Suppression of the inhibitory effect of denatured albumin on the polymerase chain reaction by sodium octanoate: application to routine clinical detection of hepatitis B virus at its infectivity threshold in serum.
Ten to fifteen percent of posttransfusion viral hepatitis cases are still caused by HBV despite mandatory third generation screening procedures for HBsAg. There is thus an urgent need for a simple, time-cost-effective, but very sensitive test for routine HBV DNA detection in serum. Nested-primed PCR has been shown to detect purified HBV DNA at its infectivity threshold in serum. Since this is too labor-intensive for routine testing, we assessed the efficiency of a Fast PCR procedure, of three pairs of primers, and of thirty-five simple serum pretreatments with the aim to achieve the same sensitivity level. Using ten-fold dilution in phosphate buffered saline as pretreatment and Fast PCR for 99 cycles, we were able to detect HBV DNA at the 2 x 10(3)/ml level in serum. Using either NaOH denaturation or sodium octanoate thermoprotection as pretreatment and Fast PCR for 99 cycles, we were able to detect HBV DNA at its infectivity threshold in serum, while the classical phenol/chloroform/isoamylic alcohol/isopropanol/ethanol DNA purification procedure enabled us to reach the 10 virus particles/ml level. These results suggest that denatured albumin is responsible for the well known inhibitory effect of serum proteins on Taq polymerase. Because of its simplicity and its lower risk of sample-to-sample cross-contamination, the sodium octanoate thermoprotection method was chosen for routine clinical detection of HBV in serum. The clinical usefulness of this approach is demonstrated by the results obtained with HBsAg-negative acute hepatitis B incubation sera and with anti HBe-positive chronic hepatitis B sera.
Virology Department, Brugmann University Hospital, Brussels, Belgium.
C
C
Vandenvelde
R
R
Scheen
M
M
Defoor
M
M
Duys
J
J
Dumon
D
D
Van Beers
D001798:Blood Proteins / Q000494:pharmacology; D002210:Caprylates / Q000494:pharmacology; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000187:drug effects; D006509:Hepatitis B / Q000097:blood*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011489:Protein Denaturation / Q000187:drug effects; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000494:pharmacology; D019914:Taq Polymerase; D013997:Time Factors
D001798:Blood Proteins; D002210:Caprylates; D004279:DNA, Viral; D012709:Serum Albumin; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; C031492:octanoic acid