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Nucleic acid combination and kit for detecting septin9 gene methylation

A methylation and kit technology, applied in the field of molecular biology, can solve the problems of decreased sample detection rate, low colorectal cancer specificity, low screening penetration rate, etc., to improve sensitivity and specificity, prevent false Positive results, the effect of shortening the test time

Inactive Publication Date: 2018-05-18
SUREXAM BIO TECH
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  • Claims
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AI Technical Summary

Problems solved by technology

This method has low specificity for colorectal cancer, and is mainly susceptible to interference from food intake. If the food contains heme or rich vitamin C, it will cause false positive results; the second is the immune fecal occult blood test iFOBT: using hemoglobin , specific antibodies to albumin or other blood components to detect intestinal bleeding. This method is not affected by food, so the detection specificity is high, but there are strict requirements on the storage time of samples, and the detection rate of samples decreases with the storage time.
2. Colonoscopy: Colonoscopy is an invasive detection method that requires adequate intestinal preparation and is cumbersome to operate. In addition, it may cause complications such as intestinal infection, bleeding, and perforation. The penetration rate of screening is very high. Low
3. The process of bisulfite conversion to DNA is prone to problems such as insufficient conversion, DNA degradation, and low recovery efficiency

Method used

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  • Nucleic acid combination and kit for detecting septin9 gene methylation
  • Nucleic acid combination and kit for detecting septin9 gene methylation
  • Nucleic acid combination and kit for detecting septin9 gene methylation

Examples

Experimental program
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Effect test

Embodiment 1

[0057] This embodiment provides a kit for detecting the methylation of the septin9 gene, which includes: a nucleic acid combination for detecting the methylation of the septin9 gene, a Blocker sequence, a pair of primers for the β-actin internal reference gene, a probe for the β-actin internal reference gene, sub- Sulfate conversion reagent set, 2×PCR Mix and taq enzyme.

[0058] Wherein, the nucleic acid combination includes: a pair of primers for detecting the methylation of the septin9 gene and a probe for detecting the methylation of the septin9 gene.

[0059] The primer pair for detecting the methylation of the septin9 gene includes: an upstream primer whose base sequence is shown in SEQ ID NO.11 and a downstream primer shown in SEQ ID NO.17.

[0060] The base sequence of the septin9 gene methylation detection probe is shown in SEQ ID NO.19.

[0061] Among them, the 5' end of the septin9 gene methylation detection probe is labeled with a fluorescent reporter group FAM, a...

experiment example 2

[0100] The septin9 methylation detection site in the present invention includes 6 methylation sites located in the third CpG island of the septin9 promoter region. In this experimental example, the forward sequence of the target sequence where the methylation site is located is used as a template to design methylation-specific reference primers and probe sequences, as shown in Table 1.

[0101] At the same time, using the β-actin gene as an internal reference, β-actin control primers and probes were designed, as shown in Table 1.

[0102] Table 1 Primer and Probe Sequences

[0103]

[0104] The nucleic acid combination (primer: SEQ ID NO.11 (septin9-F1), SEQ ID NO.17 (septin9-R3), probe: SEQ ID NO. ID NO.19 (septin9-P1)) and β-actin internal reference gene primer pair and probe (primers: SEQ ID NO.1 (β-actin-F1), SEQID NO.6 (β-actin-R3), probe Needle: SEQ ID NO.7 (β-actin-P1)) for amplification efficiency verification, as follows.

[0105] Reagents: 2×PCR Mix, taq DNA poly...

experiment example 3

[0117] Dilute the methylated septin9 synthesis plasmid to 10 -2 ng / μl, 10 -3 ng / μl, 10 -4 ng / μl, 10 -5 ng / μl, 10 -6 5 concentration gradients of ng / μl were used as DNA samples, and the copy numbers corresponding to each concentration were shown in Table 3, and the kit provided in Example 1 was used for qPCR amplification to verify the sensitivity of the kit provided in Example 1 . The result is as image 3 shown.

[0118] Table 3 Correspondence between copy number and plasmid concentration

[0119] Concentration (ng / μl)

[0120] according to Figure 6 The results showed that with the decrease of the concentration, the fluorescence value and ct value of the sample showed a downward trend. The sample concentration is 10 -6 ng / μl (10 2 copy), the amplification curve was a smooth S-shape. Continue to reduce the sample concentration, the amplification curve is not S-shaped (results not shown), which affects the result judgment. Therefore determine that the det...

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Abstract

The invention discloses a nucleic acid combination and kit for detecting septin9 gene methylation, and relates to the field of molecular biology. The nucleic acid combination includes a primer pair for detecting septin9 gene methylation. The primer pair includes an upstream primer having a base sequence shown as SEQ ID NO.11 and a downstream primer having a base sequence shown as SEQ ID NO.17. Theprimer pair can specifically amplify the target site of a septin9 gene, wherein the target site is the third CpG island containing six CpG sites in a septin9 gene promoter region, so that specific detection of septin9 gene methylation is achieved. The nucleic acid combination and kit are high in sensitivity and have the detection limit as low as 100 copies, and the difficulty that the content ofctDNA in blood plasma is low and ctDNA cannot be detected is overcome.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a nucleic acid combination and a kit for detecting methylation of septin9 gene. Background technique [0002] Colorectal cancer is one of the common digestive tract tumors. According to statistics, there were about 1.36 million new cases of colorectal cancer in the world in 2012, and the incidence rate ranked third among malignant tumors. The death rate was about 690,000, and the mortality rate ranked first among malignant tumors. fourth place. [0003] Currently, there are three main screening methods for colorectal cancer: fecal occult blood tests (FOBT), colonoscopy, and genetic testing. 1. Fecal occult blood test: According to different detection targets, FOBT is mainly divided into two categories: one is guaiac fecal occult blood test (gFOBT): to detect substances containing peroxidase activity in feces, such as heme and sub Ferrorubin, which turns blue by oxidizing guaiac...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/154C12Q2600/166
Inventor 梁金连许嘉森吴诗扬
Owner SUREXAM BIO TECH
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