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A probe pair, primer and kit for prognostic assessment of primary liver cancer

A kit and probe technology, applied in the field of genetic diagnosis, can solve problems such as increasing the risk of liver cancer, achieve the effects of reducing the probability of false positive detection, accurate experimental results, and increasing the probability of specific matching

Active Publication Date: 2017-10-31
武汉双旋生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Studies in recent years have shown that XRCC4, as a key component of DNA double-strand break repair, also plays an important role in the occurrence and development of tumors. The mutation of this gene will increase the risk of liver cancer, but so far no evidence has been found. A report on the correlation between the mutation of base 50 in the coding region of XRCC4 gene and the prognosis of primary liver cancer

Method used

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  • A probe pair, primer and kit for prognostic assessment of primary liver cancer
  • A probe pair, primer and kit for prognostic assessment of primary liver cancer
  • A probe pair, primer and kit for prognostic assessment of primary liver cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The design of embodiment 1 probe and primer

[0028] After the human XRCC4 gene was sequenced, the probe sequence was designed according to its nucleotide sequence and specific detection site (the 50th base C → T in the gene coding region): 5'-AACCCAGTATAATTC-3', and the 5' The fluorophore FAM is labeled at the end, and the quencher group MGB is labeled at the 3' end as a T probe for detecting mutant XRCC4 gene fragments; at the same time, 5'-AACCCAGTATAACTCA-3' is designed, and the 5' end is labeled with a fluorophore The group VIC and the 3' end labeled quencher group MGB are used as C probes to detect wild-type XRCC4 gene fragments.

[0029] And design the PCR amplification primers as:

[0030] Upstream primer: 5'-TGGAGAGAAAAATAAGCAGAATCCA-3'

[0031] Downstream primer: 5'-TTCCAGTGTTTTTCCCCAAGATACT-3'

[0032] Use artificially synthesized variant XRCC4 gene fragment (nucleotide sequence is SEQ ID NO: 6), wild-type XRCC4 gene fragment (nucleotide sequence is SEQ ID...

Embodiment 2

[0033] Embodiment 2 is used for detecting the kit of human XRCC4 gene mutation

[0034] A kit for detecting the C→T mutation of base 50 in the coding region of the human XRCC4 gene, the main components of which are shown in Table 1:

[0035] Table 1

[0036] serial number

Composition

Specification

quantity

1

DNA free water

1200μl

1 stick

2

2×Taqman enzyme reaction solution

1200μl

1 stick

3

10×PCR amplification solution

200μl

1 stick

4

C Probe Positive Control

10μl

1 stick

5

T probe positive control

10μl

1 stick

6

T probe and C probe double positive control

10μl

1 stick

7

negative control

10μl

1 stick

8

Optical 96-well reaction plate

Piece

1 block

9

Optical film

open

1 piece

10

manual

share

1 copy

[0037] The 2×Taqman enzyme reaction solution includes 2 kinds of fluorescentl...

Embodiment 3

[0047] Example 3 Correlation between liver cancer recurrence and XRCC4 gene mutation

[0048] Step 1: Collect liver cancer tumor samples and extract genomic DNA

[0049] A total of 399 tumor tissue specimens from patients with sporadic primary hepatocellular carcinoma were collected from the Affiliated Hospital of Youjiang Medical College for Nationalities, with an average age of 47.8±10.4 years (standard deviation), including 51 female specimens.

[0050] Tumor cells were isolated from the tumor tissue specimens collected above by proteinase K cleavage method, and sample DNA in tumor cells was extracted by phenol-chloroform method, and the sample DNA was uniformly diluted to 50 ng / μL as the DNA template to be tested.

[0051] Step 2: Detection of C→T mutation at base 50 in the XRCC4 gene coding region

[0052] Using the kit in Example 2, each DNA template to be tested was subjected to an amplification reaction with a total volume of 25 μL in a single well of a 96-well plate....

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Abstract

The invention discloses a probe pair and primer used for detecting C-to-T mutation of a 50 base of a coding region of the human XRCC4 gene. The probe pair is composed of a probe T as shown in SEQ ID No. 4 and a probe C as shown in SEQ ID No. 5. The primer is composed of an upstream primer as shown in SEQ ID No. 2 and a downstream primer as shown in SEQ ID No. 3. The invention further discloses a kit including the probe pair and the primer. The kit can be used for detecting whether C-to-T mutation of the 50 base of the coding region of the XRCC4 gene exists in a tumor tissue sample of a patient with early-stage primary liver cancer, which is favorable for prognosis evaluation of early-stage primary liver cancer.

Description

technical field [0001] The invention belongs to the field of gene diagnosis, and more specifically relates to a probe pair, a primer and a kit for evaluating the prognosis of primary liver cancer. Background technique [0002] At present, primary liver cancer is one of the most common malignant tumors in the world. The tumor is distributed regionally in the world. my country is the main place where liver cancer occurs. The number of liver cancer patients reaches 500,000, accounting for 50% of the new liver cancer patients in the world. In recent years, the incidence of liver cancer has a clear upward trend, seriously endangering human health and safety. Surgical treatment is the first choice and the most effective method for the treatment of liver cancer. But even in patients who undergo surgery, the cancer recurrence rate is high. At present, the 5-year survival rate of radical hepatectomy is only about 30%. The effect of liver transplantation in patients with early prima...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/156
Inventor 龙喜带曾智
Owner 武汉双旋生物科技有限责任公司
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