Biomarker related to colorectal cancer and application of biomarker
A technology of colorectal cancer and markers, applied in the field of biomarkers, can solve the problems of being easily affected by the sample environment, difficult to detect colorectal cancer, and high cost of colorectal cancer, so as to increase patient compliance, good application prospects and Practical significance, cost-effective effect
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Embodiment 1
[0046] Example 1, extraction of stool DNA samples.
[0047] (1) Collect individual feces samples, freeze them immediately, and put them on ice before the experiment;
[0048] (2) Weigh 200 mg of fixed feces into a 2 mL centrifuge tube, add 800 μL of fecal DNA extraction buffer, shake and mix well for 5 min, and centrifuge at 1800 g for 1 min;
[0049] (3) Take 50 μL of the suspension into a 1.5 mL centrifuge tube, add 800 μL of lysate, vortex and mix, lyse at 70°C for 5 min, centrifuge for 5 min, and transfer the supernatant to a clean 1.5 mL centrifuge tube;
[0050] (4) Add 20 μL of well-mixed magnetic beads, vortex for 20 seconds, let stand at room temperature for 4 minutes, place on a magnetic rack, let stand for 20 seconds, and absorb the supernatant;
[0051] (5) Add 500 μL of washing solution Ⅰ, vortex for 20 seconds, mix the magnetic beads, place them on a magnetic rack, let them stand for 20 seconds, and discard the supernatant;
[0052] (6) Add 750 μL of washing so...
Embodiment 2
[0057] Example 2, quantitative detection of markers.
[0058] The quantitative detection of marker 1) and marker 3) used the Taqman qPCR method, and the quantitative detection of marker 2) used the MSP (Methylmion Specific PCR) method, and the probes and primers used are shown in Table 1.
[0059] Table 1. Probes and primers for markers
[0060]
[0061] Taking the TaqMan Master Mix kit product of Suzhou Xinhai Biotechnology Co., Ltd. as an example, the specific steps for the content detection of the marker 1) and marker 3) are described below.
[0062] (1) Perform the reaction according to the qPCR reaction system shown in Table 2 to prepare the PCR reaction solution.
[0063] Table 2. qPCR reaction system
[0064]
[0065] (2) After the PCR reaction solution is prepared, mix it upside down and centrifuge, dispense it into a 96-well PCR reaction plate, centrifuge at 2000g for 2 minutes, seal it and place it in a PCR machine for reaction.
[0066] (3) The qPCR reactio...
Embodiment 3
[0071] Example 3, colorectal cancer risk assessment model.
[0072] The colorectal cancer risk assessment model uses the random forest algorithm to train and test the model using the three marker abundances of 357 healthy samples and 354 colorectal cancer patient samples, and finally establishes the colorectal cancer risk assessment model. The specific steps are as follows :
[0073] Step 1) Using the method described in Example 1 to extract fecal DNA samples from the feces of 357 healthy samples and 354 colorectal cancer samples, and divide the extracted samples into two parts, one of which was treated with bisulfate;
[0074] Step 2) Use the method described in Example 2 to detect the content of the target gene fragments of the three markers and the content of the internal reference gene in all samples, where marker 1) and marker 3) use 16S rDNA as the internal reference, and marker 2) Use the MSP product corresponding to the BMP3 gene fragment as an internal reference;
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