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Multi-gene detection primer probe set and kit for early diagnosis, screening or detection of digestive tract tumors, and applications of kit

A technology for detection of digestive tract tumors and primers, applied in the field of multi-gene detection primer probe sets, can solve the problems of difficulty in early screening or diagnosis of digestive tract tumors, low detection sensitivity, etc., and achieve early diagnosis and treatment, specific Good performance and high sensitivity

Pending Publication Date: 2019-12-10
SUZHOU VERSABIO TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the object of the present invention is to provide a multi-gene detection primer probe set, kit and application applicable to the early diagnosis, screening or detection of various gastrointestinal tumors, to solve the problem of developing a single methylated ctDNA. The detection sensitivity of the product is not high, and it is difficult to be used as an effective method for early screening or diagnosis of gastrointestinal tumors.

Method used

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  • Multi-gene detection primer probe set and kit for early diagnosis, screening or detection of digestive tract tumors, and applications of kit
  • Multi-gene detection primer probe set and kit for early diagnosis, screening or detection of digestive tract tumors, and applications of kit
  • Multi-gene detection primer probe set and kit for early diagnosis, screening or detection of digestive tract tumors, and applications of kit

Examples

Experimental program
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Effect test

Embodiment 1

[0050] 1) Select CADM1 forward primer, reverse primer, Blocker primer and probe sequences as follows: SEQ ID NO: 25, 28, 31, 34; select internal reference gene HBB, forward and reverse primers and probes of HBB such as SEQ ID NO :67~69;

[0051] 2) Add 0.4mM 4 kinds of dNTPs mixed solution, 5mM Mg2+ solution, 1.5X PCR buffer, 0.1U / μL DNA polymerase and PCR deionized water to the primers and probes selected above to detect positive methylation of digestive tract tumors sample, the result is as figure 1 As shown, the results indicate that methylated CADM1 has a good amplification effect, and it can effectively detect digestive tract tumors as a methylation marker.

Embodiment 2

[0053] 1) Select PAX1 forward primer, reverse primer, Blocker primer and probe sequences as follows: SEQ ID NO: 49, 52, 55, 58; select GADPH as an internal reference gene, GADPH forward and reverse primers and probes such as SEQ ID NO:64~66;

[0054] 2) Add 0.5mM 4 kinds of dNTPs mixed solution, 6mM Mg2+ solution, 1XPCR buffer, 0.08U / μL DNA polymerase and PCR deionized water to the primers and probes selected above to detect methylation-positive samples of digestive tract tumors, The result is as figure 2 As shown, the results indicate that the methylated PAX1 gene also has a good amplification effect, and can be used as a methylation marker to effectively detect digestive tract tumors.

Embodiment 3

[0056] 1) Select SDC2, SFRP2, PAX1 and CADM1 forward primer, reverse primer, Blocker primer and probe sequences as follows: SDC2:SEQ ID NO:2,5,8,11; SFRP2:SEQ ID NO:SEQ ID NO: 14, 17, 20, 23; PAX1: SEQ ID NO: 50, 53, 56, 59; CADM1: SEQ ID NO: 26, 29, 32, 35; ACTB was selected as the internal reference gene, forward and reverse primers and probes of ACTB As shown in SEQ ID NO:61~63;

[0057] 2) Add 0.5mM 4 kinds of dNTPs mixed solution, 5mM Mg2+ solution, 1X PCR buffer, 0.1U / μL DNA polymerase and PCR deionized water, and detect 79 gastric cancer serum samples at the same time, the volume is 3.5mL; all samples are Samples processed using the cell-free DNA extraction kit and bisulfite conversion kit manufactured by Suzhou Weishan Biotechnology Co., Ltd.

[0058] 3) At the same time, CEA, CA72-4, CA19-9, and CA242 were used to detect the serum samples of the above 79 cases of gastric cancer, and the combined detection of the four methylation markers of SDC2, SFRP2, PAX1, and CADM...

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Abstract

The present invention provides a multi-gene detection primer probe set for early diagnosis, screening or detection of digestive tract tumors, and belongs to the field of biomedicine. The primer probeset comprises at least two groups selected from the following five groups of primers and probes: (1) a forward primer, a reverse primer, a Blocker primer and a probe of a methylated SDC2 gene; (2) a forward primer, a reverse primer, a Blocker primer and a probe of a methylated SFRRP2 gene; (3) a forward primer, a reverse primer, a Blocker primer and a probe of a methylated SEP9 gene; (4) a forwardprimer, a reverse primer, a Blocker primer and a probe of a methylated CADM1 gene; and (5) a forward primer, a reverse primer, a Blocker primer and a probe of a methylated PAX1 gene. The multi-gene detection primer probe set simultaneously detects two or more methylated markers, is high in sensitivity, good in specificity, convenient in detection, and low in cost, and can be used for early screening and auxiliary diagnosis of the digestive tract tumors.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a multi-gene detection primer-probe set, kit and application thereof for early diagnosis, screening or detection of digestive tract tumors. Background technique [0002] Gastrointestinal cancer (liver cancer, pancreatic cancer, esophageal cancer, gastric cancer and colorectal cancer) are the most common malignant tumors in the Chinese population. The 2015 Chinese cancer statistics show that liver cancer, esophageal cancer, gastric cancer and colorectal cancer are all ranked first in China The top five types of cancer incidence in men and women, among which liver cancer ranks second in cancer mortality in China. At present, the proportion of early diagnosis and treatment of digestive tract cancer is less than 20%. In terms of survival, the 5-year survival rate of stage I patients can reach more than 90%, while the survival rate of stage IV patients is only slightly greater th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886
Inventor 赵国栋熊尚岷
Owner SUZHOU VERSABIO TECH INC
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