CN106399576B - Application of the mitochondrial genomes 9bp sequence gene polymorphism in evaluation benzene poisoning risk - Google Patents
Application of the mitochondrial genomes 9bp sequence gene polymorphism in evaluation benzene poisoning risk Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses application of the mitochondrial genomes 9bp sequence gene polymorphism in evaluation benzene poisoning risk.The invention discloses for detect in mitochondria with " the 9bp sequences of 2 tandem sequence repeats " still " deletion form of the 9bp sequence of 2 tandem sequence repeats " substance preparation evaluation Chronic Benzene Poisoning risk product in application；" the 9bp sequences of 2 tandem sequence repeats " are as shown in the sequence 1 of sequence table；" deletion form of the 9bp sequence of 2 tandem sequence repeats " is that " the 9bp sequences of 2 tandem sequence repeats " have lacked the 1st to 9 nucleotide.It is demonstrated experimentally that the risk that Chronic Benzene Poisoning occurs for the individual in mitochondrial genomes DNA with " deletion form of the 9bp sequence of 2 tandem sequence repeats " is higher than the individual in mitochondrial genomes DNA with " the 9bp sequences of 2 tandem sequence repeats " "." deletion form of the 9bp sequence of 2 tandem sequence repeats " can be used for evaluating the risk of Chronic Benzene Poisoning.
Description
Technical field
The present invention relates to fields of biomedicine, and in particular to mitochondrial genomes 9bp sequence gene polymorphism is in evaluation benzene
Application in risk of being poisoned.
Background technique
In recent years, benzene and benzene homologues poisoning Case report rise year by year, and cause leukaemia death also year by year after benzene poisoning
Rise, endangers very serious.Studies have shown that benzene poisoning is mainly that intermediate active metabolite acts on mitochondria by aoxidizing damage
Caused by wound.The generation of Chronic Benzene Poisoning is also related with the genetic predisposition of contactee in addition to related with the exposure concentration of benzene.
Mitochondrial cytochrome c oxidase subunit I I gene (abbreviation MTCO2 gene) and mitochondrial tRNA lysine base
Because the small fragment non-coding sequence between (abbreviation MTTK gene) contains the 9bp sequence (nucleotide sequence are as follows: 5 '-of 2 tandem sequence repeats
CCCCCTCTACCCCCTCTA-3 '), there is also insertion/deletions: the 9bp sequence of 3 tandem sequence repeats is insert type
(nucleotide sequence are as follows: 5 '-CCCCCTCTACCCCCTCTACCCCCTCTA-3 '), 1 9bp sequence are deletion form (nucleotide
Sequence are as follows: 5 '-CCCCCTCTA-3 ').Deletion form is related with the generation of a variety of genetic diseases, but has no the generation phase with benzene poisoning
The report of pass.
The main cell device of blood platelet first is that mitochondria, mitochondria are that it uniquely has the organelle of genetic function, blood
Platelet mitochondria with respect to leucocyte mitochondrial DNA it not by the interference of the other homologous sequence genes of human chromosomal, it is small to extract blood
The genomic DNA of plate can analyze advantageously mitochondrial genomes DNA.
Summary of the invention
The technical problem to be solved by the present invention is to how evaluate when person under test is engaged in benzene exposure work to occur in Chronic Benzene
The height of the risk of poison.
In order to solve the above technical problems, present invention firstly provides have " 2 tandem sequence repeats for detecting in mitochondria
The substance of 9bp sequence " still " deletion form of the 9bp sequence of 2 tandem sequence repeats " is in preparation evaluation Chronic Benzene Poisoning risk
Application in product.
In order to solve the above technical problems, the present invention also provides have " 2 tandem sequence repeats for detecting in mitochondria
9bp sequence " still " deletion form of the 9bp sequence of 2 tandem sequence repeats " substance evaluation Chronic Benzene Poisoning risk in answering
With.
If having " the 9bp sequences of 2 tandem sequence repeats " in mitochondria, person under test's Chronic Benzene Poisoning risk is lower.Such as
There is " deletion form of the 9bp sequence of 2 tandem sequence repeats " in fruit mitochondria, person under test's Chronic Benzene Poisoning risk is higher.Line grain
Lower than having in mitochondria, " 2 are gone here and there person under test's Chronic Benzene Poisoning risk with " the 9bp sequences of 2 tandem sequence repeats " in body
Join the deletion form of duplicate 9bp sequence " person under test.
It is described " to there are " the 9bp sequences of 2 tandem sequence repeats " still " 2 strings for detecting in mitochondria in above-mentioned application
Join the deletion form of duplicate 9bp sequence " substance " can be for primer pair, the primer pair is by primers F and primer R group
At；
The primers F can be following a1) or a2):
A1) single strand dna shown in the sequence 2 of sequence table；
A2) there is phase by sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2
The DNA molecular of congenerous；
The primer R can be following a3) or a4):
A3) single strand dna shown in the sequence 3 of sequence table；
A4) there is phase by sequence 3 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 3
The DNA molecular of congenerous.
Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation
Method is mutated the nucleotide sequence of primers F provided by the invention and primer R.Those by manually modified, have and this
The nucleotide sequence of the primers F and primer R of inventing design has the nucleotide of 85% or higher identity, and has identical function
Can, it is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.
In order to solve the above technical problems, the present invention also provides a kind of products for evaluating Chronic Benzene Poisoning risk.
The product of evaluation Chronic Benzene Poisoning risk provided by the present invention, it may include have " 2 in mitochondria for detecting
The substance of the 9bp sequence of a tandem sequence repeats " still " deletion form of the 9bp sequence of 2 tandem sequence repeats ".
It is described " including for detecting there are " the 9bp sequences of 2 tandem sequence repeats " still " 2 in mitochondria in the said goods
The deletion form of the 9bp sequence of a tandem sequence repeats " substance " can be the primer pair.
The product may also include the carrier for recording judgment criteria.
The judgment criteria is judgment criteria first or judgment criteria second.
Judgment criteria first: if there are " the 9bp sequences of 2 tandem sequence repeats " in mitochondria, person under test's Chronic Benzene Poisoning wind
It is dangerous lower；If there is " deletion form of the 9bp sequence of 2 tandem sequence repeats " in mitochondria, person under test's Chronic Benzene Poisoning risk
Property is higher.
Judgment criteria second: with person under test's Chronic Benzene Poisoning risk of " the 9bp sequences of 2 tandem sequence repeats " in mitochondria
Lower than the person under test in mitochondria with " deletion form of the 9bp sequence of 2 tandem sequence repeats ".
The preparation method of any of the above-described product also belongs to protection scope of the present invention.
The preparation method of any of the above-described product may include by each primer of any of the above-described special primer centering
The step of individually packing.
In order to solve the above technical problems, the present invention also provides the sides that the risk of Chronic Benzene Poisoning occurs for evaluation person under test
Method.
It is method one that the method for the risk of Chronic Benzene Poisoning, which occurs, for evaluation person under test provided by the present invention, can specifically be wrapped
It includes following steps: there is " the 9bp sequences of 2 tandem sequence repeats " still " 9bp of 2 tandem sequence repeats in detection person under test's mitochondria
The deletion form of sequence "；If in mitochondria have " the 9bp sequences of 2 tandem sequence repeats ", person under test's Chronic Benzene Poisoning risk compared with
It is low；If having " deletion form of the 9bp sequence of 2 tandem sequence repeats " in mitochondria, person under test's Chronic Benzene Poisoning risk is higher.
It is method two that the method for the risk of Chronic Benzene Poisoning, which occurs, for evaluation person under test provided by the present invention, can specifically be wrapped
It includes following steps: there is " the 9bp sequences of 2 tandem sequence repeats " still " 9bp of 2 tandem sequence repeats in detection person under test's mitochondria
The deletion form of sequence "；Person under test's Chronic Benzene Poisoning risk in mitochondria with " the 9bp sequences of 2 tandem sequence repeats " is lower than
With the person under test of " deletion form of the 9bp sequence of 2 tandem sequence repeats " in mitochondria.
" the 9bp sequences of 2 tandem sequence repeats " or " deletion form of the 9bp sequence of 2 tandem sequence repeats " are being commented as marker
Application in valence Chronic Benzene Poisoning risk also belongs to protection scope of the present invention.
The primer pair also belongs to protection scope of the present invention.
Application of the primer pair in evaluation Chronic Benzene Poisoning risk also belongs to protection scope of the present invention.
The primer pair also belongs to this in the application prepared in the kit for evaluating Chronic Benzene Poisoning risk
The protection scope of invention.
It is method three that the method for the risk of Chronic Benzene Poisoning, which occurs, for evaluation person under test provided by the present invention, can specifically be wrapped
It includes following steps: using the mitochondrial genomes DNA of person under test as template, PCR amplification being carried out using the primer pair；PCR
Amplified production is that person under test's Chronic Benzene Poisoning risk of 738bp is lower, and pcr amplification product is person under test's Chronic Benzene of 729bp
Risk of being poisoned is higher.
It is method four that the method for the risk of Chronic Benzene Poisoning, which occurs, for evaluation person under test provided by the present invention, can specifically be wrapped
It includes following steps: using the mitochondrial genomes DNA of person under test as template, PCR amplification, PCR being carried out using the primer pair
Person under test's Chronic Benzene Poisoning risk that amplified production is 738bp is lower than the person under test that pcr amplification product is 729bp.
It is any of the above-described that described to be higher than to be being higher than statistically.
It is any of the above-described described lower than can be being lower than statistically.
Any of the above-described " the 9bp sequences of 2 tandem sequence repeats " can be as shown in the sequence 1 of sequence table；
Any of the above-described " deletion form of the 9bp sequence of 2 tandem sequence repeats " can be " the 9bp sequence of 2 tandem sequence repeats
Column " have lacked the 1st to 9 nucleotide.Any of the above-described " deletion form of the 9bp sequence of 2 tandem sequence repeats " can be such as sequence table
Sequence 1 from 5 ' ends shown in the 10th to 18.
Any of the above-described Chronic Benzene Poisoning is occupational chronic benzene poisoning.
Using the patient of 27 Chronic Benzene Poisonings as case group, as a control group with 40 normal health examinees, to it
Mitochondrial genomes are studied.The result shows that with the ratio of " deletion form of the 9bp sequence of 2 tandem sequence repeats " in case group
Example (33.3%) is significantly higher than control group (10.0%), and the statistically significant (χ of difference of the two2=5.090, P=
0.024), OR value (95%CI) is 4.5 (1.21-16.62).Therefore, have " 2 tandem sequence repeats in mitochondrial genomes DNA
The risk that Chronic Benzene Poisoning occurs for the individual of the deletion form of 9bp sequence ", which is higher than in mitochondrial genomes DNA, has " 2 series connection weights
The risk of Chronic Benzene Poisoning occurs for the individual of multiple 9bp sequence ".Therefore, " deletion form of the 9bp sequence of 2 tandem sequence repeats " with
Chronic Benzene Poisoning risk has close ties, and the risk of Chronic Benzene Poisoning can be evaluated.
Detailed description of the invention
Fig. 1 is the sequencing result of control group.
Fig. 2 is the sequencing result of case group.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Its net husky serial pillar blood DNA extracts the product that reagent is Beijing day bounties Bioisystech Co., Ltd.Long PCR Master Mix is the Beijing Pu Luomaige Bioisystech Co., Ltd.
" the 9bp sequences of 2 tandem sequence repeats " are as shown in the sequence 1 of sequence table." the missing of the 9bp sequence of 2 tandem sequence repeats
Type " is that " the 9bp sequences of 2 tandem sequence repeats " have lacked the 1st to 9 nucleotide.
Each experiment in following embodiments is to carry out under the premise of the informed consent of patient, and signature is known
Letter of consent.
Application of the deletion form of the 9bp sequence of 1,2 tandem sequence repeats of embodiment in evaluation Chronic Benzene Poisoning risk
One, the selection of case
To be diagnosed as patient 27 (male 6, average age 39.8 ± 9.6 years old of Chronic Benzene Poisoning；It women 21, puts down
Equal 40.7 ± 5.9 years old age) it is studied, and number consecutively p1-p27.Using 40 normal health examinees as control (male
24, average age 42.1 ± 8.3 years old；Women 16, average age 35.3 ± 11.3 years old), and number consecutively h1-h40.
Using the patient of above-mentioned 27 Chronic Benzene Poisonings as case group, 27 chronic benzene intoxication patients are all satisfied case group and receive
Enter standard.Case group is included in standard: being diagnosed as occupational chronic benzene poisoning (GBZ68-2013 " occupational benzene poisoning diagnosis mark
It is quasi- ").
As a control group by above-mentioned 40 normal health examinees, 40 normal health examinees are all satisfied control group and are included in
Standard.Control group is included in standard: 1. at loose ends property benzene contact history；2. without smoking, history of drinking history；3. without genetic disease or family history；
4. nearly 3 months without interior without infection history, medication history；5. without X-ray or other radiation contacts histories in nearly 3 months.
Two, the preparation of primer set
Primer set is by primers F: 5 '-TATGCCCTTTTCCTAACACT-3 ' (sequence 2 in sequence table) and primer R:5 '-
TTGGGTGATGAGGAATAGTGTAAG-3 ' (sequence 3 in sequence table) composition.
In primer set, the molar ratio of primers F and primer R are 1:1.
By Sangon Biotech (Shanghai) Co., Ltd. synthetic primer F and primer R.
Three, the deletion form of the 9bp sequence of 2 tandem sequence repeats evaluates Chronic Benzene Poisoning risk
1, the patient of 27 Chronic Benzene Poisonings and the peripheral blood 5mL of 40 normal health examinees are taken respectively, are placed in EDTA
In anticoagulant tube, then 100 × g is centrifuged 15min, collects supernatant (supernatant is platelet rich plasma).
2, after completing step 1, the supernatant is taken, reagent is extracted using the net husky serial pillar blood DNA in day and extracts DNA,
Obtain mitochondrial genomes DNA.
3, using mitochondrial genomes DNA as template, PCR expansion is carried out using the primers F of step 2 synthesis and primer R as primer
Increase, obtains pcr amplification product.
Reaction system is 20 μ L, by 10 μ LLong PCR Master Mix, 1 μ L primers F, 1 μ L primer R, 2 μ L
(about 5ng) mitochondrial genomes DNA and 6 μ L distilled waters composition；In initial reaction system, the concentration of primers F is 0.5 μM, primer R
Concentration be 0.5 μM.
Reaction condition: 95 DEG C of 10s；95 DEG C of 1min, 58 DEG C of 30s, 72 DEG C of 50s, 35 circulations；72℃10min.
4, after completing step 3, pcr amplification product is taken, is sequenced by Sangon Biotech (Shanghai) Co., Ltd..
Control group sequencing result is shown in Fig. 1 (NC_012920.1 is normal person's chondrioid genome reference sequences), case group
Sequencing result is shown in Fig. 2 (NC_012920.1 is normal person's chondrioid genome reference sequences).The result shows that number p1 is extremely compiled
It is all had in the patient and number h1 of the Chronic Benzene Poisoning of number the p18 extremely pcr amplification product of the normal health examinee of number h36
The 9bp sequence of 2 tandem sequence repeats；The patient and number h37 of the Chronic Benzene Poisoning of number p19 to number p27 extremely number h40 are just
The deletion form of the 9bp sequence of 2 tandem sequence repeats is all had in the pcr amplification product of normal physical examination of healthy population.
5, statistical method
The experimental data obtained using SPSS16.0 statistical software analytical procedure 4, comparison among groups are analyzed using Chi-square Test
(being that difference is statistically significant with P < 0.05)；Compare the 9bp of case group and control group using Logistic regression analysis model
The genotype of sequence, relative advantage ratio (OR), the genotype and Chronic Benzene Poisoning of 95% confidence interval (CI) Lai Hengliang 9bp sequence
The correlation of risk.
Statistical result is shown in Table 1: with the ratio of " deletion form of the 9bp sequence of 2 tandem sequence repeats " in case group
(33.3%) it is significantly higher than control group (10.0%), and the statistically significant (χ of difference of the two2=5.090, P=0.024),
OR value (95%CI) is 4.5 (1.21-16.62).The result shows that having the " 9bp of 2 tandem sequence repeats in mitochondrial genomes DNA
The risk that Chronic Benzene Poisoning occurs for the individual of the deletion form of sequence ", which is higher than in mitochondrial genomes DNA, has " 2 tandem sequence repeats
9bp sequence " individual occur Chronic Benzene Poisoning risk；It is described to be higher than for being higher than statistically.Therefore, " 2 series connection
The deletion form of duplicate 9bp sequence " and Chronic Benzene Poisoning risk have close ties, and the risk of Chronic Benzene Poisoning can be evaluated.
Table 1
According to above-mentioned steps, step 4 is replaced with into step B, other steps are constant, and obtained statistical result and use are upper
It is almost the same to state the statistical result that step obtains.Step B are as follows: after completing step 3, take pcr amplification product, carry out Ago-Gel
Electrophoresis detection.The patient and number h1 to number h36 that testing result shows the Chronic Benzene Poisoning of number p1 to number p18 are just
The size of the pcr amplification product of normal physical examination of healthy population be 738bp, then the patient of the Chronic Benzene Poisoning of number p1 to number p18 and
There are " the 9bp sequences of 2 tandem sequence repeats " in the mitochondrial genomes DNA of the normal health examinee of number h1 to number h36；
The PCR amplification of the patient and number h37 of the Chronic Benzene Poisoning of number p19 to number the z27 extremely normal health examinee of number h40
The size of product be 729bp, then number p19 to number p27 Chronic Benzene Poisoning patient and number h37 extremely number h40 just
There is " deletion form of the 9bp sequence of 2 tandem sequence repeats " in the mitochondrial genomes DNA of normal physical examination of healthy population；Electrophoresis detection knot
The conclusion that the sequencing result of fruit and step 4 obtains is completely the same.
<110>occupational disease precaution clinic, Shenzhen
<120>application of the mitochondrial genomes 9bp sequence gene polymorphism in evaluation benzene poisoning risk
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
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<400> 1
ccccctctac cccctcta 18
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 2
tatgcccttt tcctaacact 20
<210> 3
<211> 24
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 3
ttgggtgatg aggaatagtg taag 24
Claims (3)
1. for detect in mitochondria with " the 9bp sequences of 2 tandem sequence repeats " still " the 9bp sequence of 2 tandem sequence repeats lack
Application of the substance of mistake type " in the product of preparation evaluation Chronic Benzene Poisoning risk；
" the 9bp sequences of 2 tandem sequence repeats " are as shown in the sequence 1 of sequence table；
" deletion form of the 9bp sequence of 2 tandem sequence repeats " be " the 9bp sequences of 2 tandem sequence repeats " lacked the 1st to
9 nucleotide.
2. application as described in claim 1, it is characterised in that: described " for detecting to there are " 2 tandem sequence repeats in mitochondria
9bp sequence " still " deletion form of the 9bp sequence of 2 tandem sequence repeats " substance " be primer pair；The special primer
It is right, it is made of primers F and primer R；
The primers F is single strand dna shown in the sequence 2 of sequence table；
The primer R is single strand dna shown in the sequence 3 of sequence table.
3. primer pair described in claim 2 is preparing answering in the kit for evaluating Chronic Benzene Poisoning risk
With.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245347A (en) * | 2008-01-14 | 2008-08-20 | 暨南大学 | Chronic mild benzene poisoning related antigen special TCRV alpha12 sub-family Y gene sequence |
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2016
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245347A (en) * | 2008-01-14 | 2008-08-20 | 暨南大学 | Chronic mild benzene poisoning related antigen special TCRV alpha12 sub-family Y gene sequence |
Non-Patent Citations (4)
| Title |
|---|
| Mitochondrial Insertion–Deletion Polymorphism：Role in Disease Pathology;Sreelatha Komandur et al.;《GENETIC TESTING AND MOLECULAR BIOMARKERS》;20111231;第15卷(第5期);361-364 * |
| The Mitochondrial DNA 9-bp Deletion Polymorphism is a Risk Factor for Hepatocellular Carcinoma in the Chinese Population;Yiqi Jin et al.;《GENETIC TESTING AND MOLECULAR BIOMARKERS》;20121231;第16卷(第5期);330-334 * |
| 肾透明细胞癌线粒体DNA非D-loop区微卫星不稳定性与预后的关系;耿同会 等;《中华肿瘤防治杂志》;20160131;第23卷(第1期);21-24 * |
| 血小板特异性抗原基因与职业性慢性苯中毒的相关性分析;张艳芳 等;《中国输血杂志》;20150430;第28卷(第4期);364-366 * |
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