An improved hereditary hemorrhagic telangiectasia related gene mutation detection kit

Abstract

The present invention relates to a hereditary hemorrhagic telangiectasia related gene mutation detection kit, wherein whether the coding regions and the flanking sequences of an ACVRL1 gene, an ENG gene, a SMAD4 gene and a BMP9 gene are subjected to mutation is detected so as to diagnose the generation and the type of hereditary hemorrhagic telangiectasia in an individual. The invention further relates to a kit for detecting whether mutation exists in the coding regions and the flanking sequences of the ACVRL1 gene, the ENG gene, the SMAD4 gene and the BMP9 gene of an individual so as to judgewhether the individual has arteriovenous malformation, pulmonary hypertension and other diseases related to the gene mutations.

Description

technical field

[0001] The invention belongs to the field of biotechnology, and relates to a kit for detecting genetic mutations related to hereditary hemorrhagic telangiectasia. Background technique

[0002] Hereditary hemorrhagic telangiectasia is a systemic disease with an average incidence of approximately 1 / 5000-1 / 800. Clinical manifestations include epistaxis, mucocutaneous telangiectasia, and arteriovenous malformations in the lung, liver, gastrointestinal tract, brain, and spinal cord. The age of onset of the disease ranges from childhood to tens of years, and epistaxis is the most common and earliest clinical feature. Due to the late onset and inconsistent appearance of clinical features in various tissues, hereditary hemorrhagic telangiectasia is often misdiagnosed as simple intractable epistaxis at the onset of onset, while bleeding caused by visceral arteriovenous malformations is often life-threatening Therefore, the current clinical diagnosis strategy has cer...

Examples

Embodiment 1

[0052] [Example 1] Preparation of blood sample DNA of the subject to be tested

[0053] 1. Research object

[0054] A 2-generation pedigree with 2 clinically diagnosed hereditary hemorrhagic telangiectasia was collected. The clinical features of the proband were repeated epistaxis, telangiectasia visible on the tip of the tongue, clear family history, hepatic hemangioma consistent with heredity Three of the clinical features of hemorrhagic telangiectasia can be diagnosed clinically; the son of the proband has the same disease. After signing the informed consent form, 5-10ml blood samples were collected from each person.

[0055] 2. Genomic DNA extraction

[0056] Using phenol chloroform extraction method.

[0057] first day

[0058] 1) Anticoagulant blood was diluted 1-fold with PBS.

[0059] 2) Add 2 times the volume of lymphatic separation solution (18°C to 28°C) into the centrifuge tube, spread a layer of 1 times the volume of diluted blood on top, centrifuge at 1000×g...

Embodiment 2

[0074] [Example 2] PCR amplification of ACVRL1, ENG, SMAD4 and BMP9 gene coding regions

[0075] 1. Primer sequence

[0076] The primers are the above-mentioned 37 pairs of specific primers, and the sequences are listed in Table 1.

[0077] 2. Establishment of PCR reaction system (Table 2)

[0078] Table 2 PCR reaction system

[0079]

[0080] Reaction conditions: PCR reaction is carried out on the Mastercycler thermal cycler of eppendorf company, and the 37 pairs of specific primer reaction conditions that each sample carries out are consistent (comprising temperature and time), such as figure 1 shown.

Embodiment 3

[0081] [Example 3] Purification and quantification of PCR amplification products

[0082] 1. Purification of PCR products——96-well plate method

[0083] 1. Add 50 μl sterile water to the 96-well plate containing the PCR product and mix well.

[0084] 2. Transfer it to the Millipore purification plate, put it on the vacuum pump for about 3 minutes, and see that there is no water in the purification plate.

[0085] 3. Add 50 μl of deionized water to the purification plate again, and continue to filter until there is no water in the purification plate.

[0086] 4. Remove the purification plate from the vacuum pump, add 20 μl of deionized water to the plate, let it rest for 15 minutes, shake it for another 15 minutes, and then suck it into a new 96-well plate.

[0087] 5. Store in a -20°C refrigerator.

[0088] 2. Quantification by electrophoresis

[0089] 1. Sample preparation

[0090] Take a 96-well spotting plate, add 6 μl of sample buffer to each well, remove the PCR prod...

technical field

[0001] The invention belongs to the field of biotechnology, and relates to a kit for detecting genetic mutations related to hereditary hemorrhagic telangiectasia. Background technique

[0002] Hereditary hemorrhagic telangiectasia is a systemic disease with an average incidence of approximately 1 / 5000-1 / 800. Clinical manifestations include epistaxis, mucocutaneous telangiectasia, and arteriovenous malformations in the lung, liver, gastrointestinal tract, brain, and spinal cord. The age of onset of the disease ranges from childhood to tens of years, and epistaxis is the most common and earliest clinical feature. Due to the late onset and inconsistent appearance of clinical features in various tissues, hereditary hemorrhagic telangiectasia is often misdiagnosed as simple intractable epistaxis at the onset of onset, while bleeding caused by visceral arteriovenous malformations is often life-threatening Therefore, the current clinical diagnosis strategy has cer...

Examples

Embodiment 1

[0052] [Example 1] Preparation of blood sample DNA of the subject to be tested

[0053] 1. Research object

[0054] A 2-generation pedigree with 2 clinically diagnosed hereditary hemorrhagic telangiectasia was collected. The clinical features of the proband were repeated epistaxis, telangiectasia visible on the tip of the tongue, clear family history, hepatic hemangioma consistent with heredity Three of the clinical features of hemorrhagic telangiectasia can be diagnosed clinically; the son of the proband has the same disease. After signing the informed consent form, 5-10ml blood samples were collected from each person.

[0055] 2. Genomic DNA extraction

[0056] Using phenol chloroform extraction method.

[0057] first day

[0058] 1) Anticoagulant blood was diluted 1-fold with PBS.

[0059] 2) Add 2 times the volume of lymphatic separation solution (18°C to 28°C) into the centrifuge tube, spread a layer of 1 times the volume of diluted blood on top, centrifuge at 1000×g...

Embodiment 2

[0074] [Example 2] PCR amplification of ACVRL1, ENG, SMAD4 and BMP9 gene coding regions

[0075] 1. Primer sequence

[0076] The primers are the above-mentioned 37 pairs of specific primers, and the sequences are listed in Table 1.

[0077] 2. Establishment of PCR reaction system (Table 2)

[0078] Table 2 PCR reaction system

[0079]

[0080] Reaction conditions: PCR reaction is carried out on the Mastercycler thermal cycler of eppendorf company, and the 37 pairs of specific primer reaction conditions that each sample carries out are consistent (comprising temperature and time), such as figure 1 shown.

Embodiment 3

[0081] [Example 3] Purification and quantification of PCR amplification products

[0082] 1. Purification of PCR products——96-well plate method

[0083] 1. Add 50 μl sterile water to the 96-well plate containing the PCR product and mix well.

[0084] 2. Transfer it to the Millipore purification plate, put it on the vacuum pump for about 3 minutes, and see that there is no water in the purification plate.

[0085] 3. Add 50 μl of deionized water to the purification plate again, and continue to filter until there is no water in the purification plate.

[0086] 4. Remove the purification plate from the vacuum pump, add 20 μl of deionized water to the plate, let it rest for 15 minutes, shake it for another 15 minutes, and then suck it into a new 96-well plate.

[0087] 5. Store in a -20°C refrigerator.

[0088] 2. Quantification by electrophoresis

[0089] 1. Sample preparation

[0090] Take a 96-well spotting plate, add 6 μl of sample buffer to each well, remove the PCR prod...

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