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Reagent system and kit for NDRG4 gene methylation detection and application thereof

A methylation and kit technology, applied in the field of genetic engineering, can solve the problems of no reagent system and kit for detecting the methylation status of NDRG4 gene, difficulty in early diagnosis of colorectal cancer, low sensitivity of detection methods, etc., to achieve Fast detection speed, intuitive results, high sensitivity and specificity

Inactive Publication Date: 2017-03-22
刘鹏飞
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem solved by the present invention is: the early diagnosis of colorectal cancer is difficult, the detection method in the prior art is low in sensitivity, low in accuracy and inconvenient, and there is no reagent system and reagent that can quickly and easily detect the methylation state of NDRG4 gene box

Method used

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  • Reagent system and kit for NDRG4 gene methylation detection and application thereof
  • Reagent system and kit for NDRG4 gene methylation detection and application thereof
  • Reagent system and kit for NDRG4 gene methylation detection and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0065] The preparation of embodiment 1 kit

[0066] The kit prepared in this example includes a positive standard containing the methylation sequence of the NDRG4 gene, a negative standard containing a blank plasmid, a reaction solution and a DNA polymerase. Wherein, the volume of the test tube containing the DNA polymerase is 500 μL, and the tube contains 22 μL of Taq DNA polymerase at a concentration of 5 U / μL.

[0067] (1) Preparation of positive standard and negative standard

[0068] By comparing and analyzing the DNA sequences of a large number of clinical colorectal cancer patients and normal people, a sequence related to colorectal cancer was designed. The NDRG4 gene methylation sequence is SEQ ID NO.4, and the sequence of SEQ ID NO.4 is as follows :

[0069]

[0070] Taking the sequence of SEQ ID NO.4 as the target gene, the primer sequences SEQ ID NO.5 and SEQ ID NO.6 were designed for conventional PCR amplification. The PCR reaction system is shown in Table 1. ...

Embodiment 2

[0095] The preparation of embodiment 2 kit

[0096] The kit prepared in this example is the same as the positive standard and the negative standard in the kit prepared in Example 1. The difference between this example and Example 1 is that the formula of the reaction solution in this example is as shown in Table 4. Wherein, the primers SEQ ID NO.1 and SEQ ID NO.2, and the specific probe SEQ ID NO.3 are the same as those in Example 1. In this example, the enzyme in the DNA polymerase tube is Taq DNA polymerase, its concentration is 4 U / μL, and the volume is 30 μL.

[0097] Table 4 The formula of the reaction solution in this embodiment

[0098] composition Volume (μL) 10x Buffer 2 SEQ ID NO.1 0.5(300nM) SEQ ID NO.2 0.5(300nM) SEQ ID NO.3 0.3(300nM) dNTP 1.0(200mM) h 2 o

13.7 total capacity 18

[0099] Wherein, the composition of the buffer (buffer) solution is: Tris-HCl 150mM, KCl 600mM, MgCl 2 20mM, where the...

Embodiment 3

[0101] The preparation of embodiment 3 kits

[0102] The kit prepared in this example is the same as the positive standard and negative standard in the kit prepared in Example 1. The difference between this example and Example 1 is that the formula of the reaction solution in this example is as shown in Table 5. Wherein, the primers SEQ ID NO.1 and SEQ ID NO.2, and the specific probe SEQ ID NO.3 are the same as in Example 1, but the fluorescent reporter group on the probe is HEX, and the fluorescent quencher group is BHQ; In this example, the enzyme in the DNA polymerase tube is Taq DNA polymerase, its concentration is 5 U / μL, and the volume is 30 μL.

[0103] Table 5 The formula of the reaction solution in this embodiment

[0104] composition Volume (μL) 10x Buffer 3 SEQ ID NO.1 0.4(250nM) SEQ ID NO.2 0.4(250nM) SEQ ID NO.3 0.3(250nM) dNTP 1.0(200mM) h 2 o

12.9 total capacity 18

[0105] Wherein, the composit...

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Abstract

The invention relates to a reagent system and kit for NDRG4 gene methylation detection and application thereof. The reagent system or the kit comprises specific primers SEQ ID NO.1 and SEQ NO.2 and a specific probe SEQ ID NO.3, and a report group and a quenching group are connected to the probe. The reagent system further comprises a positive standard substance, a negative standard substance, a buffer solution, dNTPs and DNA polymerase. A use method of the kit comprises the steps that 1, methylation treatment is conducted on a sample DNA; 2, a fluorescent quantitative PCR reaction is conducted on the sample DNA obtained after methylation treatment in the step 1 by means of the specific primers SEQ ID NO.1 and SEQ NO.2 and the specific probe SEQ ID NO.3, a fluorescent quantitative PCR reaction is conducted on the positive standard substance and the negative standard substance simultaneously, and the methylation state of a gene is judged by analyzing fluorescence alteration of the PCR process. The preparation method of the kit is simple, the kit is convenient to use and high in speed, and the detection result is accurate, visual and high in sensitivity.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a NDRG4 gene methylation detection reagent system, kit and application thereof. Background technique [0002] Colorectal cancer is one of the major human malignant tumors, and its morbidity and mortality rank third and fourth, respectively. Each year, 1.2 million new cases are diagnosed and more than 600,000 patients die from colorectal cancer. The incidence of colorectal cancer is low in the under-50 age group, but increases with age. The median age at onset of colorectal cancer in developed countries is 70 years. The early symptoms of colorectal cancer are not obvious. As the tumor grows, symptoms such as changes in bowel habits, blood in the stool, diarrhea, alternating diarrhea and constipation, and local abdominal pain appear. In the late stage, systemic symptoms such as anemia and weight loss appear. Colorectal cancer can occur in any part of the colon or rectum, but t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6886C12Q2600/154C12Q2523/125C12Q2563/107C12Q2545/113
Inventor 刘鹏飞王菁蕊
Owner 刘鹏飞
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