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US 8,076,081 B2Granted
Systems, methods, and compositions for detection of human papilloma virus in biological samples
AssigneeThe Regents of the University of Michigan
InventorsDavid M. Kurnit, Michael D. Kane
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Abstract
The present invention comprises, without limitation, systems, methods, and compositions for the detection, identification, and quantification, down to the single copy level, of human papillomavirus (HPV) in biological samples, including but not limited to, mammalian body fluids and cervix scrapings, for purposes of detection, treatment and/or management of cancer and dysplasia.
Claims11
A method for detecting, identifying, and/or quantifying HPV DNA in a mammalian biological sample, comprising the steps of:
- extracting DNA from a mammalian biological sample;
- conducting a first amplification by PCR of at least a portion of the extracted DNA in the presence of at least one competitor sequence, said competitor sequence comprising a polynucleotide substantially homologous to a polynucleotide in a DNA sequence of a known HPV type, said competitor sequence having a nucleotide substitution not present in said HPV DNA sequence;
- conducting a second amplification by PCR in the presence of at least one extension primer for said known HPV type and at least two different dideoxynucleotides;
- and
- determining the level of any amplified extension primer for said known HPV type by mass spectrometry;
- wherein the first amplification comprises at least one matched set of forward and reverse primer sequences for a known HPV type substantially matching at least one competitor sequence and wherein at least one extension primer relates to the same known HPV type;
- wherein the first amplification comprises the extracted DNA in the presence of at least one competitor sequence comprising a polynucleotide substantially homologous to a polynucleotide in a DNA sequence of gene erbB-2, said competitor sequence having a nucleotide substitution not present in said gene erbB-2.
4 more elements
A method for quantifying HPV DNA in a urine sample, comprising the steps of:
- extracting DNA from said urine sample;
- conducting a first amplification by PCR of at least a portion of the extracted DNA in the presence of at least one competitor sequence, said competitor sequence comprising a polynucleotide substantially homologous to a polynucleotide in a DNA sequence of a known HPV type, said competitor sequence having a nucleotide substitution not present in said HPV DNA sequence;
- conducting an extension reaction by PCR in the presence of at least one extension primer for said known HPV type and at least two different dideoxynucleotides;
- and
- determining the level of any amplified extension primer for said known HPV type by mass spectrometry.
2 more elements
Dependencies are inferred from the claim text.