A nonradioisotopic reverse phase dipstick hybridization method for detection of polymerase chain reaction amplified product.
A rapid and simple nonradioisotopic method has been developed for detection of polymerase chain reaction (PCR) amplified product. Digoxigenin-11-dUTP (DIG-11-dUTP) was incorporated in the amplified product by including it in the PCR reaction mixture. The PCR product was detected colorimetrically either directly or by reverse phase hybridization method where an unlabelled oligo-nucleotide probe was immobilized on a nitrocellulose dipstick and the digoxigenin labelled PCR product was in the liquid phase. With this system the PCR product could be detected even after 10 cycles of amplification by both direct and hybridization methods. The method was applied on the amplified product of DNA from peripheral blood mononuclear cells from 10 HIV-1 ELISA positive and 8 ELISA negative individuals. PCR was positive in all ELISA positive, Western blot positive individuals from whom HIV-1 was also isolated. PCR was negative in all ELISA negative individuals.
Bawa
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N
Department of Microbiology, All India Institute of Medical Sciences, New Delhi.
Broor
S
S
Seth
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P
D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D015483:HIV Antibodies / Q000032:analysis; D015497:HIV-1 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D004279:DNA, Viral; D015483:HIV Antibodies