Competitive polymerase chain reaction using an internal standard: application to the quantitation of viral DNA.
A general strategy for the construction of an internal standard for the polymerase chain reaction (PCR) is described together with its application in the evaluation of clinical samples. This internal standard is a plasmid containing a modified target sequence that is co-amplified with the native target using the same set of primers. The co-amplification reaction will generate two fragments of different size that are readily separated without the need for restriction enzyme digestion. Thereafter, they are detected and quantitated by hybridization to the same probe. Detection of HIV proviral DNA was chosen as a model for this competitive PCR. The assay proved to be a sensitive tool for the detection of PCR inhibitors and allowed quantitation of HIV with a 20-30% variation coefficient. Despite limitations that appear inherent to the amplification process, internal standards appear to be useful tools for quantitative analysis by PCR.
Telenti
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Institute of Medical Microbiology, University of Berne, Switzerland.
Imboden
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Germann
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D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000032:analysis*; D015658:HIV Infections / Q000175:diagnosis; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*
D004279:DNA, Viral