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AssigneesUniversidade Federal de Uberlândia, Fundacao De Amparo A Pesquisa Do Estado De Minas Gerais-Fapemig
InventorLuiz Goulart
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The present invention relates to the use of nucleic acid amplification technology through two-step (duplicated) polymerase chain reactions with multiple markers, also known as multiplex nested PCR, using general consensus oligonucleotides followed by multiple specific oligonucleotides and the components thereof (reagents and reaction conditions) to simultaneously detect 40 types of human papillomavirus present in fluids and samples of human tissue, and also carry-out a semi-quantitative analysis between viral types, determining the viral dominance relationship (viral load) in multiple infections. The sequences of the general consensus-degenerate primers (specific initiators or oligonucleotides, SEQ ID NOS: 1 to 93) (1st PCR reaction) and combined primers are disclosed, as well as specific combined primers (2nd nested PCR reaction) for 40 viral types, which include non-oncogenic, indeterminate and oncogenic types.
An oligonucleotide comprising one of the sequences of SEQ ID NO:
A method for detecting and typing human papillomavirus, comprising the combined use of the two-step (duplicate) multiplex nested PCR technique with multiple markers using general consensus oligonucleotides (first stage) followed by multiple specific oligonucleotides (second stage) and their components (reagents and reaction conditions) for simultaneously detecting 40 human papillomavirus types present in fluids and human tissue samples, in a single reaction tube for capillary electrophoresis detection.
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