Simultaneous screening for HBV DNA and HCV RNA genomes in blood donations using a novel TaqMan PCR assay.
The risk of contracting hepatitis from blood transfusions is estimated to be 1 in 63000 in the case of hepatitis B virus (HBV) and 1 in 103000 in the case of hepatitis C virus (HCV). In some countries (Germany, USA and England, for example), molecular protocols are evaluated to detect viral genomes in blood donations in order to reduce the seroconversion period. However, no such method is available currently to screen large series samples for HBV and HCV. While strategies involving the pooling of plasma samples have been proposed and tested in Germany, there is the question of sensitivity. We developed a novel approach to screen for HBV and HCV based on the TaqMan technology that allows for the quantification of an amplified fragment during PCR analysis (Lee et al., 1993). This approach is more sensitive than other quantification methods. As a first step primers and probes were designed to detect the different sub-types of HBV and HCV genomes. We then optimized the reaction conditions in order to screen for the two viruses at the same time. The observed sensitivity is less than 50 molecules per ml for HBV and less than 50 molecules per ml for HCV. This assay is, to our knowledge, the first that allows the simultaneous detection of DNA and RNA viral genomes. In conclusion, this TaqMan approach could be used as a single test to screen for HBV and HCV genomes in a series of 96 samples in less than 5 h. Such an approach is a first step for development of automation allowing a systematic screening of blood donations.
Etablissement de Transfusion Sanguine de Bretagne Occidentale, Brest, France. Bernard.mercier@univ-brest.fr
B
B
Mercier
L
L
Burlot
C
C
Férec
D001782:Blood Donors; D015342:DNA Probes / Q000235:genetics; D004279:DNA, Viral / Q000097:blood*; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008403:Mass Screening; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004279:DNA, Viral; D012367:RNA, Viral