A primer and probe set for gastrointestinal cancer diagnosis, detection or screening
A digestive tract cancer and probe group technology, applied in the field of biomedicine, can solve the problems of low patient acceptance, low early detection rate of digestive tract cancer, and difficulty in popularization
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Embodiment 1
[0026] The genome sequence of the cell line with the known SFRP2 gene fully methylated was used as the DNA template. The DNA template was treated with bisulfite before the PCR reaction, and the ammonium bisulfite solution with a bisulfite concentration of 8M was used. The reaction conditions Heat at 70°C for 60 minutes, then use Axygen gel purification kit to purify the transformed DNA template, and then perform PCR.
[0027] SEQ ID NO. 1, SEQ ID NO. 17 and SEQ ID NO. 37 were used to carry out quantitative PCR reaction, wherein the fluorescence labeled by SEQ ID NO. 37 was ROX; the PCR system and reaction conditions were shown in Table 2 and Table 3.
[0028] Table 2 PCR components of Example 1
[0029] component Final concentration PCR reaction buffer 1X DNA polymerase 0.1U / μL dNTPs mix 0.2mM Mg2+ 5mM SEQ ID NO: 1 0.2μM SEQ ID NO: 17 0.2μM SEQ ID NO: 37 0.1μM Deionized water Replenish water to 15μL Post-t...
Embodiment 2
[0035] Colorectal cancer tissue and matched paracancerous tissue were used as detection objects. After DNA was extracted by Qiagen's DNeasy Blood&Tissue Kit, 10M bisulfite was used for treatment. The bisulfite solution used was 6M ammonium bisulfite, A mixed solution of 3M sodium bisulfite and a mixed solution of 1M anhydrous sodium sulfate, the reaction conditions were heating at 80°C for 40 minutes, and then PCR was performed after purification using Axygen gel purification kit.
[0036] Using SEQ ID NO.2, SEQ ID NO.22 and SEQ ID NO.31, wherein the fluorescence marked by SEQ ID NO.31 is JOE, 16 cases of colorectal cancer tissue and its adjacent tissue samples were detected by fluorescence quantitative PCR reaction. The PCR system and reaction conditions are shown in Table 4 and Table 5.
[0037] Table 4 PCR components of Example 2
[0038] component Final concentration PCR reaction buffer 1X DNA polymerase 0.08U / μL dNTPs mix 0.4mM Mg2+ ...
Embodiment 3
[0043] The test samples were serum cfDNA from 10 patients with esophageal cancer, serum cfDNA from 31 patients with gastric cancer, and serum cfDNA from 36 patients without obvious gastrointestinal diseases, and the serum volume was 1 mL. After DNA extraction was carried out using the cfDNA extraction kit from Suzhou Weishan Biotechnology Co., Ltd., 9M bisulfite was used for treatment. The bisulfite solution used was 9M ammonium bisulfite solution, and the reaction conditions were heating at 90°C for 30 min, followed by PCR after purification using the Axygen gel purification kit.
[0044] Using SEQ ID NO.5, SEQ ID NO.23 and SEQ ID NO.34, wherein the fluorescence marked by SEQ ID NO.34 is FAM, the fluorescence quantitative PCR reaction detection is carried out, and the PCR reaction system and reaction conditions are shown in Table 6 and Table 7 The test results are shown in Table 8.
[0045] Table 6 PCR components of Example 3
[0046] component Final concentrati...
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