Comparison of hepatitis B virus DNA extractions from serum by the QIAamp blood kit, GeneReleaser, and the phenol-chloroform method.
The abilities of GeneReleaser and QIAamp to extract the hepatitis B virus (HBV) DNA template from serum for amplification by PCR were evaluated and compared with that of the standard phenol-chloroform method. Differences in the sensitivities of the three methods were revealed by nested PCR of HBV DNA extracted from serially diluted hepatitis B e antigen (HBeAg)-positive (high-titer) serum. Phenol-chloroform was found to be the most sensitive extraction method but was time-consuming and labor intensive, and the many steps required increased the possibility of contamination. In a titration of HBeAg-negative (low-titer) serum, all three methods coupled with nested PCR were capable of detecting low levels of HBV DNA. In the case of QIAamp and GeneReleaser, the extraction was relatively simple and rapid. The higher quantity of serum (200 microliters) used in the QIAamp extraction did not provide higher sensitivity, possibly because of incomplete removal of Taq polymerase inhibitors from the serum or inadequate disruption of the virion. GeneReleaser was more efficient because it gave the same detection limit in low-titer serum as phenol-chloroform even though it utilizes only 5 microliters of serum. However, it did not produce consistent amplifications of HBV DNA, giving false-negative results in 7 of the 50 cases (14%) in one experiment. Use of a larger volume of serum and replicate extractions may overcome this problem. Advantages thus exist in each of the extraction methods, and these should be weighed against the disadvantages when deciding which extraction method is appropriate.
Medical Research Council-University Molecular Hepatology Research Unit, Department of Medicine, University of the Witwatersrand, Parktown, Johannesburg, South Africa.
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Kramvis
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Bukofzer
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Kew
D001483:Base Sequence; D002725:Chloroform; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics*; D005069:Evaluation Studies as Topic; D005188:False Negative Reactions; D006509:Hepatitis B / Q000175:diagnosis / Q000821:virology; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D019800:Phenol; D010636:Phenols; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data
D017931:DNA Primers; D004279:DNA, Viral; D010636:Phenols; D019800:Phenol; D002725:Chloroform