Colourimetric point mutation assay: for detection of precore mutants of hepatitis B.
A colourimetric assay for the analysis of point mutations in PCR amplified DNA fragments from hepatitis B virus (HBV) is described. The method was applied for analysis of the single point mutation in codon 28 of the precore gene of HBV, which inhibits expression of HBe antigen. The assay, which uses a microtitre plate formate, incorporates fluorescein-labelled dideoxynucleotides as opposed to radioactively-labelled deoxynucleotides used in methods described previously. Synthetic control wild type and mutant oligonucleotides were tested to optimise the reaction conditions. The assay was thus shown to yield both qualitative and quantitative data on the relative proportions of wild type and mutant sequences within a given sample. Amplicons from clinical specimens of known sequence were analysed to validate the assay. Sixteen chronic carriers of HBV were tested using the codon 28 point mutation assay, and the results were confirmed by direct sequencing. The method described is suitable for applications where point mutations are of interest.
Ballard
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AL
Public Health Laboratory, Birmingham Heartlands and Solihull NHS Trust, Heartlands Hospital, UK.
Boxall
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EH
D003124:Colorimetry / Q000379:methods; D004252:DNA Mutational Analysis; D006509:Hepatitis B / Q000235:genetics / Q000821:virology; D006515:Hepatitis B virus / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity