Primer probe set and kit for early diagnosis, detection or screening of colorectal cancer and precancerous lesions thereof
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A technology for precancerous lesions, colorectal cancer, applied in the field of biomedicine
Pending Publication Date: 2019-05-28
SUZHOU VERSABIO TECH INC
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[0005] The object of the present invention is to provide a stool polygene combined detection primer probe set and kit for early diagnosis, detection
Method used
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Embodiment 1
[0042] Example 1 Verification of the validity of the detection of polymethylation markers
[0043] 1) The sequences of the forward primers, reverse primers, blocker primers and probes of the methylated SDC2 gene, SFRP2 gene, SFRP1 gene, SEPT9 gene and PRIMA1 gene are as follows:
[0044] SDC2: SEQ ID NO: 1, 4, 7, 10; SFRP2: SEQ ID NO: 13, 16, 19, 22; SFRP1: SEQ ID NO: 25, 28, 31, 34; SEPT9: SEQ ID NO: 37, 40, 43, 46; PRIMA1: SEQ ID NO: 49, 52, 55, 58;
[0045] 2) In the primers and probes selected above, add the forward primer, reverse primer and probe of the internal reference gene ACTB of the sequence shown in SEQ ID NO: 61-63; then add 0.4mM 4 kinds of dNTPs mixed solution, 5mM Mg2+ solution, 1.2X PCR reaction buffer, 0.1U / uL DNA polymerase and PCR deionized water, simultaneously detect colorectal cancer methylation positive samples and negative samples (colorectal cancer methylation positive samples and negative samples are both is an existing sample), the result is as f...
Embodiment 2
[0046] Example 2 Detection of colorectal cancer and precancerous lesion stool samples
[0047] 1) Select the forward primer, reverse primer, blocker primer and probe sequences of five methylated SDC2 gene, SFRP2 gene, SFRP1 gene, SEPT9 gene and PRIMA1 gene as follows:
[0048] SDC2: SEQ ID NO: 2, 5, 8, 11; SFRP2: SEQ ID NO: 14, 17, 20, 23; SFRP1: SEQ ID NO: 26, 29, 32, 35; SEPT9: SEQ ID NO: 38, 41, 44, 47; PRIMA1: SEQ ID NO: 50, 53, 56, 59;
[0049] 2) In the primers and probes selected above, add the forward primer, reverse primer and probe of the internal reference gene ACTB of the sequence shown in SEQ ID NO: 61-63; then add 0.5mM 4 kinds of dNTPs mixed solution, 5mM Mg 2+ Solution, 1XPCR buffer, 0.1U / μL DNA polymerase and PCR deionized water were used to simultaneously detect colorectal cancer and precancerous lesion stool samples, and the detection results are shown in Table 2. Among them, all the stool samples of colorectal cancer and precancerous lesions were extract...
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Abstract
The invention belongs to the field of biomedicine and relates to a primer probe set for early diagnosis, detection or screening of colorectal cancer and precancerous lesions thereof, comprising at least two groups selected from the following groups of primers and probes: a) a forward primer, a reverse primer, a blocker primer and a probe for a methylated SDC2 gene, b) a forward primer, a reverse primer, a blocker primer and a probe for a methylated SFRP2 gene, c) a forward primer, a reverse primer, a blocker primer and a probe for a methylated SFRP1 gene, d) a forward primer, a reverse primer,a blocker primer and a probe for a methylated SEPT9 gene, and e) a forward primer, a reverse primer, a blocker primer and a probe for a methylated PRIMA1 gene. In the invention, the primer probe setand kit simultaneously detect a plurality of methylation markers specific to colorectal cancer and colorectal advanced adenoma, which can improve the detection sensitivity of colorectal cancer and precancerous lesions thereof and minimize the complexity of operation to the greatest extent.
Description
technical field [0001] The invention belongs to the field of biomedicine, and relates to a stool multi-gene combined detection primer probe set and kit for early diagnosis, detection or screening of colorectal cancer and its precancerous lesions. Background technique [0002] Colorectal cancer (CRC, or colorectal cancer) is a major health problem in Western countries. According to the estimates of the American Cancer Society, the death toll of CRC in the United States is about 50,000, and nearly 130,000 new cases were diagnosed in 2016. It is the third most common cancer, while in China, the incidence of colorectal cancer is high, And showing an upward trend. Currently, the proportion of colorectal cancer stage for initial treatment is as follows: stage I accounts for 15%; stage II accounts for 20%-30%; stage III accounts for 30%-40%; stage IV accounts for 20%-25%. In terms of survival, the 5-year survival rate of patients with stage I can reach more than 90%, while the su...
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