CN107475397A - A kind of method and its kit for detecting susceptible occupational noise deaf gene - Google Patents
A kind of method and its kit for detecting susceptible occupational noise deaf gene Download PDFInfo
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Abstract
The invention discloses a kind of method for detecting susceptible occupational noise deaf gene, it is included in the case group obtained in productive noise colony and control group, gather the DNA of its peripheral blood, mutational site GSTT1, GSTM1 and the GSTP1 common to three of GST carry out Classification Identification again, obtain susceptible occupational noise deaf gene i.e. GSTM1 deletion forms.Meanwhile the invention also discloses the kit of the susceptible occupational noise deaf gene, including GSTM1 amplimers and Albumin amplimers.The present invention carries out examination in Occupational Health Monitoring before worker carries out hilllock, finds, to the susceptible worker of noise, to avoid worker from being engaged in Noise Exposure post, to reach the purpose precisely prevented.
Description
Technical field
The invention belongs to biological gene detection technique field, more particularly to a kind of susceptible occupational noise deaf gene of detection
Method and its kit.
Background technology
At present, productive noise is one of occupational hazard the most serious, and exposed population group is extensive, and harm is serious,
With invisible and chronicity, be particularly easy to it is ignored, moreover, engineering prevention and control and individual protection are more difficult.Occupational noise
Caused hearing loss (NIHL) has turned into the occupational illness of the numerous labourers' health in serious threat China.Nearly 5 years
National report of occupational disease shows that occupational noise deaf case load occupies the 3rd in national occupational disease, is only second to pneumoconiosis and duty
Industry is poisoned, and in increased trend year by year.Due to the irreversibility of occupational noise deaf, lack effective remedy measures in addition,
Rehabilitation is expensive, therefore of crucial importance for the primary prevention of noise induced hearing loss.
The primary prevention method of occupational noise deaf mainly includes innovation technique, and using low noise acoustic equipment, installation sound insulation disappears
Sound facility, strengthen occupational health management, carry out personal protection etc., but because most enterprise's production technologies can not change, look forward to
The reasons such as industry occupational health management is not in place, it is bad for the preventive effect of occupational noise deaf.
The factor such as occupational noise deaf and environment, individual habits and customs and heredity has relation.Twin study is prompted,
Effect maximum of the inherent cause (referring mainly to genetic predisposition) in hearing loss can account for 70%~75%, and environmental factor accounts for 25%
~30%；The contribution of Noise Exposure accounts for 1/3 again in environmental factor.So if NIHL tumor susceptibility genes are can confirm that, just can be susceptible
Individual is screened before being exposed to high-noise environment, effectively reduces NIHL generation, this combination susceptible gene site
Primary prevention means so that the prevention to noise Susceptible population is more targeted, can mitigate family, enterprise, society significantly
Guarantee burden.
The content of the invention
The first object of the present invention is to provide a kind of method for detecting susceptible occupational noise deaf gene, first obtains and is exposed to
The case group and control group of productive noise colony, gather the DNA of its peripheral blood, then three to GST common mutational sites
GSTT1 (NM_000853, rs10549055), GSTM1 (NM_000561, rs10712361) and GSTP1 (NM_000852,
Rs1695 Classification Identification) is carried out, susceptible occupational noise deaf gene i.e. GSTM1 deletion forms is obtained, is made an uproar so as to realize from occupational
The worker susceptible to noise is being found on the deaf susceptible gene of sound, and examination is carried out in Occupational Health Monitoring, is making susceptible worker
Noise is avoided contact with, to reach the incidence of disease for reducing occupational noise deaf, protects the purpose of workers ' health.
The second object of the present invention is to provide a kind of kit for detecting susceptible occupational noise deaf gene, is surveyed using gene
Examination kit carries out examination in Occupational Health Monitoring before worker carries out hilllock, finds, to the susceptible worker of noise, to avoid worker from being engaged in
Noise Exposure post, to reach the purpose precisely prevented.
Technical scheme is as follows：
A kind of method for detecting susceptible occupational noise deaf gene, comprises the following steps：
(1) Screening Samples, case group and control group are obtained respectively；
(2) DNA sample of leucocyte is gathered respectively, and Classification Identification is carried out to GSTT1, GSTM1 and GSTP1, and primer is such as
Shown in lower, enter performing PCR amplification；
(3) after PCR amplifications, reimaging after electrophoresis；
(4) identification of amplified production, wherein, using 350bp fragment as albumin internal reference, there are 350bp fragment and 480bp
Fragment, then it is GSTT1 to illustrate the genotype；There are 350bp fragment and 480bp fragment, then illustrate that the genotype is
GSTM1；There are 329bp and 113bp fragment, then explanation is GSTP1 wild types, the fragment for having 216bp, 113bp, and 107bp, then
It is homozygous for GSTP1；There are 329bp, 216bp, 113bp and 107bp fragment, be then GSTP1 heterozygous；
(5) database is established using Epidate softwares, the calculating and analysis of all data use SAS softwares (9.1.3),
The analysis of continuous variable is examined using sided t, and the analysis of classified variable use bilateral Chi-square Test, and calculate its OR value with
95%CI, influence of the reciprocation of genotypic and environment factor to occupational noise deaf neurological susceptibility is analyzed with Multiple-Factor Model,
It is susceptible occupational noise deaf gene to obtain GSTM1 deletion forms.
Preferably, the experimental group of step (1) is that the ears high frequency threshold of audibility is more than or equal to 40dB, and the ears speech frequency threshold of audibility is more than or equal to
25dB, and meet the feature of typical noise induced hearing loss (Dosimeter is more serious than speech frequency hearing impairment, in " V " word
Type sink).
Preferably, the control group of step (1) is that the ears high-frequency average threshold of audibility is less than 40dB, and age and sex and case group
The worker of matching, in addition, case group worker and control group worker are substantially the shift workers with post.
Preferably, the DNA sample of step (2) is the DNA sample of EDTA anticoagulation cirumferential bloods,
Preferably, the PCR amplification system of step (3) is：
Preferably, the PCR amplification conditions of step (3) are：
Preferably, the GSTP1 digestion systems of step (2) are：
The invention also discloses a kind of kit for detecting susceptible occupational noise deaf gene, including GSTM1PCR amplifications to draw
Thing and Albumin pcr amplification primer things, described GSTM1PCR amplimers include the-GAACTCCCTGAAAAGC of forward primer 5 '
TAAAGC-3 ' and-the GTTGGGCTCAAATATACGGTGG-3 ' of reverse primer 5 '；Described Albumin pcr amplification primer things include
- the GCCCTCTGCTAACAAGTCCTAC-3 ' of the forward primer 5 ' and-GCCCTAAAAAGAAAATCGCCAATC-3 ' of reverse primer 5 '.
Compared with prior art, beneficial effects of the present invention are as follows：
The primary prevention of existing occupational noise hearing loss be mainly focused on control to noise (environmental factor) and
Prevention, avoids crowd's contact noise, the inadequate master of the odjective cause and enterprise's attention degree that can not change due to production technology
Reason is seen, the primary prevention effect to occupational noise deaf is unsatisfactory, and new cases are in ascendant trend, while many right year by year
The susceptible worker of noise is exposed to noise circumstance due to not passing through examination, causes the incidence of disease further to increase, especially prominent
, it is existing be not bound with accounted in occupational noise deaf occurrence cause 70% hereditary factors, many works susceptible to noise
People has also been engaged in Noise Exposure, and the present invention utilizes the Gene Susceptibility biomarker-GSTM1 deletion forms pair of occupational noise deaf
Occupational noise deaf is precisely prevented, and is a kind of primary prevention method of brand-new occupational noise deaf, while prepared
Into kit to carry out examination to Occupational Health Monitoring crowd in Occupational Health Monitoring, to find neurological susceptibility worker, reach essence
Quasi- prevention.
Brief description of the drawings
Fig. 1 is the gel electrophoresis figure of GSTT1 genotype, GSTM1 genotype and GSTP1 genotype.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate this hair
It is bright, rather than limit protection scope of the present invention.Those skilled in the art change according to what the present invention made in actual applications
Enter and adjust, still fall within protection scope of the present invention.
Cochlea is the very active organ of an energetic supersession, and when exposed to noise, cochlea needs substantial amounts of energy, but together
When can also produce substantial amounts of active oxy group (ROS).Active oxygen has damage to DNA, albumen, cell membrane etc., in general feelings
Endogenous anti-oxidative system (including superoxide dismutase, glutathione S-transferase, glutathione peroxide in condition servant's body
Compound enzyme, glutathione reductase and catalase) internal active oxygen can be removed, so as to maintain the normal work(of cochlea
Energy.When the endogenous anti-oxidative system miopragia in human body, hair cell is highly susceptible to injure, and Henderson D etc. are ground
Study carefully and also indicate that, the cellular oxidation that noise can be mediated by ROS stress mechanism damage inner ear hair cells so as to cause hearing loss.
Glutathione S-transferase (GST) is the important enzyme for being catalyzed xenobiotics phase II metabolic, can be catalyzed gluathione
Peptide is combined with electrophilic substrates, and so as to eliminate electrophilic xenobiontics and the damaging effect caused by oxidative stress, human body can
The GST of dissolubility at least four kinds of isodynamic enzymes：α, μ, P and δ.When α GST (GSTT1) or μ GST (GSTM1), P GST (GSTP1) base
During because of missing, it is impossible to corresponding enzyme is expressed, so as to influence its anti-oxidation function.The present invention is in the Chinese Han Population of more large sample size
The relation between GST three kinds of Common genes polymorphisms (GSTM1, GSTT1, GSTP1) and occupational noise deaf neurological susceptibility is inquired into,
Foundation is provided for the primary prevention of occupational noise deaf.
1) object and method
1.1 research object
We are with certain big machinery manufactory in unified inclusive criteria selection Jiangsu Province and the noise of certain chemical fibre group
Exposure 2344, wherein male worker 2047, women worker 297, inclusive criteria are：(1) contact noise 3 years with
On Han nationality worker；(2) now suffered from the disease without hypertension, high fat of blood etc., the Medical history such as no tympanitis, craniocerebral injury, earless
Drug toxicity takes history, without familial hereditary hearing impairment history and explosion deafness history；(3) high temperature, vibrations etc. are not contacted during operation
The chemically adverse factor such as physical adverse factor and organic solvent, heavy metal；(4) safeguard procedures such as earplug are not used.It is selected
The research object overwhelming majority entered is all to be worked always in the two factories, the health supervision data of resurrectionist's last decade and operation field
Institute's monitoring materials are all fairly perfect.The computational methods in worker's Noise Exposure length of service are time when worker departs from noise post simultaneously
(having disengaged from noise post) or the now time subtract (without departing from noise post is crossed) enters the progress operation of noise post for the first time
Time.
1.2 questionnaire
Using questionnaire is formatted, content includes：(1) general demographic data；(2) smoking, drink, ototoxic drug
Take history and everyday exposure situation；(3) occupational exposure (total length of service, Noise Exposure length of service etc.) and the pendant of safeguard procedures
Wear situation；(4) hypertension, high fat of blood, diabetes etc. now suffer from the disease；(5) medical history such as tympanitis, craniocerebral injury；(6) pinking
Property deaf and familial deafness history.All application forms (are informed by this problem group membership in the informed consent for obtaining research object
The basic conditions such as the research contents of content including this research, the biological specimen needed, personal information privacy) and signature after use face
The mode on opposite is filled in after inquiring.Effective 2140 parts of questionnaire is finally obtained altogether.
The hearing test of 1.3 research objects and on-site noise measurement
By ear nose larynx doctor with reference to GBZ 49-2007《On The Diagnostic Criterion of Occupational Noise Induced Deafness》And Specification will
Ask, in soundproof room of the background values noise less than 25dB (A), all objects are carried out with left and right ear 500,1,000,2,000,3,
000th, 4,000, the pure tone air conduction hearing threshold test of 6,000Hz totally 6 frequencies, all objects are required to depart from noise circumstance before being examined
At least more than 16 hours, pure tone test result pressed GB 7582-87《The AC threshold of acoustics-otology normal person and age and sex
Relation》Requirement carry out age and sex amendment, and calculate and listen threshold value.Because the worker of low frequency hearing impairment is according to state
Family's pertinent regulations are all by distancing Noise Exposure post, therefore the occupational noise deaf worker collected by this research is height
Frequency hearing loss.According to above-mentioned standard, research object of the ears high-frequency average threshold of audibility more than or equal to 40dB is defined as high frequency hearing
Worker's (object of observation) of loss, other workers are then defined as the normal worker of hearing.According to GBZ/T 189.8-2007《Work
Place physical factor measurement-noise》Requirement, the measurement of noise carried out in the case of normal production, and microphone should be placed on
The height (stance 1.5m, sitting posture 1.0m) of labourer ear, point to the direction of sound source.It is transaudient when wind speed is more than 3m/s
Device should wear fan housing, while avoid the interference of electromagnetic field as far as possible.Quest SoundPro type sound level meters are used in one-point measurement
(Quest, USA), three measuring points are selected when sound-filed simulation is uniform in workplace, and each measuring point measurement three times, is averaged
Value, workplace is divided into several sound level areas when sound-filed simulation is uneven, each sound level area selects two measuring points, each
Measuring point measures three times, averages.Quest Noise Pro-DL type multifunctional personals are used when measuring personal noise dose
Noise dose meter (Quest, USA), and worn using the method choice labourer of sampling, sampling requires to be in target sample
The contact noise that different posies should be included endanger highest and contact noise time most long worker, remaining worker random selection, often
It should all elect target sample when the labourer in individual post is less than 3 people as, 2 people are selected during 3-5 people as target sample, during 6-10 people
3 people are selected as target sample, select 4 people as target sample during more than 10 people.Measurement terminates rear immediate record measured value.Root
According to above-mentioned inclusive criteria, with reference to hearing test data and live Noise Exposure data, the last complete occupational of data collection altogether
Noise-induced deafness worker 444 (case groups), compare and matched for the ears high-frequency average threshold of audibility less than 40dB and age and sex with case
Worker, 445 altogether, case group worker and control group worker are substantially the shift workers with post.
1.4 DNA extraction and Genotyping
Using EDTA anticoagulant tubes collection research object peripheral blood 2ml, 3000r/min, centrifuge 5min, washed corpuscles and
Blood plasma, respectively -20 DEG C and -80 DEG C preservations.DNA is extracted from leucocyte with Tiangeng RNA isolation kit (non-centrifugation column type).And to GST
Three common mutational site GSTT1 (NM_000853, rs10549055), GSTM1 (NM_000561, rs10712361) and
GSTP1 (NM_000852, rs1695) carries out Classification Identification.The method of parting uses PCR-RFLP (PCRs-limit
Property fragment length polymorphism processed), design of primers is separately designed using software Primer premier 6.0 and with reference to pertinent literature
The corresponding primer of GSTTl, GSTMl, GSTP1 gene, then the primer is evaluated with Oligo6.0, draw on each primer
The nature parameters such as G/C content, Tm values, hairpin structure and primer dimer.Shanghai introvigen companies carry out primer synthesis and (drawn
One) thing sequence and primer size are shown in Table, it is 10uM/L that then each primer, which adds deionized water dissolving into concentration, and -20 DEG C of preservations are standby
With.Simultaneously we extracted in experiment sample 10% by an other people to genotyping result carry out blind checking, twice experiment knot
Fruit is completely the same.
Table one GSTT1, GSTM1, GSTP1 primer sequence and primer size
1.5 DNA amplification and digestion
The PCR amplification system (10ul systems) of one DNA sample
The mixture system prepared first vibrates mixing, rear to centrifuge the 3-5 seconds, and then withdraw mix 9.4ul adds 0.5ml's
In EP pipes, 0.6ul DNA sample to be measured is added, after the completion of sample-adding, cover EP pipes, number in sequence, be then placed in PCR
Amplification instrument carries out cyclic amplification.The condition of amplification is as follows：
The preparation of Ago-Gel.The Ago-Gel generally with 2% is verified, 2g agaroses+100mlTBE (1 ×), is put
Enter in flask and heated in micro-wave oven, generally require boiling three times, add 5ul ethidium bromides, then pour into gel grinding tool rapidly
In, glue tooth and glue comb are extracted after condensation, you can be loaded.
Electrophoresis：110V,15min.
Imaging：Gel is put into gel imaging system, ultraviolet photoetching 5s, preserves picture.GSTT1 genes and GSTM1 bases
Because can directly be judged according to band, if GSTP1 band is more satisfactory, remaining product can carry out digestion, if not
It is preferable then need to be loaded amplification again.
The preparation of GSTP1 digestion systems：
37 DEG C of oven overnights are put into after the digestion system prepared is numbered.
Glue：Prepared by 3% Ago-Gel, 3g agarose+100ml TBE (1 ×), be put into flask in micro-wave oven
Middle heating, generally require boiling three times, when having transparent bubble generation, add 5ul ethidium bromides, then pour into gel grinding tool rapidly
In, glue tooth and glue comb are extracted after condensation, you can be loaded.If old glue need not then add ethidium bromide.
Electrophoresis：90V, 20min.
Imaging：Gel is put into gel imaging system, ultraviolet photoetching 5s, preserves picture.
The identification of 1.6 amplified productions
The identification of GSTT1 genotype：350bp fragment is albumin internal reference, if 350bp fragment and 480bp
Fragment then illustrates that the genotype has type for GSTT1, if only 350bp fragment, for GSTT1 deletion forms, if all without
Then illustrate that DNA sample or experimental implementation are problematic.
The identification of GSTM1 genotype：350bp fragment is albumin internal reference, if 350bp fragment and 480bp
Fragment then illustrates that the genotype has type for GSTM1, if only 350bp fragment, for GSTM1 deletion forms, if all without
Then illustrate that DNA sample or experimental implementation are problematic.
The identification of GSTP1 genotype：If 329bp and 113bp fragment then illustrates for GSTP1 wild types (AA types)；
If 216bp, 113bp, and 107bp fragment are then homozygous (the GG types) of mutation；If 329bp, 216bp, 113bp and
It is then GSTP1 heterozygous (AG types) that 107bp fragment, which all exists,.Because 113bp and 107bp fragment difference is too small, this examination
Test middle gel electrophoresis to fail to separate, but remain to distinguish GSTP1 three kinds of genotype.
1.7 statistical analysis
Database is established using Epidate softwares, the calculating and analysis of all data use SAS softwares (9.1.3).Continuously
Property variable analysis using sided t examine, the analysis of classified variable uses bilateral Chi-square Test, and calculates its OR value and 95%
CI, influence of the reciprocation of genotypic and environment factor to occupational noise deaf neurological susceptibility is analyzed with Multiple-Factor Model.
1.8 result
By study find GSTM1 deletion forms be occupational noise deaf risk factors, when with environmental risk factors knot
The generation of occupational noise deaf can be aggravated after conjunction, be the susceptibility biomarkers of occupational noise deaf, therefore can be drawn using this
The method screening Susceptible population is utilized during the preceding physical examination on duty of thing genetic test box.
Present invention disclosed above preferred embodiment is only intended to help and illustrates the present invention.Preferred embodiment is not detailed
All details are described, it is only described embodiment also not limit the invention.Obviously, according to the content of this specification,
It can make many modifications and variations.This specification is chosen and specifically describes these embodiments, is to preferably explain the present invention
Principle and practical application so that skilled artisan can be best understood by and utilize the present invention.The present invention is only
Limited by claims and its four corner and equivalent.
This project is between -2015 years 2014 in the occupational health prison before will contacting hilllock of Nanjing occupational disease precaution clinic
In shield, filter out about more than 500 example workers and be not suitable for being engaged in Noise Exposure, protect the health of worker.
Claims (8)
- A kind of 1. method for detecting susceptible occupational noise deaf gene, it is characterised in that comprise the following steps：(1) Screening Samples, case group and control group are obtained respectively；(2) DNA sample of leucocyte is gathered respectively, and Classification Identification, the following institute of primer are carried out to GSTT1, GSTM1 and GSTP1 Show, enter performing PCR amplification；(3) after PCR amplifications, reimaging after electrophoresis；(4) identification of amplified production, wherein, using 350bp fragment as albumin internal reference, there are 350bp fragment and 480bp piece Section, then it is GSTT1 to illustrate the genotype；There are 350bp fragment and 480bp fragment, then it is GSTM1 to illustrate the genotype；Have 329bp and 113bp fragment, then explanation is GSTP1 wild types, and the fragment for having 216bp, 113bp, and 107bp, then be GSTP1 It is homozygous；There are 329bp, 216bp, 113bp and 107bp fragment, be then GSTP1 heterozygous；(5) database is established using Epidate softwares, the calculating and analysis of all data use SAS softwares (9.1.3), continuously Property variable analysis using sided t examine, the analysis of classified variable uses bilateral Chi-square Test, and calculates its OR value and 95% CI, influence of the reciprocation of genotypic and environment factor to occupational noise deaf neurological susceptibility is analyzed with Multiple-Factor Model, is obtained GSTM1 deletion forms are susceptible occupational noise deaf genes.
- 2. the method for the susceptible occupational noise deaf gene of detection according to claim 1, it is characterised in that step (1) Experimental group is that the ears high frequency threshold of audibility is more than or equal to 40dB, and the ears speech frequency threshold of audibility is more than or equal to 25dB, and meets typical noise-induced The feature of hearing loss.
- 3. the method for the susceptible occupational noise deaf gene of detection according to claim 2, it is characterised in that step (1) Control group is that the ears high-frequency average threshold of audibility is less than 40dB, and the worker that age and sex match with case group, in addition, case group work People and control group worker are substantially the shift workers with post.
- 4. the method for the susceptible occupational noise deaf gene of detection according to claim 1, it is characterised in that step (2) DNA sample is the DNA sample of EDTA anticoagulation cirumferential bloods.
- 5. the method for the susceptible occupational noise deaf gene of detection according to claim 1, it is characterised in that step (3) PCR amplification system is：。
- 6. the method for the susceptible occupational noise deaf gene of detection according to claim 1, it is characterised in that step (3) PCR amplification conditions are：。
- 7. the method for the susceptible occupational noise deaf gene of detection according to claim 1, it is characterised in thatThe GSTP1 digestion systems of step (2) are：。
- A kind of 8. kit for detecting susceptible occupational noise deaf gene, it is characterised in that including GSTM1 pcr amplification primers thing and Albumin pcr amplification primer things, described GSTM1 pcr amplification primers thing include the-GAACTCCCTGAAAAGCTAA of forward primer 5 ' AGC-3 ' and-the GTTGGGCTCAAATATACGGTGG-3 ' of reverse primer 5 '；Described Albumin pcr amplification primers thing is included just To-the GCCCTCTGCTAACAAGTCCTAC-3 ' of primer 5 ' and-GCCCTAAAAAGAAAATCGCCAATC-3 ' of reverse primer 5 '.
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| CN113033962A (en) * | 2021-03-01 | 2021-06-25 | 四川大学华西第四医院 | Occupational disease and danger early warning method, system and terminal based on environment real-time big data |
| CN113736872A (en) * | 2020-05-29 | 2021-12-03 | 上海交通大学医学院附属第九人民医院 | Biomarker for predicting susceptibility risk of noise-induced hearing loss and application |
| CN116356014A (en) * | 2023-04-18 | 2023-06-30 | 江苏省疾病预防控制中心(江苏省公共卫生研究院) | Biological marker for noise susceptibility hearing impairment of workers and application thereof |
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| 沈欢喜等: "谷胱甘肽硫转移酶T1、M1、P1基因多态性与汉族人群职业性噪声聋易感性的关系", 《环境与职业医学》 * |
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| CN113736872A (en) * | 2020-05-29 | 2021-12-03 | 上海交通大学医学院附属第九人民医院 | Biomarker for predicting susceptibility risk of noise-induced hearing loss and application |
| CN113736872B (en) * | 2020-05-29 | 2023-07-14 | 上海交通大学医学院附属第九人民医院 | Biomarker for predicting susceptibility risk of noise hearing loss and application |
| CN113033962A (en) * | 2021-03-01 | 2021-06-25 | 四川大学华西第四医院 | Occupational disease and danger early warning method, system and terminal based on environment real-time big data |
| CN113033962B (en) * | 2021-03-01 | 2023-11-24 | 四川大学华西第四医院 | Occupational disease hazard early warning method, system and terminal based on environment real-time big data |
| CN116356014A (en) * | 2023-04-18 | 2023-06-30 | 江苏省疾病预防控制中心(江苏省公共卫生研究院) | Biological marker for noise susceptibility hearing impairment of workers and application thereof |
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