Biomarker combinations, kits and applications for early screening of prostate cancer
A biomarker, prostate cancer technology, applied in the field of biomarker combination for early screening of prostate cancer, can solve the problems of poor specificity, increased pain and injury, low sensitivity of prostate cancer detection, etc., and achieves good stability and specificity. Strong and efficient effect
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Embodiment 1
[0050] Example 1 Kit for Early Screening of Prostate Cancer
[0051] Including Qiagen urine free DNA extraction kit, HinP1I, HpaII, AciI, HpyCH4IV methylation-sensitive endonuclease system, 5 pairs of target-specific primers and internal reference primers, 5 pairs of specific primer probes and internal reference primer probes;
[0052] Wherein the sequences of the 5 pairs of target-specific primers are as follows:
[0053] Seq ID NO.1F ACCGAGAAGAAAAGGCCGTACTC,
[0054] Seq ID NO. 1R TGTGAACCTTTCGCGGCTCGAC;
[0055] Seq ID NO. 2F AGTTGCTGACGCAGTTCCTTC,
[0056] Seq ID NO.2R CCACGCCAGACTGTAGAGCAC;
[0057] Seq ID NO. 3F GGAATTCTTTTGCTGCGCTG,
[0058] Seq ID NO. 3R CAAGGCCCGGCACTTTTCA;
[0059] Seq ID NO. 4F CCGAAGCCCACGTAGCCTG,
[0060]Seq ID NO.4R CACCGGACCCTCCTGGACTC;
[0061] Seq ID NO.5F CAAAGCCGCCGCTGCCAAAG,
[0062] Seq ID NO.5R CTCGGGGTCCAATAGTAGCGGGTAC;
[0063] The sequences of internal reference primers are as follows:
[0064] Seq ID NO.6F CCTACGAAAACCTCACGG...
Embodiment 2
[0074] Example 2 Method for detecting changes in ctDNA methylation level
[0075] 1. Take 10 mL of urine and use Qiagen Urine Free DNA Extraction Kit (Cat: 954154) to extract free cfDNA;
[0076] 2. Take 20 ng cfDNA and 20 μL HinP1I, HpaII, AciI, HpyCH4IV four methylation-sensitive endonuclease systems (final concentration 10 U / μL), incubate at 37°C for 16 h, and inactivate the enzyme at 80°C for 20 min;
[0077] 3. Use all the incubated products as templates, 5 pairs of target-specific primers (50 nM each) plus 1 internal reference primer (10 nM) to configure the system, set the following table 1 to set the program and perform the amplification program in a common PCR instrument ;
[0078] Table 1
[0079]
[0080] 4. Take 1 μL of the amplification product from the previous step as a template, add 5 pairs of target-specific primers (0.25 μM each), 5 specific primer probes (0.1 μM each), plus 1 internal reference primer (0.05 μM) and internal reference Primer probe (0.02...
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