Primer pair, probe and kit used for noninvasive polygene methylation combination detection for early stage colorectal cancer and applications thereof
A colorectal cancer, combined detection technology, applied in the determination/examination of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of limited early diagnosis and low sensitivity of CRC, and achieve intuitive results and reduced The effect of contamination, detection process optimization
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Embodiment 1
[0062] This example is a primer pair and probe for non-invasive multi-gene methylation joint detection of early colorectal cancer, which includes primer pairs and probes for detecting Spetin9, NDRG4, BMP3, THBD and SDC2 gene methylation respectively. The primer pair and probe of probe and internal reference ACTB; Wherein
[0063] The primer pair used to detect the methylation of the Spetin9 gene is the sequence shown in SEQ ID No: 1 and SEQ ID No: 2, and the probe is the sequence shown in SEQ ID No: 3;
[0064] The primer pair used to detect NDRG4 gene methylation is the sequence shown in SEQ ID No: 4 and SEQ ID No: 5, and the probe is the sequence shown in SEQ ID No: 6;
[0065] The pair of primers used to detect the methylation of the BMP3 gene is the sequence shown in SEQ ID No: 7 and SEQ ID No: 8, and the probe is the sequence shown in SEQ ID No: 9;
[0066] The primer pair used to detect THBD gene methylation is the sequence shown in SEQ ID No: 10 and SEQ ID No: 11, and ...
Embodiment 2
[0071] This example is a kit for the non-invasive multi-gene methylation combined detection of early colorectal cancer containing the primer pair and probe described in Example 1.
[0072] The above kits include free DNA extraction reagents, sulfite conversion reagents, real-time fluorescent quantitative PCR amplification reaction reagents, positive control substances, and negative control substances; wherein, free DNA extraction reagents and sulfite conversion reagents are all commercially available reagents box, the free DNA extraction reagent specifically includes lysate, protease mixed solution, magnetic beads, washing solution 1, washing solution 2 and eluent, and the real-time fluorescent quantitative PCR amplification reaction reagent includes PCR amplification buffer, such as The primers and probes described in SEQ ID No: 1-SEQID No: 18, UNG enzyme and DNA polymerase, the corresponding relationship between primers and probes is specifically shown in Table 1; the positiv...
Embodiment 3
[0074] This example is a method for detecting early colorectal cancer using the kit described in Example 2.
[0075] 1. Materials, reagents, instruments
[0076] Free DNA extraction kit, sulfite conversion kit, and fluorescent quantitative PCR instrument are ABI7500.
[0077] 2. Sample Preparation
[0078] The positive control uses bovine serum albumin and human genomic DNA, and then dilutes it with DEPC water, and the negative control sample is DEPC H 2 O; The sample to be processed is the patient's peripheral blood.
[0079] 3. Extraction of cell-free DNA and sulfite conversion
[0080] Extract free DNA from 15ml samples according to the instructions of the free DNA extraction kit, which includes DNA cleavage and binding, DNA washing and elution, and the DNA obtained by eluting with 60 μl eluent at the end is the DNA to be extracted; Sulfate conversion kit performs sulfite conversion of DNA samples, which includes sulfite conversion, binding step, first wash, desulfo, se...
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