CN105200045B - The nucleotides special to vibrio fluvialis O11, O14, O16 and O17 and its application - Google Patents
The nucleotides special to vibrio fluvialis O11, O14, O16 and O17 and its application Download PDFInfo
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Abstract
The present invention relates to include SEQ ID NO to vibrio fluvialis O11, O14, the special nucleotides of O16 and O17 serotypes and its application, the nucleotides:At least one of nucleotides shown in 18.These nucleotides can be used for preparing detection vibrio fluvialis PCR kit and genetic chip.This laboratory is to vibrio fluvialis O2, O4, O13, O15 and O18 serotype specific nucleotide applications patent, number of patent application CN201410158813.The present invention to vibrio fluvialis O11, O14, the special nucleotides of O16 and O17 serotypes and PCR kit comprising the nucleotides, genetic chip improve sensitivity and the efficiency of detection because product length greatly shortens, it is practical, PCR kit compound method is easy, and detection cycle is short, speed is fast, workable, it is easy to industrialization production, and testing cost is relatively low；Accuracy is high；High sensitivity.
Description
Technical field
The present invention relates to vibrio fluvialis O11, O14, the special nucleotides of O16 and O17 serotypes, more particularly to river
The special nucleotides of individual gene and its application in vibrios O11, O14, O16 and O17 serotype O antigen gene clusters.
Background technology
Vibrio fluvialis is Gram-negative bacteria, is that the main of marine environment inhabites one of bacterium, is widely present in river and goes out
Haikou environment waters, and one of the mankind and the main bacterial pathogen of aquatile, can play a variety of cultivation such as fish, shrimp, shellfish
The disease of animal, serious economic loss is brought to aquaculture.Vibrio fluvialis can also cause the mankind serious by various foods
Epidemic diarrhea, be the kinds of pathogenic vibrio that comma bacillus and vibrio parahaemolytious are only second in vibrio, it is considered to be Yi Zhongquan
The new pathogen of the Zoonosis of ball.Its parting and identification are one of important prerequisites that ocean strain library is established.
Typing of bacteria mainly has traditional phenotypic approach, serological method and method for identifying molecules with authentication method.So
And with the development of molecular biology, traditional serotype and authentication method there is it is certain the problem of, as serotype this
Kind of diagnostic method needs substantial amounts of antiserum, and antiserum general classes are not complete, lazy weight, substantial amounts of antiserum preparing and
It is difficult there is also some in storage.Time-consuming, sensitivity is low, loss is high for another aspect serotype method, accuracy is poor, no
Cross reaction is frequently present of between antiserum caused by same O antigens.Therefore, the serum mirror based on Protocols in Molecular Biology is established
Determining method turns into developing direction.
The Molecular Identification of vibrio fluvialis is increasingly valued by people, and turns into vibrio fluvialis strain and the weight of plant type identification
Will foundation, therefore many new bacterium also produce.For vibrios, biochemical reaction is mainly the performance of various zymograms, belongs to outside shape
The content of state, what the similitude of nucleic acid was only vibrios kind defines most basic, most direct feature.Therefore, the parting of vibrio fluvialis
With identifying that most effective, most direct mode should trigger from the research of nucleic acid, by comparing nucleic acid（Including genome and nucleic acid piece
Section）Similitude determine the ownership of vibrio fluvialis.
In recent years, increasing molecular engineering is used for parting, identification, detection and the disease screening of pathogen, including turns
Record spacer region (ITS) sequence analysis, random amplification length polymorphism (RAPD) is analyzed, rDNA Restriction Fragment Length is more
State property (RFLP) analysis etc..Molecular biology method cannot be only used for the rapid serum parting examination of vibrio fluvialis, stable mirror
The deficiency of phenotypic characteristic authentication method can be made up by determining result.Compared with traditional sensing techniques, it is anti-that these are based on polymerase chain type
Answer (PCR) molecular detection technology, it is not necessary to by processes such as the separation of pathogen, pure cultures, and with it is quick, sensitive,
The advantages that high specificity.
Polymerase chain reaction technology (Polymerase chain reaction, abbreviation round pcr) is examined as microorganism
Survey technology is currently accepted and promoted, and the technology has high flux, detection speed fast, specific relative to traditional method
By force, the advantages that high sensitivity, simply pre- increasing bacterium need to be only carried out to sample or increases bacterium process, then is prepared carefully by centrifuging and cracking
Bacterium DNA profiling, it is possible to expand target sequence during the PCR under the mediation of high specific primer, reaching in detection sample is
The no purpose containing invasive organism to be measured.PCR amplification procedure only needs 1 and a half hours.This to inspection and quarantine department and
Clinical examination undoubtedly greatly improves operating rate and reduces job costs.
No matter from the perspective of internal and international, serum type is quickly and accurately identified, is the prevention and control of vibrio fluvialis
It is highly important to provide effective technical support.
The content of the invention
Object of the present invention is to provide the present invention relates to special to vibrio fluvialis O11, O14, O16 and O17 serotypes
Nucleotides, it is characterised in that described nucleotides has：
1）SEQ ID NO:At least one of nucleotides shown in 1-8；
2）With SEQ ID NO:At least one of nucleotides of nucleotide complementary shown in 1-8；Described SEQ ID
NO:1-8 is as follows：
It is described present invention also offers a kind of PCR kit, including PCR primer, dNTP, buffer solution and archaeal dna polymerase
PCR primer is such as SEQ ID NO:At least one of nucleotides shown in 1-12.Described kit, in addition to following examination
Agent：10 mM dNTP 30μl；The μ l of 10 × enzyme spcificity reaction buffer 50；The μ l of 5U/ μ l hot resistant DNA polymerases 5；Primer mixes
The μ l of thing 10；The μ l of positive reference substance 10；The μ l of negative controls 10；ddH2O 5ml。
Wherein described PCR primer is preferably described such as SEQ ID NO:At least one of nucleotides shown in 1-8.
The present invention further discloses to vibrio fluvialis O11, O14, the special SEQ ID NO of O16 and O17 serotypes:1-8
Nucleotides is preparing PCR kit, gene core for detecting vibrio fluvialis vibrio fluvialis O11, O14, O16 and O17 serotype
Application in terms of piece or microarray.
Vibrio fluvialis of the present invention can sample in running water, river water, seawater culture crude extract, or river
Flow crude extract of pure culture of vibrios etc..
Collecting vibrio fluvialis extraction genome is prepared using conventional method.
For the PCR kit of vibrio fluvialis, whole detecting step includes sample pretreatment-amplification-electrophoresis detection knot
Fruit.Reagent required for primer and PCR reaction systems has been previously added in amplification pipe, and user only need to be by pretreated sample
Add amplification pipe and start amplified reaction, simple and quick completion detection work.
The present invention also provides a kind of genetic chip, including solid phase carrier is visited with the oligonucleotides being fixed on solid phase carrier
Pin, wherein the oligonucleotide probe includes above-mentioned nucleotides；Wherein described nucleotides is preferably such as SEQ ID NO:1-8 institutes
The nucleotides shown.
The present invention also provides a kind of micro- array, and it includes above-mentioned nucleotides；Wherein described nucleotides is preferably such as SEQ
ID NO:Nucleotides shown in 1-8.
The present invention further discloses prepared by vibrio fluvialis O11, O14, the special nucleotides of O16 and O17 serotypes
Application in terms of for detecting vibrio fluvialis PCR kit.Answering in terms of preparation is used to detect the genetic chip of vibrio fluvialis
With.Application in terms of preparation is used to detect vibrio fluvialis microarray.Described detection vibrio fluvialis refers to that detection causes sternly
Suffer from diarrhoea again, septicaemia, enterocolitis, meningitis, entophthamia, the bacterium of marine organisms infection.
It is disclosed by the invention to vibrio fluvialis O11, O14, the special nucleotides of O16 and O17 serotypes and prior art phase
Than, the invention has the advantages that：
（1）It is practical
A kind of PCR reaction systems that the present invention establishes, it can detect vibrio fluvialis, there is provided the used spy of serotype detection
Different primer, clinical samples can be detected using the PCR method.
（2）Accuracy is high
The present invention is reacted by the PCR of the special gene of each serotype to vibrio fluvialis, and each sample obtains an entry
Band, purpose fragment will be obtained and compared with known length, it is possible to obtain the serotype belonging to vibrio fluvialis.
（3）Testing cost is relatively low
Food Hygiene Surveillance, environmental monitoring, the still field such as product supervision and inspection quarantine, and for other not can be applied to
Technology mode is provided with pathogenic bacteria detection combination.
Brief description of the drawings
Fig. 1 represents O11 serotypes of the present inventionWZYOther serotype reference cultures of gene P1 and P2 primer detection vibrio fluvialis
Electrophoresis result figure,WZY genes P11 and P12The screening of primer, purpose band 109bp, remaining serotype do not have any
Band, specific bacterial strain information are shown in Table 2；
Fig. 2 represents O11 serotypes of the present inventionWZY genes P1 and P2The species specific identification electrophoresis result figure of primer, wherein
WithWZY genes P1 and P2Primer detection 6 plants of vibrios and 1 plant of detection of Salmonella, 1 plant of Escherichia coli, without any band, specific bacterium
Strain information is shown in Table 2；
Fig. 3 represents other serotype reference cultures of O14 serotypes WZY gene P3 and P4 primer detections vibrio fluvialis of the present invention
Electrophoresis result figure, the screening of WZY gene P7 and P8 primers, purpose band 105bp, remaining serotype do not have any band.
Specific bacterial strain information is shown in Table 2
Fig. 4 represents the species specific identification electrophoresis result figure of O14 serotypes WZY gene P3 and P4 primers of the present invention, wherein
6 plants of vibrios and 1 plant of detection of Salmonella, 1 plant of Escherichia coli are have detected, without any band.Specific bacterial strain information is shown in Table 2.
Fig. 5 represents other serotype reference cultures of O16 serotypes WZY gene P5 and P6 primer detections vibrio fluvialis of the present invention
Electrophoresis result figure, the screening of WZY gene P5 and P6 primers, purpose band 100bp, remaining serotype do not have any band,
Specific bacterial strain information is shown in Table 2；
Fig. 6 represents the species specific identification electrophoresis result figure of O16 serotypes WZY gene P5 and P6 primers of the present invention, wherein
6 plants of vibrios and 1 plant of detection of Salmonella, 1 plant of Escherichia coli with WZY gene P5 and P6 primer detections, without any band, specific bacterium
Strain information is shown in Table 2；
Fig. 7 represents other serotype reference cultures of O17 serotypes WZY gene P7 and P8 primer detections vibrio fluvialis of the present invention
Electrophoresis result figure, the screening of WZY gene P7 and P8 primers, purpose band 114bp, remaining serotype do not have any band,
Specific bacterial strain information is shown in Table 2；
Fig. 8 represents O17 serotypes of the present inventionWZY genes P7 and P8The species specific identification electrophoresis result figure of primer, wherein
WithWZY genes P11 and P12Primer detection 6 plants of vibrios and 1 plant of detection of Salmonella, 1 plant of Escherichia coli, without any band, specifically
Bacterial strain information is shown in Table 2；
O11, O14 are used in Fig. 9 expressions respectively, and the amplification of O16 and O17 special primers corresponds to the electrophoresis result figure of serotype, specifically
Bacterial strain information is shown in Table 2；
Wherein：O11, O14, O16 and O17 and negative control (-) represent corresponding serotype primer amplification, first swimming lane
H and last swimming lane are 2000bp ladder marker.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as Sambrook et al., molecular cloning：Laboratory manual（New York: Cold Spring Harbor Laboratory
Press,1989）Described in condition.Wherein vibrio fluvialis derives from Japan Collection of Microorganisms
（JCM）.
Embodiment 1：The extraction of genome
37 DEG C of nutrient broth medium culture vibrio fluvialises, collect bacterium, and extraction genome comprises the following steps that：
Cell is resuspended with 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, 37 DEG C incubate 20 minutes,
Then the lysozyme for adding 10ul 10mg/ml continues insulation 20 minutes.3ul 20mg/ml Proteinase K, 15ul is added afterwards
10%SDS, 50 DEG C incubate 2 hours, add 3 ul 10mg/ml RNase, and 65 DEG C incubate 30 minutes.Isometric phenol is added to extract
Mixture, takes supernatant, then with isometric phenol：Chloroform：Isoamyl alcohol（25：24：1）Solution extracts twice, takes supernatant, then use
Isometric ether extraction with remove remnants phenol.Supernatant precipitates DNA with 2 times of volume ethanols, rolls out DNA with glass fiber and is used in combination
70% ethanol washes DNA, and finally DNA is resuspended in 30ul TE.Genomic DNA is detected by 0.4% agarose gel electrophoresis.
Embodiment 2:Sequence is decoded
The genome of vibrio fluvialis O11, O14, O16 and O17 serotype reference culture is extracted, passes through Solexa pair-
End sequencing technologies carry out the sequence that genome sequencing obtains the serotype to each serotype genome of vibrio fluvialis, use
Blast and PSI-Blast carries out sequence alignment, carries out transmembrane structure prediction using the program of TMHMM 2.0, uses
ClustalW program carry out sequence alignment and screening is guarded and specific gene fragment, final to obtain each serum of vibrio fluvialis
The O antigen gene clusters sequence and decoding result of type.
Embodiment 3：Design of primers
The O antigen gene cluster sequences of each serotypes of vibrio fluvialis O11, O14, O16 and O17 are that this laboratory is tested oneself,
Analyzed by comparing, we choose the relatively low gene specific area of Blast comparison result identity and similarity values
Section design primer.Wherein O11 serotypeswzyGene comparison result identity values and similarity values are 48% and 70%；O14
SerotypewzyGene comparison result identity values and similarity values are 55% and 74%；O16 serotypeswzyGene
Comparison result identity values and similarity values are 44% and 63%, O17 serotypewzyGene comparison result identity values
It is 41% and 63% with similarity values.So each serotype chooses above-mentioned corresponding spy of the gene as the serotype respectively
Different target gene, special primer is separately designed for the gene specific section of each serotype.
Design of primers is the core of the invention.Said gene importing Primer Premier 5 are carried out into primer to set
Meter, each gene design pair of primers, there is single goal band.
BLAST is carried out after design of primers in Genbank, the primer of design can not be with the sequence phase of other nearly edge bacteriums
It is too high like property, so ensure that the primer is only expanded in the precalculated position of oneself, without with other nearly edge bacterium or
Nearly edge bacterium in the environment of collect specimen does not produce positive reaction.This point is for avoiding the generation and experiment of non-specific band
Success or failure are particularly significant.
The primer designed is as shown in table 1.
Table 1 is used for PCR primer sequence
Embodiment 4：The screening of special primer
It has collected the reference culture of vibrio fluvialis O11, O14, O16 and O17 serotype and the standard of other 14 kinds of serotypes
Each one plant of bacterial strain, the vibrio fluvialis of 11 plants of non-partings, 6 plants of vibrio other bacterial strains, 1 plant of Salmonella strain and 1 plant of large intestine
Bacillus strain verifies the specificity of primer, and strain number and source are shown in Table 2.
Table 2 is used for the bacterial strain of specific detection
The PCR system that identified for genes primer screening is used is 5 μM of μ l of primer 0.4,10 × enzyme spcificity reaction buffers 2.5
μ l, the μ l of 10mM dNTP 0.25, the μ l of 5U/ μ l hot resistant DNA polymerases 0.2 and 3 μ l testing sample template to 0.2ml thin-walled
In PCR pipe, last ddH2O is supplied to 25 μ l.All primers all obtain positive findings in each corresponding serotype, at it
Any PCR primer band is not obtained in his group.
The temperature and time of denaturation of the reaction cycle parameter in PCR instrument including DNA, renaturation, extension in the step,
Cycle-index, it is specially：
Early stage is denaturation is reached required temperature and a circulation of required processing procedure early stage is 95 DEG C, 5
Minute；
Denaturation temperature and time are 95 DEG C, 45 seconds；
Renaturation temperature and time is 55 DEG C/68 DEG C, 1 minute；
Elongating temperature and time are 72 DEG C, 1 minute；
Denaturation, renaturation, the cycle-index of extension are 35 circulations；
The temperature and time that a circulation is carried out to stablize amplified production is 72 DEG C, 5 minutes
Wherein, O2 uses 55-58 DEG C using 61 DEG C of amplifications, O13 using 68 DEG C of amplifications, O4 using 55-65 DEG C of amplification, O15
Amplification, O18 use 65 DEG C of amplifications.
Above-mentioned steps electrophoresis amplified production in electrophoresis equipment, record concretely comprising the following steps for result：
2~5 μ l amplified productions and 6 × bromophenol blue sample-loading buffer are taken with 1：1 volume ratio mixing；
Mixed liquor is splined on 1.0% Ago-Gel；
By agarose gel electrophoresis 120v voltage stabilizings electrophoresis about 20 minutes, compareed with DL500 Marker；
Observe and record result.
The work of primer screening terminates substantially after being reacted by basic PC R, and necessary length adjustment is to W-response bar
Part is influenceed less, and the primer sequence used in the present invention is all summarised in table 1.
Table 1 is used for PCR primer sequence
Embodiment 5：
The preparation of PCR detection kit and application (it should be noted that：The mixing of all primers refers both to detect in this patent
The mixing of the upstream and downstream primer of a certain specific serovar.That is P1 is O11. P3 and P4 mixing with the P2 mixing PCR products come out
Detection is O14, P5 and P6 hybrid detections go out O16, P7 and P8 hybrid detections go out O17）；
1st, the composition of PCR kit：
dNTP(10mM) 30μl；
The μ l of 10 × Buffer (10 × enzyme spcificity reaction buffer) 50；
Taq polymerase（5U/ μ l hot resistant DNA polymerases） 5μl；
PCR primer mixture（5μM） 10μl；
The μ l of positive reference substance (KP) 10；
Negative controls（KN） 10μl；
ddH2O 5ml；
Each kit can be used for 10 samples of detection.
Wherein 10 × Buffer, dNTP, Taq polymerase are provided by precious bioengineering Co., Ltd；Primer mixture is certainly
The sequence of row design is supplied to Shanghai Ying Jun biotech companies to synthesize；Positive reference substance, negative controls and ddH2O is by us
Voluntarily prepare.
2nd, instrument and equipment
Wherein 10 × Buffer, dNTP, Taq polymerase are provided by Shanghai life work；Primer mixture is the sequence of designed, designed
Row are supplied to Shanghai Ying Jun biotech companies to synthesize；Positive reference substance, negative controls and ddH2O are voluntarily prepared by us.
The equipment PCR instrument of experiment（Also known as DNA thermal cycling amplification instrument）, electrophoresis equipment（Including electrophoresis apparatus and electrophoresis tank）, gel imaging
Instrument, -20 DEG C of refrigerators, supercentrifuge, micropipettor and 0.2ml PCR light-wall pipes.
3rd, the use instantiation of PCR kit
PCR detection method using above-mentioned PCR kit detection vibrio fluvialis comprises the following steps：
（1）Extract environmental sample template to be measured；
（2）In PCR light-wall pipes addition, dNTP, 10 × Buffer, Taq polymerase, primer, testing sample template and
DdH2O is mixed；
（3）The mixture mixed in thin-walled PCR pipe is expanded in PCR instrument；
（4）The electrophoresis amplified production in electrophoresis equipment, record result；
（5）Analyze and carry out result judgement.
Environmental sample template in above-mentioned steps (1) is the crude extract of the culture of running water, river water, seawater etc., or
It is the crude extract or pure dna of the pure culture of vibrio fluvialis, or positive reference substance and negative controls.
Above-mentioned steps（1）In the extracting method of environmental sample template be：
1.5ml cultures are taken, is centrifuged 1 minute under the conditions of 12000rpm, removes supernatant；
Take 500 μ l ddH2Precipitation is resuspended in O, is centrifuged 5 minutes under the conditions of 8000rpm, removes supernatant, dries；
Take 100 μ lddH2Precipitation, water-bath 10 minutes in 100 DEG C of boiling water are resuspended in O；
It is placed on ice after 10 minutes, is centrifuged 2 minutes under the conditions of 12000rpm again；
5. 3 μ l middle layer supernatants are taken as pcr template
Above-mentioned steps（3）In PCR instrument on the denaturation of reaction cycle parameter including DNA, renaturation, the temperature of extension and when
Between, cycle-index, be specially：
Early stage is denaturation is reached required temperature and a circulation of required processing procedure early stage is 95 DEG C, 5
Minute；
Denaturation temperature and time are 95 DEG C, 45 seconds；
Renaturation temperature and time is 55 DEG C/68 DEG C, 1 minute；
Elongating temperature and time are 72 DEG C, 1 minute；
Denaturation, renaturation, the cycle-index of extension are 35 circulations；
The temperature and time that a circulation is carried out to stablize amplified production is 72 DEG C, 5 minutes.
Above-mentioned steps（4）The electrophoresis amplified production in electrophoresis equipment, record concretely comprising the following steps for result：
2~5 μ l amplified productions and 6 × bromophenol blue sample-loading buffer are taken with 5：1 volume ratio mixing；
Mixed liquor is splined on 1.0% Ago-Gel；
By agarose gel electrophoresis 120v voltage stabilizings electrophoresis about 20 minutes, compareed with DL500 Marker；
Observe and record result.
The present invention by configure a kind of detectable vibrio fluvialis can industrialization production PCR kit, by PCR detection sides
The component that method needs to use is combined, in use, extraction testing sample, while can by relatively simple operation sequence
To carry out quick, sensitive, easy detection, the dosage of each component and concentration are experiment gained in kit, with the kit
Testing equipment used in detection vibrio fluvialis is simple, and testing cost is low.
The use of positive and negative controls purpose is to be used for Quality Control whole operation process, to draw accurate judgement.
If containing vibrio fluvialis purpose O antigenic types, the bar with positive reference substance same position is observed that from electrophoresis result
Band；If not containing vibrio fluvialis purpose O antigenic types, do not have this band as negative controls.
Amount of reagent in kit used in one-time detection experiment of the present invention see the table below shown in 3, and DNA profiling amount is 3 μ l
Amount of reagent in kit used in the experiment of the one-time detection of table 3
Hot resistant DNA polymerase in the present invention is Taq enzyme.
Above-mentioned positive reference substance be have determined that be each O antigenic types of vibrio fluvialis sample, negative controls are then
It is not the sample of vibrio fluvialis through laboratory determination.
If this PCR kit bacteria suspension of vibrio fluvialis enters performing PCR amplification, with extracted obtained DNA as template
Acquired results are always.Susceptibility and specific indifference, so, the extraction step of template DNA can be saved, enables operating method
Simplify.Meanwhile compared to routine biochemistry detection method for, used by this method testing sample can directly be clinical sample training
Nutrient solution, or simple separation culture is carried out to detection sample and can be carried out detecting, thus save manpower and materials.
4th, the offer of testing sample have collected vibrio fluvialis O11, O14, O16 and O17 serotype reference culture, 6 plants of vibrios
Belong to the specificity of other bacterial strains, 1 plant of Salmonella strain and 1 plant of coli strain checking primer, strain number and source
It is shown in Table 2.
SEQUENCE LISTING
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Claims (3)
1. the nucleotide combination special to vibrio fluvialis O11, O14, O16 and O17 serotype, it is characterised in that described nucleotides
It is combined as：SEQ ID NO:Nucleotides shown in 1-8；Nucleotides therein is used to prepare detection vibrio fluvialis O11, O14, O16
With O17 serotype PCR kits；The vibrio fluvialis sample in running water, river water, seawater culture crude extract,
Or the crude extract of the pure culture of vibrio fluvialis.
2. one kind is included as claimed in claim 1 to vibrio fluvialis O11, O14, the special nucleotides group of O16 and O17 serotypes
The PCR kit of conjunction, it is characterised in that including PCR primer, dNTP, buffer solution and archaeal dna polymerase, the PCR primer is such as SEQ
ID NO:Nucleotides shown in 1-8.
3. a kind of making to vibrio fluvialis O11, O14, the special nucleotide combination of O16 and O17 serotypes as claimed in claim 1
It is ready for use on the application in terms of detection vibrio fluvialis microarray；Wherein described detection vibrio fluvialis refers to that detection causes serious abdomen
Rush down, septicaemia, enterocolitis, meningitis, entophthamia, the bacterium of marine organisms infection.
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| CN101558168A (en) * | 2006-10-03 | 2009-10-14 | 国立大学法人岐阜大学 | Detection of bacterium by utilizing DnaJ gene and use thereof |
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| 两种重要食源性致病菌0血清型分子分型系统的建立、两株不同宿主来源的大肠杆菌比较基因组学和转录组学研究;郭丹;《中国博士学位论文全文数据库（电子期刊）医药卫生科技辑》;20140615;全文 * |
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