Assay for screening for potential cervical cancer
Inventors
Millar, Douglas Spencer • Melki, John R. • Miklos, George L. Gabor
Assignees
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Publication Number
US-8343738-B2
Publication Date
2013-01-01
Expiration Date
Abstract
The invention relates to an assay for determining a health state of a subject using a combination of detecting the presence of a virus and detecting the presence of a genomic target or marker indicative of a health state.
Core Innovation
An assay for screening for potential cervical cancer in a human subject is performed by treating a single sample with a bisulfite reagent under conditions that cause unmethylated cytosines in human papilloma virus (HPV) nucleic acid and human genomic nucleic acid to be converted to uracil to form derivative HPV nucleic acid and derivative human genomic nucleic acid. The treated sample is contacted with primers capable of binding to regions of the derivative HPV nucleic acid to allow amplification of a desired HPV-specific nucleic acid molecule from the derivative HPV nucleic acid.
The treated single sample is also contacted with primers capable of binding to a region of the derivative human genomic nucleic acid to allow amplification to yield a target genomic specific nucleic acid amplification product. The region of derivative human genomic nucleic acid is derivatized from a region of the human genome in which the methylation status is known to be associated with the potential for cervical cancer in a human subject.
An amplification reaction is carried out on the treated single sample containing derivative HPV nucleic acid, derivative human genomic nucleic acid, and the primers. Potential for cervical cancer in the human subject is determined by assaying for the presence of an amplified HPV-specific nucleic acid product and the target genomic specific nucleic acid amplification product, wherein the presence of both amplified products is indicative of a level of risk for cervical cancer.
Claims Coverage
The partial content includes two independent claims. Both independent claims cover an assay that combines bisulfite conversion of unmethylated cytosines with dual amplification: an HPV-specific amplified product plus an amplified genomic target derived from regions with known methylation associations to cervical cancer risk, using presence of both products as an indication of risk.
Bisulfite conversion of HPV and human genomic nucleic acids in a single sample
Treating a single sample from the human subject with a bisulfite reagent under conditions that cause unmethylated cytosines in HPV nucleic acid and human genomic nucleic acid in the sample to be converted to uracil to form derivative HPV nucleic acid and derivative human genomic nucleic acid.
Amplification of derivative HPV nucleic acid using HPV-specific primers
Contacting the treated sample with primers capable of binding to regions of derivative HPV nucleic acid, the primers being capable of allowing amplification of a desired HPV-specific nucleic acid molecule of the derivative HPV nucleic acid.
Amplification of methylation-status-associated derivative human genomic region
Contacting the treated single sample with primers capable of binding to a region of derivative human genomic nucleic acid, the primers being capable of allowing amplification of the region of derivative human genomic nucleic acid to yield a target genomic specific nucleic acid amplification product, wherein the region of derivative human genomic nucleic acid is derivatized from a region of the human genome in which the methylation status is known to be associated with the potential for cervical cancer in a human subject.
Risk determination based on presence of both amplification products
Carrying out an amplification reaction on the treated single sample containing derivative HPV nucleic acid, the derivative human genomic nucleic acid, and the primers; and determining potential for cervical cancer in the human subject by assaying for the presence of an amplified HPV-specific nucleic acid product and said target genomic specific nucleic acid amplification product wherein the presence of both an amplified HPV-specific nucleic acid and said target genomic specific nucleic acid amplification product are indicative of a level of risk for cervical cancer.
Bisulfite conversion of nucleic acid to derivative nucleic acids
Contacting a single nucleic acid sample from a human subject with a bisulfite reagent under conditions that cause unmethylated cytosines to be converted to uracil, thereby generating derivative nucleic acids in which said unmethylated cytosines have been converted to uracil.
HPV strain-associated amplification using HPV-specific primers
Performing an amplification reaction on said derivative nucleic acids using primers complementary to portions of derivative nucleic acid from an HPV strain in which unmethylated cytosines have been converted to uracil, wherein said HPV strain is an HPV strain which is associated with an increased risk of cervical cancer and wherein the primers yield an HPV-specific amplification product if said HPV strain is present in said nucleic acid sample.
Methylation-associated target human derivative sequence amplification
Performing an amplification reaction on said derivative nucleic acids using primers complementary to portions of a target human derivative sequence in which unmethylated cytosines have been converted to uracil, wherein the target human derivative sequence is derivatized from a region of the human genome in which the methylation status is known to be associated with the potential for cervical cancer in a human subject.
Risk determination based on presence of HPV-specific and human-derivative amplification products
Determining potential for cervical cancer in the human subject by assaying for the presence of an HPV-specific amplification product and an amplification product from said target human derivative sequence wherein the presence of both an HPV-specific amplification product and an amplification product from said target human derivative sequence is indicative of a level of risk for cervical cancer.
Across both independent claims, the inventive coverage is directed to a bisulfite-based assay of a single sample that generates derivative HPV nucleic acid and derivative human genomic nucleic acid, amplifies an HPV-specific product using HPV-associated primers and amplifies a genomic target derived from regions with known methylation status associated with cervical cancer potential, and determines cervical cancer risk by assaying for the presence of both amplified products.
Stated Advantages
Assaying for a level of risk for cervical cancer based on the presence of both an amplified HPV-specific nucleic acid product and a target genomic specific nucleic acid amplification product.
Documented Applications
Screening for potential cervical cancer in a human subject.
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