Detection of HIV1 DNA in biological samples by an homogeneous assay: fluorescence measurement of double-stranded RNA synthesized from amplified DNA.
A nonisotopic homogeneous detection of nucleic acid sequences after amplification is described. We show that a DNA fragment bearing T7 RNA polymerase promoters on each extremity is able to be transcribed in two complementary RNAs, leading to a high yield direct synthesis of double-stranded RNA (dsRNA). Thus, this dsRNA can be easily detected and quantified in solution by fluorescence in the presence of propidium iodide. This reaction, used as a postamplification step, has been associated with a nested polymerase chain reaction (PCR); the second PCR round allowing the incorporation of the T7 promoters. This leads to a very efficient homogeneous assay. The fluorescence signal is proportional to the concentration of PCR product and is highly specific. This method can be easily carried out with currently available reagents and with unsophisticated instrumentation. This homogeneous procedure has been evaluated for the detection of HIV1 in blood samples; the sensitivity and the specificity appear to be equivalent to that of the radioactive method.
Livache
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T
Cis Bio International Laboratoire des Sondes Moléculaires, Gif sur Yvette, France.
Fouque
B
B
Teoule
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R
D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis* / Q000097:blood / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods; D005784:Gene Amplification; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012330:RNA, Double-Stranded / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014764:Viral Proteins
D017931:DNA Primers; D004279:DNA, Viral; D012330:RNA, Double-Stranded; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases