Systems, methods, and compositions for detection of human papilloma virus in biological samples
View Patent ↗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.
1. 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.
2. The method of claim 1 , wherein the first amplification comprises at least one matched set of forward and reverse primer sequences relating to said known HPV type, each such sequence comprised of at least one inosine base.
3. The method of claim 1 , wherein at least one extension primer is comprised of at least one inosine base.
4. The method of claim 1 , wherein said determining step comprises detection of any amplified extension primer at a concentration below, or about, 200 attomolar.
5. The method of claim 1 , wherein the first amplification comprises the presence of a plurality of competitor sequence types each comprising a polynucleotide substantially homologous to a polynucleotide in a DNA sequence of a different known HPV type, said competitor sequence type having a nucleotide substitution not present in said respective HPV DNA sequence and wherein the second amplification comprises the presence of a plurality of external primer types each for a different known HPV type.
6. The method of claim 5 , wherein the plurality of known HPV types comprises two or more of HPV types 16, 18, 23, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82.
7. The method of claim 5 , wherein the plurality of known HPV types comprises at least HPV types 16, 18, 23, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82.
8. 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.
9. The method of claim 8 , wherein said known HPV type is selected from the group consisting of one or more of HPV types 16, 18, 23, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82.
10. The method of claim 8 , wherein at least one extension primer is comprised of a spacer molecule affixed to the primer's 5′ end, such spacer selected from the group consisting of phosphorylation, a C3 spacer, a D spacer, an amino modifier C12, a spacer 18, and an amino modifier C6 dT.
11. The method of claim 8 , wherein said quantifying of said HPV DNA is used to identify a subject having or at risk for bladder cancer.