Biomarker combination for early screening of esophageal cancer, kit and application
A biomarker and esophageal cancer technology, applied in the field of biomarker combinations for early screening of esophageal cancer, can solve the problems of poor specificity and low detection sensitivity of esophageal cancer, and achieve objective interpretation, simple detection, and stability Good results
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Embodiment 1
[0047] Example 1 Kit for Early Screening of Esophageal Cancer
[0048] Including Qiagen plasma cell-free DNA extraction kit, methylation-sensitive endonuclease system including HinP1I, HpaII, AciI, HpyCH4IV, 4 pairs of target-specific primers and internal reference primers, 4 pairs of specific primer probes, internal reference primer probes and internal reference primer probe;
[0049] Wherein the sequences of the 4 pairs of target-specific primers are as follows:
[0050] Seq ID NO.1: F-CCCGGCGCGATTGCAAAGTTTTTCGT,
[0051] Seq ID NO.2: R-CCGCCCCGTCGCCGAGTCC;
[0052] Seq ID NO.3: F-GTCAGCAGCACCCCCGAGCCTT,
[0053] Seq ID NO.4: R-CGCACTCTTGGGGCGGGGTCCTATCTC;
[0054] Seq ID NO.5: F-GGTACTGTGAAGGGTCCGGGTC,
[0055] Seq ID NO.6: R-AAGTCGGGGTCGAAAGTCCTC;
[0056] Seq ID NO.7: F-ACGTTTGCACGCAGGTTCA,
[0057] Seq ID NO.8: R-CCGGGATCGTGTCCGTAAGC;
[0058]The sequences of internal reference primers are as follows:
[0059] Seq ID NO.9: F-GGTGCCAGATTTTCTCCATGTCGTC,
[0060] ...
Embodiment 2
[0068] Example 2 Method for detecting changes in ctDNA methylation level
[0069] 1. Take 5 mL of plasma, and use Qiagen plasma cell-free DNA extraction kit (Cat: 55204) to extract cell-free cfDNA;
[0070] 2. Take 20ng cfDNA and 20μHinP1I, HpaII, AciI, HpyCH4IV four methylation-sensitive endonuclease systems (final concentration 10U / μL), incubate at 37°C for 16h, and inactivate the enzyme at 80°C for 20min. The system is shown in Table 1 below ;
[0071] Table 1
[0072]
[0073]
[0074] Use all the products after incubation as templates, 4 pairs of target-specific primers (50nM each) plus 1 internal reference primer (10nM) to configure the system (see Table 3), set the following table 4 to set the program and perform amplification in a common PCR instrument program;
[0075] table 3
[0076]
[0077] Table 4
[0078]
[0079] 4. Take 1 μL of the amplification product of the previous step as a template, add 4 pairs of target-specific primers (0.25 μM each), ...
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