Peripheral blood BRAF gene detection kit and application thereof in preparation of papillary craniopharyngeal tubuloma early diagnosis preparation

Abstract

The invention discloses a peripheral blood BRAF gene detection kit and application of the peripheral blood BRAF gene detection kit in preparation of a papillary craniopharyngeal tubuloma early diagnosis preparation. The kit contains a primer BR-F and a primer BR-R which are used for digital PCR (Polymerase Chain Reaction), and a probe BR-WP and a probe BR-MP. The kit can be used for directly detecting BRAF V600E gene mutation by taking peripheral blood as a template, the peripheral blood does not need to be extracted, and the operation is convenient and rapid. The kit can be used for detecting papillary craniopharyngeal tubuloma in peripheral blood, non-invasive detection is realized, and the risk of tissue sampling is avoided.

Description

Technical Field

[0001] The present invention belongs to the field of medical biotechnology, and particularly relates to a kit for detecting BRAF gene in peripheral blood and its application in the preparation of early diagnostic agents for papillary craniopharyngioma. Background Art

[0002] Craniopharyngioma (CP) occurs in 30% - 50% of children and adolescents. Although the tumor is benign, its mass effect affects the structures and functions of the hypothalamus, pituitary gland, optic chiasm, etc., leading to obvious neuroendocrine dysfunction. The clinical manifestations are increased intracranial pressure, visual field disorders, and pituitary-target gland axis dysfunction.

[0003] CP is divided into two different subtypes: papillary type (papillary craniopharyngiomas, PCP) and adamantinomatous type (adamantinomatous craniopharyngioma, ACP). The mutation sites of the two subtypes are mutually exclusive, and they are valuable molecular diagnostic genetic markers for clinical differentiation between the two. Compared with ACP, the neuroendocrine dysfunction of the PCP subtype is more significant at the time of diagnosis. BRAF V600E gene mutation exists in more than about 95% of PCPs, which is the main pathogenic driving factor of PCP. Currently, the detection of BRAF V600E gene mutation in clinical surgical pathological tissues is used as the main basis for diagnosing PCP. However, obtaining biopsy tissues through surgery is traumatic, and the damage to the structure and function of the sellar region during surgery further aggravates the irreversible damage to the hypothalamus-pituitary-target gland axis. The growth, development and metabolism of patients are severely affected, and they need lifelong hormone replacement therapy, with poor quality of life and prognosis. If a non-invasive method can be found to make a clear diagnosis before surgery, it will be of great significance for the condition assessment and treatment method selection of CP. Domestic and foreign research in this field is still ongoing. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a kit for detecting BRAF gene in peripheral blood.

[0005] Another purpose of the present invention is to provide the application of the above-mentioned kit for detecting BRAF gene in peripheral blood in the preparation of early diagnostic agents for papillary craniopharyngioma.

[0006] The purpose of the present invention is achieved by the following technical solutions: A kit for detecting BRAF gene in peripheral blood, containing primers and probes for digital PCR, specifically as follows:

[0007] BR-F: 5'-catgaagacctcacag-3';

[0008] BR-R: 5'-ccagacaactgttcaaa-3';

[0009] BR-WP: 5'-HEX-ctagctacagtgaaatctcgatg-3';

[0010] BR-MP: 5'-FAM-ctagctacagagaaatctcgatg-3'.

[0011] The described peripheral blood BRAF gene detection kit further contains at least one of enzyme, chip, water, Low TE buffer and instruction manual.

[0012] The enzyme is an enzyme that can be used for digital PCR; preferably QuantStudio TM 3D Digital PCR MasterMix v2.

[0013] The chip is preferably a chip that can be used in conjunction with QuantStudio TM 3D Digital PCR Master Mix v2; more preferably QuantStudio TM 3D Digital PCR 20K chip.

[0014] The water is preferably deionized water or pure water.

[0015] The composition of the Low TE buffer is as follows: 10 mM Tris-HCl, 0.1 mM EDTA, pH = 8.0.

[0016] The instruction manual records the detection procedure, specifically as follows:

[0017] (1) Prepare the digital PCR reaction system: Dilute the primers and probes to 10 μM with Low TE buffer respectively, and prepare the reaction system according to the following table:

[0018] Name Volume <![CDATA[QuantStudio TM 3D Digital PCR Master Mix v2]]> 7.5 μL BR-F 1.2 μL BR-R 1.2 μL BR-WP 0.3 μL BR-MP 0.3 μL Template 2 μL Water To a final volume of 15 μL

[0019] Then, add 14.5 μL to the chip to obtain the detection chip;

[0020] (2) Perform digital PCR reaction, and the reaction conditions are as follows: 96 °C for 10 min; 57 °C for 2 min, 98 °C for 30 s, 39 cycles; 57 °C for 2 min; 10 °C ∞.

[0021] Application of the above-mentioned peripheral blood BRAF gene detection kit in the preparation of early diagnosis agents for papillary craniopharyngioma.

[0022] The present invention has the following advantages and effects compared with the prior art:

[0023] (1) The peripheral blood BRAF gene detection kit provided by the present invention can directly use peripheral blood as a template for the detection of BRAF V600E gene mutations, without the need for extraction from peripheral blood, and is convenient and fast to operate.

[0024] (2) The peripheral blood BRAF gene detection kit provided by the present invention can detect papillary craniopharyngioma in peripheral blood, achieving non-invasive detection and avoiding the risk of tissue sampling. Specific embodiments

[0025] The present invention will be further described in detail below in conjunction with embodiments, but the embodiments of the present invention are not limited thereto.

[0026] Embodiment 1

[0027] (1) Design primers and probes for digital PCR of BRAF V600E to obtain the following primers and probes:

[0028] BR-F: 5'-catgaagacctcacag-3';

[0029] BR-R: 5'-ccagacaactgttcaaa-3';

[0030] BR-WP: 5'-HEX-ctagctacagtgaaatctcgatg-3';

[0031] BR-MP: 5'-FAM-ctagctacagagaaatctcgatg-3'.

[0032] (2) Samples:

[0033] Take the peripheral blood of 10 patients with papillary craniopharyngioma (from Shenzhen, Guangdong Province) as the experimental group, and at the same time use the peripheral blood of healthy people as the negative control group.

[0034] The detection results are shown in the following table:

[0035]

[0036] It can be seen that the accuracy rate of the kit provided by the present invention reaches 70%.

[0037] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A kit for detecting BRAF gene in peripheral blood, characterized in that: Containing primers and probes for digital PCR, specifically as follows: BR-F: 5'-catgaagacctcacag-3'; BR-R: 5'-ccagacaactgttcaaa-3'; BR-WP: 5'-HEX-ctagctacagtgaaatctcgatg-3'; BR-MP: 5'-FAM-ctagctacagagaaatctcgatg-3'.

2. The kit for detecting BRAF gene in peripheral blood according to claim 1, wherein: The kit for detecting BRAF gene in peripheral blood further contains at least one of enzyme, chip, water, Low TE buffer and instruction manual.

3. The kit for detecting BRAF gene in peripheral blood according to claim 1, wherein: The enzyme mentioned is QuantStudio TM 3D Digital PCR Master Mix v2; The chip described is a chip that can be used in conjunction with QuantStudio TM 3D Digital PCR Master Mix v2; The water is deionized water or pure water; The composition of the Low TE buffer is as follows: 10 mM Tris-HCl, 0.1 mM EDTA, pH = 8.0; The instruction manual records the detection procedure, specifically as follows: (1) Prepare the digital PCR reaction system: Dilute the primers and probes to 10 μM with Low TE buffer respectively; The reaction system is as follows: QuantStudio TM 3D Digital PCR Master Mix v2 7.5 μL, BR-F1 1.2 μL, BR-R 1.2 μL, BR-WP 0.3 μL, BR-MP 0.3 μL, template 2 μL, made up to 15 μL with water. Then, 14.5 μL of the reaction system was added to the chip to obtain the detection chip; (2) Perform digital PCR reaction, and the reaction conditions are as follows: 96 °C for 10 min; 57 °C for 2 min, 98 °C for 30 s, 39 cycles; 57 °C for 2 min; 10 °C ∞.

4. The kit for detecting BRAF gene in peripheral blood according to claim 3, wherein: The chip described above is QuantStudio TM 3D Digital PCR 20K chip.

5. Use of the kit for detecting BRAF gene in peripheral blood according to any one of claims 1 to 4 in the preparation of early diagnosis agents for papillary craniopharyngioma.

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