CN106148523A - The method determining the gene mutation site of child's interstitial lung pneumonia - Google Patents
The method determining the gene mutation site of child's interstitial lung pneumonia Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 29
- 206010064571 Gene mutation Diseases 0.000 title claims abstract description 9
- 210000004072 lung Anatomy 0.000 title description 7
- 206010035664 Pneumonia Diseases 0.000 title description 5
- 238000012163 sequencing technique Methods 0.000 claims abstract description 25
- 208000029523 Interstitial Lung disease Diseases 0.000 claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 238000012408 PCR amplification Methods 0.000 claims abstract description 13
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- 102100039061 Cytokine receptor common subunit beta Human genes 0.000 claims description 5
- 102100020856 Forkhead box protein F1 Human genes 0.000 claims description 5
- 102100028113 Granulocyte-macrophage colony-stimulating factor receptor subunit alpha Human genes 0.000 claims description 5
- 101001033280 Homo sapiens Cytokine receptor common subunit beta Proteins 0.000 claims description 5
- 101000916625 Homo sapiens Granulocyte-macrophage colony-stimulating factor receptor subunit alpha Proteins 0.000 claims description 5
- 101000801640 Homo sapiens Phospholipid-transporting ATPase ABCA3 Proteins 0.000 claims description 5
- 101001086862 Homo sapiens Pulmonary surfactant-associated protein B Proteins 0.000 claims description 5
- 101000612671 Homo sapiens Pulmonary surfactant-associated protein C Proteins 0.000 claims description 5
- 101150111110 NKX2-1 gene Proteins 0.000 claims description 5
- 102100033623 Phospholipid-transporting ATPase ABCA3 Human genes 0.000 claims description 5
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Abstract
A kind of method that the invention discloses gene point mutation determining childhood interstitial pneumonitis, obtains peripheral blood including (1), and described peripheral blood comes from the individuality that clinical diagnosis is childhood interstitial pneumonitis；(2) from described peripheral blood, genomic DNA is extracted；(3) use specificity amplification primer to expand described genomic DNA to by PCR, thus obtain pcr amplification product；(4) described pcr amplification product is carried out the first order-checking, in order to obtain the first sequencing result；(5) described first sequencing result is filtered, in order to obtain the first sequencing result through filtering；(6) described the first sequencing result through filtering is compared with reference sequences, and determine the gene mutation site relevant to childhood interstitial pneumonitis based on comparison result；(7) the described gene containing mutational site is carried out the second order-checking, in order to verify.Method used herein is secondary order-checking, and a relatively conventional generation checks order, and flux is big, the time is short, degree of accuracy is high, informative.
Description
Technical field
The present invention relates to biological technical field, more particularly to the method for the gene mutation site determining a kind of disease, more
The method being specifically related to determine the gene mutation site of child's interstitial lung pneumonia.
Background technology
Interstitial diseases/Diffuse-type gastric carcinoma (ILD/DPLD) is that one group of cause of disease is different, but clinical, iconography and tissue are sick
Similar different substantiality disease of science, it is now recognized that include more than 200 kind diseases.Child, particularly infant ILD/DPLD (chILD/
ChDPLD) with the existing similarity of adult, the cause of disease of specificity, such as chILD and prognosis is had again to exist significantly with adult
Difference, the primary pulmonary interstitial fibrosis (IPF) that usual adult generally diagnoses there's almost no child, and chILD
Prognosis is the best compared with adult.
The sickness rate of child's interstitial diseases is 0.3/100000, and mortality rate is higher.
It is diffusivity, mist illiteracy sample, clouded glass infiltration that infant has Radiologic imaging, and when there is no obvious cause,
Interstitial diseases should be suspected clinically.
But, the method for the present stage gene mutation site to determining child's interstitial lung pneumonia need further to grind
Study carefully.
At infantile period, the chILD caused by inherited genetic factors occupies an important position, and Abroad in Recent Years enters this one side
Exhibition is very fast, and this field domestic is still within space state.
The detection method of this gene is relevant with interstitial diseases, but the direct purpose of this detection is not Diagnosis of pulmonary
Interstitial disease, and simply detect the intermediate parameters of interstitial diseases
Summary of the invention
Present invention seek to address that one of technical problem present in prior art, to this end, it is an object of the present invention to
A kind of method providing gene mutation site determining child's interstitial lung pneumonia, this method can be to interstitial diseases relevant people
The coding region total length of 8 genes and near transcribe splice site and carry out mutation analysis.Cover the point mutation in the range of this, compare
Little insertion and deletion form sudden change.Method used herein is secondary order-checking, and a relatively conventional generation checks order, and flux is big, the time
Short, degree of accuracy is high, informative, at short notice 8 genes that interstitial lung is relevant can be accurately positioned and analyze,
And the analysis cost saved in a large number.
According to an aspect of the present invention, the invention provides a kind of gene point mutation determining childhood interstitial pneumonitis
Method.According to embodiments of the invention, the method includes:
(1) obtaining peripheral blood, described peripheral blood comes from the individuality that clinical diagnosis is childhood interstitial pneumonitis；
(2) from described peripheral blood, genomic DNA is extracted；
(3) use specificity amplification primer to expand described genomic DNA to by PCR, thus obtain pcr amplification product；
(4) described pcr amplification product is carried out the first order-checking, in order to obtain the first sequencing result；
(5) described first sequencing result is filtered, in order to obtain the first sequencing result through filtering；
(6) described the first sequencing result through filtering is compared with reference sequences, and true based on comparison result
The fixed gene mutation site relevant to childhood interstitial pneumonitis；
(7) the described gene containing mutational site is carried out the second order-checking, in order to verify.
Further, the nucleotide sequence of described specificity amplification primer pair is SEQ ID NO:1 and 76, SEQ ID NO:
2 and 77, SEQ ID NO:2 and 77, SEQ ID NO:3 and 78, SEQ ID NO:4 and 79, SEQ ID NO:5 and 80, SEQ ID
NO:6 and 81, SEQ ID NO:7 and 82, SEQ ID NO:8 and 83, SEQ ID NO:9 and 84, SEQ ID NO:10 and 85, SEQ
ID NO:11 and 86, SEQ ID NO:12 and 87, SEQ ID NO:13 and 88, SEQ ID NO:14 and 89, SEQ ID NO:15
With 90, SEQ ID NO:16 and 91, SEQ ID NO:17 and 92, SEQ ID NO:18 and 93, SEQ ID NO:19 and 94, SEQ
ID NO:20 and 95, SEQ ID NO:21 and 96, SEQ ID NO:22 and 97, SEQ ID NO:23 and 98, SEQ ID NO:24
With 99, SEQ ID NO:25 and 100, SEQ ID NO:26 and 101, SEQ ID NO:27 and 102, SEQ ID NO:28 and 103,
SEQ ID NO:29 and 104, SEQ ID NO:30 and 105, SEQ ID NO:31 and 106, SEQ ID NO:32 and 107, SEQ
ID NO:33 and 108, SEQ ID NO:34 and 109, SEQ ID NO:35 and 110, SEQ ID NO:36 and 111, SEQ ID
NO:37 and 112, SEQ ID NO:38 and 113, SEQ ID NO:39 and 114, SEQ ID NO:40 and 115, SEQ ID NO:41
With 116, SEQ ID NO:42 and 117, SEQ ID NO:43 and 118, SEQ ID NO:44 and 119, SEQ ID NO:45 and
120, SEQ ID NO:46 and 121, SEQ ID NO:47 and 122, SEQ ID NO:48 and 123, SEQ ID NO:49 and 124,
SEQ ID NO:50 and 125, SEQ ID NO:51 and 126, SEQ ID NO:52 and 127, SEQ ID NO:53 and 128, SEQ
ID NO:54 and 129, SEQ ID NO:55 and 130, SEQ ID NO:56 and 131, SEQ ID NO:57 and 132, SEQ ID
NO:58 and 133, SEQ ID NO:59 and 134, SEQ ID NO:60 and 135, SEQ ID NO:61 and 136, SEQ ID NO:62
With 137, SEQ ID NO:63 and 138, SEQ ID NO:64 and 139, SEQ ID NO:65 and 140, SEQ ID NO:66 and
141, SEQ ID NO:67 and 142, SEQ ID NO:68 and 143, SEQ ID NO:69 and 144, SEQ ID NO:70 and 145,
SEQ ID NO:71 and 146, SEQ ID NO:72 and 147, SEQ ID NO:73 and 148, SEQ ID NO:74 and 149, SEQ
ID NO:75 and 150.
Further, the gene loci that described childhood interstitial pneumonitis is relevant is ABCA3, SFTPB, SFTPC, CSF2RA,
CSF2RB, NKX2.1, FOXF1 and SLC7A7 gene coding region and intron are sheared and are connect site.
Further, in above-mentioned steps (2), the concentration of genomic DNA is 20 μ g/ μ L-100 μ g/ μ L.
Further, during described pcr amplification product is carried out the first order-checking, build Insert Fragment be 175～
The sequencing library of 275bp.
Further, calculating according to 10 μ L reaction systems, in above-mentioned steps (3), the reaction system of PCR amplification is:
| Reagent | Volume |
| LA enzyme (5U/uL) | 0.2μL |
| ddH2O | 6.2μL |
| Primer working solution | 1μL |
| Sample DNA | 1μL |
| dNTP | 0.4μL |
| buffer | 1μL |
| DMSO | 0.2μL |
Optionally, described primer working solution is that amplimer adds ultra-pure water by its quality and is made into the mother that concentration is 100 μm ol
Liquid, according still further to the pair principle of amplimer pair, is made into the working solution of final concentration of 5 μm ol.
Optionally, the response procedures of described PCR amplification is:
Further, described first order-checking utilizes Illumina miseq platform to carry out double end sequencing.
Further, the reference sequences in above-mentioned steps (4) is ABCA3:NG_011790.1；SFTPB:NG_
016967.1；SFTPC:NG_029659；CSF2RA:NG_012280.1；CSF2RB:NG_008040.1；NKX2.1:NG_
013365.1；FOXF1:NG_016273.1；SLC7A7:NG_012851.2, optionally, described reference sequences derives from NCBI.
Further, described second order-checking utilizes Sanger method to carry out.
Further, described sequencing library uses Illumina Nextera XT to build storehouse test kit and prepare.
According to embodiments of the invention, the advantage of the method that the present invention provides can detect relevant to interstitial lung simultaneously
It is also analyzed, low cost by 8 genes, and flux is high, and the time is short, degree of accuracy is high.
The additional aspect of the present invention and advantage will part be given in the following description, and part will become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Accompanying drawing explanation
Fig. 1 is 2% gel electricity of the pcr amplified fragment expanding 75 exons obtained according to one embodiment of the invention
Swimming figure
Fig. 2 is to exist c.218T on Sanger sequence verification SFTPC gene > schematic diagram that suddenlys change of C (p.Ile73Thr)
Detailed description of the invention
Embodiments of the invention are described below in detail.The embodiment described below with reference to accompanying drawing is exemplary, only
For explaining the present invention, and it is not considered as limiting the invention.
Embodiment 1
(1) preparation of sample DNA
Sample information: China man patient, in 1 years old November, clinical diagnosis is interstitial pneumonia.
Collect the peripheral blood specimen of patient, use Shanghai to fly prompt biological whole blood DNA extraction test kit and extract DNA, use
NanoDrop 2000 spectrophotometer DNA to having extracted carries out concentration mensuration.Mensuration concentration is 56.2ng/ul, reaches detection
Standard.
(2) design of primer and amplification
Use Primer5 software to carry out the design of primer for the coding region of 8 genes and adnexa intron region, set altogether
75 pairs of primers are counted.
PCR amplification is carried out by following amplification system,
| LA enzyme | 0.2μL |
| ddH2O | 6.2μL |
| Primer working solution | 1μL |
| Sample DNA | 1μL |
| dNTP | 0.4μL |
| buffer | 1μL |
| DMSO | 0.2μL |
| Total | 10μL |
PCR amplification program is as follows:
Amplification obtains 75 DNA fragmentations, carries out gel electrophoresis and sees Fig. 1, it may be determined that will carry out the purpose fragment checked order
The most effectively expanded.
(2) structure in library and order-checking
The preparation in library: use Illumina Nextera XT to build storehouse test kit and build the sample of Insert Fragment 175-275bp
This library, concrete operations are shown in that test kit description is prepared in library.Then use Illumina miseq platform to carry out double end to survey
Sequence.
(3) data analysis
Filtering low quality initial data, blood samples of patients sample obtains the monoploid coverage of 3950 multipliers.
Sequencing result passes through MiSeq Report and corresponding reference sequences (ABCA3:NG_011790.1；SFTPB:NG_
016967.1；SFTPC:NG_029659；CSF2RA:NG_012280.1；CSF2RB:NG_008040.1；NKX2.1:NG_
013365.1；FOXF1:NG_016273.1；SLC7A7:NG_012851.2, derives from NCBI) contrast, based on vcf.out file
Being analyzed, the genovariation data obtaining this patient are as follows:
In conjunction with the related gene data base on clinical information and NCBI, document, above-listed variation information is analyzed screening,
Remove pleomorphism site and non-pathologic site.
Patient detects and exists c.218T on SFTPC gene > C (p.Ile73Thr), this sudden change is considered and interstitial lung disease
Sick relevant, and have pertinent literature support.
(4) positive site checking
The positive site of patient's SFTPC gene is carried out Sanger method sequence verification.
Use forward primer: GTAAAACGACGGCCAGTGGAGGGAAGCGCATTTGAGTA
Downstream primer: GGAAACAGCTATGACCATGAGCCAATGAGGAACAGTGCT
Expanding patient's specimen, amplification program and system are with before.
Amplified production is carried out generation order-checking, and sequencing primer is:
Forward sequencing primer: GTAAAACGACGGCCAGTG
Reverse sequencing primer: GGAAACAGCTATGACCATG
Generation order-checking uses ABI3500, and sequencing procedures is shown in instrument description.
Sequencing result is analyzed:
Use software FinchTV machine data lower to generation order-checking to be analyzed, from NCBI, download SFTPC gene reference sequence
Row, contrast, and find that heterozygous mutant is c.218T > C (p.Ile73Thr), it was demonstrated that secondary sequencing result is accurate, refers to Fig. 2.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " specifically show
Example " or the description of " some examples " etc. means to combine this embodiment or example describes specific features, structure, material or spy
Point is contained at least one embodiment or the example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.And, the specific features of description, structure, material or feature can be any
One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
These embodiments can be carried out multiple change in the case of departing from the principle of the present invention and objective, revise, replace and modification, this
The scope of invention is limited by claim and equivalent thereof.
Claims (10)
1. the method for the gene point mutation determining childhood interstitial pneumonitis, it is characterised in that include
(1) obtaining peripheral blood, described peripheral blood comes from the individuality that clinical diagnosis is childhood interstitial pneumonitis；
(2) from described peripheral blood, genomic DNA is extracted；
(3) use specificity amplification primer to expand described genomic DNA to by PCR, thus obtain pcr amplification product；
(4) described pcr amplification product is carried out the first order-checking, in order to obtain the first sequencing result；
(5) described first sequencing result is filtered, in order to obtain the first sequencing result through filtering；
(6) described the first sequencing result through filtering is compared with reference sequences, and determine based on comparison result and
The gene mutation site that childhood interstitial pneumonitis is relevant；
(7) the described gene containing mutational site is carried out the second order-checking, in order to verify.
Method the most according to claim 1, it is characterised in that the nucleotide sequence of described specificity amplification primer pair is
SEQ ID NO:1 and 76, SEQ ID NO:2 and 77, SEQ ID NO:2 and 77, SEQ ID NO:3 and 78, SEQ ID NO:4 and
79, SEQ ID NO:5 and 80, SEQ ID NO:6 and 81, SEQ ID NO:7 and 82, SEQ ID NO:8 and 83, SEQ ID NO:
9 and 84, SEQ ID NO:10 and 85, SEQ ID NO:11 and 86, SEQ ID NO:12 and 87, SEQ ID NO:13 and 88, SEQ
ID NO:14 and 89, SEQ ID NO:15 and 90, SEQ ID NO:16 and 91, SEQ ID NO:17 and 92, SEQ ID NO:18
With 93, SEQ ID NO:19 and 94, SEQ ID NO:20 and 95, SEQ ID NO:21 and 96, SEQ ID NO:22 and 97, SEQ
ID NO:23 and 98, SEQ ID NO:24 and 99, SEQ ID NO:25 and 100, SEQ ID NO:26 and 101, SEQ ID NO:
27 and 102, SEQ ID NO:28 and 103, SEQ ID NO:29 and 104, SEQ ID NO:30 and 105, SEQ ID NO:31 and
106, SEQ ID NO:32 and 107, SEQ ID NO:33 and 108, SEQ ID NO:34 and 109, SEQ ID NO:35 and 110,
SEQ ID NO:36 and 111, SEQ ID NO:37 and 112, SEQ ID NO:38 and 113, SEQ ID NO:39 and 114, SEQ
ID NO:40 and 115, SEQ ID NO:41 and 116, SEQ ID NO:42 and 117, SEQ ID NO:43 and 118, SEQ ID
NO:44 and 119, SEQ ID NO:45 and 120, SEQ ID NO:46 and 121, SEQ ID NO:47 and 122, SEQ ID NO:48
With 123, SEQ ID NO:49 and 124, SEQ ID NO:50 and 125, SEQ ID NO:51 and 126, SEQ ID NO:52 and
127, SEQ ID NO:53 and 128, SEQ ID NO:54 and 129, SEQ ID NO:55 and 130, SEQ ID NO:56 and 131,
SEQ ID NO:57 and 132, SEQ ID NO:58 and 133, SEQ ID NO:59 and 134, SEQ ID NO:60 and 135, SEQ
ID NO:61 and 136, SEQ ID NO:62 and 137, SEQ ID NO:63 and 138, SEQ ID NO:64 and 139, SEQ ID
NO:65 and 140, SEQ ID NO:66 and 141, SEQ ID NO:67 and 142, SEQ ID NO:68 and 143, SEQ ID NO:69
With 144, SEQ ID NO:70 and 145, SEQ ID NO:71 and 146, SEQ ID NO:72 and 147, SEQ ID NO:73 and
148, SEQ ID NO:74 and 149, SEQ ID NO:75 and 150.
Method the most according to claim 1, it is characterised in that the gene loci that described childhood interstitial pneumonitis is relevant is
ABCA3, SFTPB, SFTPC, CSF2RA, CSF2RB, NKX2.1, FOXF1 and SLC7A7 gene coding region and intron are sheared and are connect
Site.
Method the most according to claim 1, it is characterised in that in described step (2), the concentration of genomic DNA is 20 μ g/ μ
L-100μg/μL。
Method the most according to claim 1, it is characterised in that in described step (4), is entering described pcr amplification product
During row first checks order, build the sequencing library that Insert Fragment is 175～275bp；.
Method the most according to claim 1, it is characterised in that calculate according to 10 μ L reaction systems, in described step (3)
The reaction system of PCR amplification is:
Optionally, described primer working solution is that amplimer adds ultra-pure water by its quality and is made into the mother solution that concentration is 100 μm ol, then
According to the pair principle of amplimer pair, it is made into the working solution of final concentration of 5 μm ol.
Optionally, the response procedures of described PCR amplification is:
Method the most according to claim 1, it is characterised in that described first order-checking utilizes Illumina miseq platform to enter
The double end sequencing of row.
Method the most according to claim 1, it is characterised in that the reference sequences in described step (4) is ABCA3:NG_
011790.1；SFTPB:NG_016967.1；SFTPC:NG_029659；CSF2RA:NG_012280.1；CSF2RB:NG_
008040.1；NKX2.1:NG_013365.1；FOXF1:NG_016273.1；SLC7A7:NG_012851.2, optionally, described
Reference sequences derives from NCBI.
Method the most according to claim 1, it is characterised in that described second order-checking utilizes Sanger method to carry out.
Method the most according to claim 4, it is characterised in that described sequencing library is to use Illumina Nextera
XT builds prepared by storehouse test kit.
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| CN201610541039.3A CN106148523B (en) | 2016-07-11 | 2016-07-11 | Method for determining gene mutation site of children interstitial pneumonia |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106990177A (en) * | 2017-03-29 | 2017-07-28 | 山东大学 | Purposes of the glutamine of 617 generation mass shifts of AKAP4 albumen in the few weak smart diagnostic reagent of severe is prepared |
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Non-Patent Citations (4)
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| AKIMOTO T等: "Hereditary interstitial lung diseases manifesting in early childhood in Japan", 《PEDIATRIC RESEARCH》 * |
| R EPAUD等: "Pathologies génétiques du surfactantGenetic disorders of surfactant", 《ARCHIVES DE PÉDIATRIE》 * |
| SUZUKI T等: "Pulmonary macrophage transplantation therapy", 《NATURE》 * |
| VALIMAHAMED-MITHA S等: "Lung involvement in children with lysinuric protein intolerance", 《J INHERIT METAB DIS》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106990177A (en) * | 2017-03-29 | 2017-07-28 | 山东大学 | Purposes of the glutamine of 617 generation mass shifts of AKAP4 albumen in the few weak smart diagnostic reagent of severe is prepared |
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Denomination of invention: Method for identifying gene mutation sites in children with interstitial pneumonia Granted publication date: 20200529 Pledgee: Pudong Shanghai Development Bank Limited by Share Ltd. Wuhan branch Pledgor: WUHAN KINDSTAR MEDICAL TESTING INSTITUTE CO.,LTD. Registration number: Y2024980003351 |