. The frequency 
      of cross-contamination using the automated procedure was 0.1%. Five specimens 
      have been found repeatedly reactive for HCV-RNA, four of these were anti-HCV 
      positive, one sample from a repeat donor was negative in anti-HCV assays. A 
      seroconversion was detectable at his next presentation, 6 months later. 
      CONCLUSION: In this pilot study, we demonstrate that automated HCV-RT-PCR testing 
      is practicable for individual donations in high-throughput. Additionally, the 
      described PCR approach could easily be adapted to the detection of other viral 
      genomes by the use of specific primers.
FAU - Legler, T J
AU  - Legler TJ
AD  - Department of Transfusion Medicine, University of Gottingen, Germany.
FAU - Kohler, M
AU  - Kohler M
FAU - Heermann, K H
AU  - Heermann KH
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Netherlands
TA  - J Clin Virol
JT  - Journal of clinical virology : the official publication of the Pan American 
      Society for Clinical Virology
JID - 9815671
RN  - 0 (RNA, Viral)
SB  - IM
MH  - Automation
MH  - *Blood Donors
MH  - Gene Amplification
MH  - Hepacivirus/genetics/*isolation & purification
MH  - Humans
MH  - Pilot Projects
MH  - RNA, Viral/*analysis
MH  - Reverse Transcriptase Polymerase Chain Reaction/*methods
MH  - Sensitivity and Specificity
MH  - Time Factors
EDAT- 1999/07/16 00:00
MHDA- 1999/07/16 00:01
CRDT- 1999/07/16 00:00
PHST- 1999/07/16 00:00 [pubmed]
PHST- 1999/07/16 00:01 [medline]
PHST- 1999/07/16 00:00 [entrez]
AID - S1386-6532(99)00003-7 [pii]
AID - 10.1016/s1386-6532(99)00003-7 [doi]
PST - ppublish
SO  - J Clin Virol. 1999 Jun;13(1-2):95-103. doi: 10.1016/s1386-6532(99)00003-7.