Genomic imprinting subtype hemangioblastoma diagnosis kit

A technology of hemangioblastoma and diagnostic kit, applied in the field of gene imprinted subtype hemangioblastoma diagnostic kit

Pending Publication Date: 2019-07-09
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the diagnosis of gene imprinted subtype hemangioblastoma has not been realized in the prior art

Method used

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  • Genomic imprinting subtype hemangioblastoma diagnosis kit
  • Genomic imprinting subtype hemangioblastoma diagnosis kit
  • Genomic imprinting subtype hemangioblastoma diagnosis kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] This embodiment provides a gene imprinted subtype hemangioblastoma diagnostic kit, which includes: reagents for bisulfite treatment of genomic DNA, reagents for VHL in methylation-specific PCR A primer set for amplification of the promoter, PCR buffer, and deoxynucleotide triphosphates and Taq DNA polymerase.

[0019] Wherein, the reagents for treating the genomic DNA with bisulfite include: 2M NaOH, 3M sodium bisulfite and 10 mM hydroquinone. These three reagents are contained in different containers respectively.

[0020] The primer set includes a first primer, a second primer, a third primer and a fourth primer.

[0021] The first primer includes a first upstream primer and a first downstream primer, wherein the sequence of the first upstream primer is shown in SEQ NO:1, and the sequence of the first downstream primer is shown in SEQ NO:2.

[0022] The second primer includes a second upstream primer and a second downstream primer, wherein the sequence of the second...

Embodiment 2

[0030] This embodiment provides a method for diagnosing gene imprinted subtype hemangioblastoma in human tissue or blood using the gene imprinted subtype hemangioblastoma diagnostic kit in Example 1.

[0031] The method includes the following steps:

[0032] Step 1. Genomic DNA extraction.

[0033] Genomic DNA was extracted from tissues or peripheral blood of subjects using QIAamp DNA mini Kit (Qiagen Inc, Chatsworth, CA), and then purified using Wizard Genomic DNA Purification Kit (Promega, Madison, WI, U.S.A.).

[0034] Step 2, bisulfite treatment.

[0035] Take 1 μg of purified genomic DNA, denature it with 2M NaOH, then incubate in 3M sodium bisulfite and 10 mM hydroquinone at 55°C for 17 hours, and then use the EpiTect Bisulfite kit (Axygen) to treat the genomic DNA with bisulfite Get modified DNA.

[0036] Step 3, methylation-specific PCR.

[0037] The PCR system includes: 1×PCR buffer (New England Biolabs), deoxynucleotide triphosphate (2.5mM each), primer set (0.8m...

Embodiment 3

[0044] This example uses the gene imprinted subtype hemangioblastoma diagnostic kit in Example 1 and performs the gene imprinted subtype hemangioblastoma diagnostic test on the tissues or blood of three groups of subjects according to the diagnostic method in Example 2 .

[0045] Three groups of subjects include: experimental group, positive control group and negative control group.

[0046]Among them, the experimental group included three patients with confirmed hemangioblastoma due to the imprinted subtype, all of whom had no VHL gene mutation and all had hemangioblastoma. The positive control group included three hemangioblastoma patients with confirmed VHL gene mutations. The negative control group included three healthy volunteers.

[0047] In this embodiment, 5 mL of blood from three groups of subjects were collected as experimental samples for experiments.

[0048] The collection of samples from all the above subjects was approved by the Ethics Committee of Huashan H...

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Abstract

The invention provides a hemangioblastoma typing diagnostic kit. The diagnostic kit is characterized by comprising a genomic imprinting subtype hemangioblastoma diagnostic reagent, a hemangioblastomarecurrence tracking diagnostic reagent and a gene mutation subtype hemangioblastoma diagnostic reagent, wherein the genomic imprinting subtype hemangioblastoma diagnostic reagent comprises a first primer (SEQ NO:1 and SEQ NO:2), a second primer (SEQ NO:3 and SEQ NO:4), a third primer (SEQ NO:5 and SEQ NO:6) and a fourth primer (SEQ NO:7 and SEQ NO:8); the hemangioblastoma recurrence tracking diagnostic reagent comprises a fifth primer (SEQ NO:9) for detecting microRNA-365 and a sixth primer (SEQ NO:10) for detecting microRNA-517.

Description

technical field [0001] The invention relates to a tumor diagnosis kit, in particular to a gene imprint subtype hemangioblastoma diagnosis kit. Background technique [0002] Von Hippel-Lindau (VHL) disease (OMIM 193300) is an autosomal dominant syndrome with the potential to develop a variety of benign and malignant neoplasms in various organ systems. Its inheritance results from germline mutations in the chromosomally located VHL gene and exhibits significant phenotypic variability, age-dependent penetrance, and interfamilial variation. Tumors characteristic of VHL include: central nervous system (CNS) and retinal hemangioblastomas (HBs), clear cell renal carcinoma, neuroendocrine tumors, pancreatic cysts, pheochromocytoma, etc. Most patients with VHL will be infected with CNS-HBs, with an incidence ranging from 34.3% to 72%. [0003] VHL disease occurs in all ethnic groups around the world. Both sexes are equally affected, with a prevalence rate of 1:30,000 to 1:50,000 (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/154
Inventor 马德选王莹
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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