P53 assay for a urine test for HCC screening
A rapid and sensitive assay to detect p53 mutations in urine has been developed for use in screening cancer patients. The method uses a locked nucleic acid (LNA) clamp mediated one-step PCR-based assay with a sensitivity of up to a single copy and can be used not only in urine, but also other biological samples. The assay is particularly useful for hepatocellular carcinoma, colon cancer, breast cancer, lung cancer, prostate cancer, ovarian cancer, bladder cancer, lymphoma, and stomach cancer.
1. A method for detecting hepatocellular carcinoma in a subject comprising:
amplifying a low molecular weight p53 249T mutation in a urine sample from said subject, wherein the amplification comprises:
suppressing the amplification of wildtype p53 nucleic acid sequences using a Locked Nucleic Acid clamp formed of GGAGGCC, wherein the Locked Nucleic Acid clamp binds wildtype p53 but not p53 249T nucleic acid sequences, and
amplifying the sample with primers consisting of SEQ ID NO: 1 and SEQ ID NO: 3; and
detecting the presence of p53 249T mutant using an oligonucleotide probe, wherein the presence of p53 249T mutant nucleic acid sequences is indicative of hepatocellular carcinoma.
2. The method of claim 1 , wherein the low molecular weight p53 249T mutation is a DNA sequence of 50 nucleotides or less.
3. The method of claim 1 , wherein the Locked Nucleic Acid clamp is a chemically modified RNA nucleotide having a ribose modified with a methylene bridge connecting a 2′-oxygen and 4′-carbon and wherein the Locked Nucleic Acid clamp is complementary to wild type p53.
4. The method of claim 1 , comprising an additional first step of isolating a low molecular weight nucleic acid from the biological sample prior to amplifying and detecting the presence of p53 249T.
5. A kit for detecting a circulating LMW p53 249T mutant DNA sequence in a urine sample from a patient being tested for hepatocellular carcinoma comprising (a) a LNA clamp formed of GGAGGCC; (b) primers to amplify a DNA sequence encoding for codons 248-250 of p53, wherein said primers consist of SEQ ID NO: 1 and SEQ ID NO: 3; and (c) a Fluorescein-labeled p53 249T oligonucleotide probe, wherein the probe specifically hybridizes to a p53 249T mutant sequence and emits more fluorescence than it does when it is not hybridized to the p53 249T mutant.
6. The kit of claim 5 , wherein (c) consists of SEQ ID NO: 4 and SEQ ID NO: 5.
7. The method of claim 1 , wherein the absence of p53 249T mutant nucleic acid sequences is indicative of an absence of hepatocellular carcinoma.