CN103602753A - Method and kit for detecting single nucleotide polymorphism site of LBP (Lipopolysaccharide-Binding Protein) genetic label - Google Patents
Method and kit for detecting single nucleotide polymorphism site of LBP (Lipopolysaccharide-Binding Protein) genetic label Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology and particularly relates to a method and a kit for detecting a single nucleotide polymorphism site of an LBP (Lipopolysaccharide-Binding Protein) genetic label. The method comprises the following steps: (1) designing a specific amplification primer and a sequencing primer; (2) carrying out PCR (Polymerase Chain Reaction) amplification on DNA (Deoxyribonucleic Acid) of a specimen to be detected by using the specific amplification primer; (3) sequencing by using a pyrosequencing method. According to the method and the kit, an SNP (Single Nucleotide Polymorphism) gene type of the LBP genetic label can be detected in a rapid, simple and convenient, accurate, efficient, practical and economical manner; the requirements of actual work of clinical examination can be met, so as to be good for detecting and predicating happening and developing risks of diseases by SNP of the LBP genetic label.
Description
Technical field
The invention belongs to biology field, be specifically related to be applicable to pcr amplification and in conjunction with tetra-sodium sequencing technologies, detect method and the test kit of LBP gene label list nucleic acid pleomorphism site.
Background technology
Tobias in 1986 first separated from rabbit anteserum, be purified into lipopolysaccharide binding protein (Lipopolysaccharide binding protein, LBP), LBP belongs to I type acute phase reactive protein, people LBP albumen is comprised of 481 amino acid, and by typical 25 signal peptides that amino acid forms of secreted protein.LBP albumen relative molecular mass is 60ku, and protein portion is comprised of the single chain polypeptide of a 50ku, and its plasma concentration is 2～7mg/L, and after LPS induction acute reaction, blood plasma LBP concentration can obviously improve.LBP is mainly synthetic at liver, there are some researches show, the extrahepatic tissues such as lung, intestines, kidney, spleen are synthetic LBP also, and wherein lung is the maximum organ of extrahepatic tissue LBP mrna expression amount.In adult respiratory distress syndrome (ARDS) patient's bronchoalveolar lavage fluid, LBP concentration is 64 times of normal people.LBP is not only a kind of plasma proteins, also can be used as a kind of transmembrane protein.This film can embed in monocytic after birth in conjunction with LBP itself, promote lipopolysaccharides (LPS) to embed wherein simultaneously, anti-LBP monoclonal antibody does not suppress the embedding of LBP, but can suppress the generation of the embedding of LPS and the TNF-α of LPS induction, so film is considered to an important step of LBP mediation LPS activated mononuclear cell in conjunction with the embedding of LBP and LPS.
Single nucleic acid polymorphism (SNP) refers to DNA sequence polymorphism in the colony that on karyomit(e), single nucleotide diversity causes, have that density is high, inheritance stability, be easy to the features such as automated analysis, be considered to the third generation genetic marker after restriction fragment length polymorphism, microsatellite polymorphism, can be used for the aspects such as the assignment of genes gene mapping, polymorphism analysis, is the common counter of study of disease genetic predisposition, medical diagnosis on disease, drug screening etc.Detection and the somatotype of LBP gene label list nucleic acid pleomorphism site, to LBP gene at the genetic characteristics of healthy population and with disease, occur, the genetic association research of development is significant.
Detection method about SNP, having developed at present more than 20 plants, as direct sequencing, probe hybridization method, flight mass spectrum analytical method, restriction fragment length polymorphism analytical method etc., but because of the shortcoming of the aspects such as expense, accuracy and applicable scale of these methods, limited to the genetic association research of gene pleiomorphism and disease generation, development and prognosis.And tetra-sodium sequencing technologies is the DNA sequence analysis technology of a new generation based on enzyme cascade, under the synergy of archaeal dna polymerase, ATP sulfurylase, luciferase and 4 kinds of enzymes of apyrase, each dNTP polymerization on DNA single chain and first order fluorescence signal release coupling are got up, by detecting release and the intensity of fluorescence, reach the object of the real time measure DNA sequence dna.This technology has its unique advantage, without electrophoresis, DNA fragmentation without fluorescent mark; There is easy and simple to handle, the feature such as testing cost is low, required sample size is little, quick, accurate, high-throughput, sensitivity height, meet Big Clinical Samples testing requirement.
Summary of the invention
In view of this, one of object of the present invention is to provide a kind of method of the LBP of detection gene label list nucleic acid pleomorphism site, and it can simple and direct, intuitive and accurately detect and interpretation LBP Tag SNP genotype.
For achieving the above object, technical scheme of the present invention is:
The method that detects LBP gene label list nucleic acid pleomorphism site, comprises the following steps:
1) design of Auele Specific Primer: according to confirmed LBP gene label list nucleic acid pleomorphism site, design specificity amplification primer and sequencing primer, wherein, in described specificity amplification primer one with biotin labeling; 2) pcr amplification: the DNA of sample to be measured of take is template, carries out PCR reaction with the specificity amplification primer described in step 1), obtains pcr amplification thing; 3) tetra-sodium order-checking: by step 2) gained pcr amplification thing adopts alkaline denaturation to make it be separated into strand, get wherein by after biotin labeled strand pcr amplification product purifying, carry out hybridization with sequencing primer described in step 1), and analytical results is to judge whether the target list nucleic acid of described sample to be measured possesses polymorphism.The innovative point of the technical program is mainly adopting tetra-sodium sequencing to detect single nucleic acid polymorphism of LBP gene first.
Further, described LBP gene label list nucleic acid pleomorphism site comprises: one or more in rs1780623, rs11536972 and rs2232618.Above-mentioned site obtains by the following method: utilize HapMap(http: //www.hapmap.org) snp database (HapMap Data Rel 28 Phase II+III, Aug10, on NCBI B36 assembly, dbSNP b126) download BeiJing, China's Chinese Han Population (CHB) SNP data in LBP gene and upstream and downstream 10kb thereof: 1) frame retrieval is inputted gene name and checked that each gene locates at HapMap, and upstream and downstream is respectively extended 10kb, 2) in the option of " Reports & Analysis ", select: Download SNP genotype data.3) in " config ", select BeiJing, China's Chinese Han Population (Chinese Han Beijing, CHB), obtain all SNP site in each gene of BeiJing, China Chinese Han Population SOCS family and elongated area thereof.
Further, the nucleotide sequence of the upstream amplimer in described rs1780623 site is as shown in SEQ ID NO:1, and the nucleotide sequence of downstream amplimer is as shown in SEQ ID NO:2, and sequencing primer is as shown in SEQ ID NO:3; The nucleotide sequence of the upstream amplimer in described rs11536972 site is as shown in SEQ ID NO:4, and the nucleotide sequence of downstream amplimer is as shown in SEQ ID NO:5, and sequencing primer is as shown in SEQ ID NO:6; The nucleotide sequence of the upstream amplimer in described rs2232618 site is as shown in SEQ ID NO:7, and the nucleotide sequence of downstream amplimer is as shown in SEQ ID NO:8, and sequencing primer is as shown in SEQ ID NO:9.Refer to following table:
Note: bio-represents 5 ' end biotin modification.
Further, described rs1780623 site, rs11536972 site and rs2232618 Sites Combination are carried out triple PCR amplification.Method of the present invention, the triple PCR amplification in the substance PCR in arbitrary site, the double PCR in any two sites or three sites is all feasible.Wherein, preferably triple PCR amplification, confirms through embodiment, and under the synergy of three site amplifications, amplification efficiency is high, and the DNA band of acquisition is impartial bright, single.
Another object of the present invention is to provide a kind of test kit for detection of LBP gene label list nucleic acid pleomorphism site, it can realize quick, easy, accurate, efficient, practical, economic detection LBP gene label SNP genotype, the needs of Clinical Laboratory real work be can meet, generation, developing risk that LBP gene label SNP detects predictive disease are beneficial to.
For achieving the above object, technical scheme of the present invention is:
Test kit for detection of LBP gene label list nucleic acid pleomorphism site, described test kit comprises the specificity amplification primer of LBP gene label list nucleic acid pleomorphism site, the tetra-sodium sequencing primer of LBP gene label list nucleic acid pleomorphism site, and PCR reaction solution, PCR product screening liquid, tetra-sodium sequencing reaction liquid.
Further, described specificity amplification primer is as shown in SEQ ID NO:1-2, and/or as shown in SEQ ID NO:4-5, and/or as shown in SEQ ID NO:7-8.
Further, described sequencing primer is as shown in SEQ ID NO:3, and/or as shown in SEQ ID NO:6, and/or as shown in SEQ ID NO:9.
Further, described test kit also comprises the vitamin H of amplimer described in mark.
The present invention also has an object to be to be provided for detecting the Auele Specific Primer of LBP gene label list nucleic acid pleomorphism site, this primer amplification efficiency is high, technical scheme is: described Auele Specific Primer is as shown in SEQ ID NO:1-2, and/or as shown in SEQ ID NO:4-5, and/or as shown in SEQ ID NO:7-8.
Useful technique effect of the present invention is:
Method of the present invention and test kit, by adopting the combination of high specific primer and proper method, can realize quick, easy, accurate, efficient, practical, economic detection LBP gene label SNP genotype, can be used for LBP gene at the association study of disease genetic susceptibility, the needs of Clinical Laboratory real work be can meet, generation, developing risk that LBP gene label SNP detects predictive disease are beneficial to.Compared with prior art, its application tetra-sodium sequencing technologies can carry out short dna sequential analysis quickly and accurately, is convenient to build normalizing operation flow process, has the advantages such as height is accurate, high-throughput, low cost; Pcr amplification primer efficiency is high, and product can directly carry out tetra-sodium order-checking, and without carrying out the secondary treatments such as product purification, easy and simple to handle, amount of samples is few.
Through embodiment, confirm: (1) PCR primer three re-constituted amplification object fragments provided by the invention, electrophoresis result shows that two target DNA bands under three re-constituteds are impartial bright, single, illustrate that triple assembly PCR amplimer efficiency provided by the invention is high, three pairs of primer amplification efficiency are suitable; (2) sample LBP gene label SNP polymorphism tetra-sodium sequencing result (see figure 1) shows, adopts test kit of the present invention and method, can simple and direct, intuitive and accurate LBP Tag SNP genotype be detected and interpretation.
Accompanying drawing explanation
The result figure of Fig. 1 for using triple PCR to carry out gene type assay to the detection sample in LBP gene rs1780623, rs11536972 and rs2232618 site.
Embodiment
Illustrated embodiment is in order better content of the present invention to be described, but is not that content of the present invention only limits to illustrated embodiment.So those of ordinary skill in the art carry out nonessential improvement and adjustment according to foregoing invention content to embodiment, still belong to protection scope of the present invention.
The screening of embodiment 1 candidate gene label list nucleic acid pleomorphism site
Utilize HapMap(http: //www.hapmap.org) snp database (HapMap Data Rel 28 Phase II+III, Aug10, on NCBI B36assembly, dbSNP b126) download BeiJing, China's Chinese Han Population (CHB) SNP data in LBP gene and upstream and downstream 10kb thereof: 1) frame retrieval is inputted gene name and checked that each gene locates at HapMap, and upstream and downstream is respectively extended 10kb, 2) select in the option of " Reports & Analysis ": Download SNP genotype data.3) in " config ", select BeiJing, China's Chinese Han Population (Chinese Han Beijing, CHB), obtain all SNP site in each gene of BeiJing, China Chinese Han Population SOCS family and elongated area thereof.
Under TAGster software package catalogue, open program human window.According to building bin and selecting Tag SNP standard (linkage relationship r2 >=0.8 between minimum gene frequency >=0.05 and SNP) parameters,, can there is the bin figure of survey region and the Tag SNP of corresponding bin in action command " fconvert ", " TAGster ".
Utilize NCBI to download the sequence in each gene promoter (upstream region of gene 3kb) SNP site, region.The sequence of wild-type and saltant type is submitted to respectively to transcription factor forecast database TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html), obtain the nuclear factor of combination with it, and the relatively impact of SNP site on transcription factor combination.
Between comprehensive SNP, linkage strength and bioinformatic analysis finally determine that 3 Tag SNP sites of LBP gene of detecting are as table 1.
3 tagged single-nucleotide polymorphic loci information of table 1
The single nucleic acid pleomorphism site of embodiment 2 pcr amplification primer and the design of tetra-sodium sequencing primer are with synthetic
Utilize NCBI (www.ncbi.nlm.nih.gov) GenBank to download each Tag SNP site and near about 500bp genome sequence thereof.Utilize tetra-sodium sequenator bundled software " Assay Design " to design single nucleic acid pleomorphism site pcr amplification primer and tetra-sodium sequencing primer, select the highest primer pair of scoring.The primer designing entrusts Shanghai biotechnology company limited synthetic, and carries out a wherein PCR primer 5 ' end biotin modification.3 label list nucleic acid pleomorphism site amplimers and the sequencing primer of design refer to table 2.
3 label list nucleic acid pleomorphism site amplimers of table 2 and sequencing primer
Note: bio-represents 5 ' end biotin modification.
Embodiment 3 Whole Blood Genomic DNA preparations
Check Whole Blood Genomic DNA purification kit (take U.S. Promega company produce reagent be example), tetra-sodium sequencing reagent quality guaranteed period and guarantee to have added in Wash Buffer ethanol, and carry out mark at bottle cap; (2) Virahol and 75% ethanol; (3) the 1.5ml Eppendorf(EP in autoclaving validity period) pipe and various model pipettor and rifle head.
From-80 refrigerators, take out the trauma patient peripheral blood that EDTA anticoagulant tube is collected, under room temperature, thawing and turn upside down mixes for several times; Pipette respectively 300ul whole blood and 900ul Cell Lysis Solution and add the 1.5ml EP pipe of carrying out corresponding sample labeling, incubated at room 10 minutes, put upside down and mixes once every 2-3 minute; 10000g, 4 ℃ are centrifugal 1 minute; Take out EP pipe, observe white precipitate, discard red supernatant; Repetitive operation 2-3 time, until redfree agglomerate in precipitation; Add 300ul Nuclei Lysis Solution, fully vibration mixes, and with 1ml or 200ul pipettor, precipitation is broken up completely, and room temperature is placed 10 minutes with abundant lysing cell; Add 100ul Protein Precipitation Solution, 20 seconds visible particle shape precipitations of thermal agitation; 12000g, 4 ℃ are centrifugal 3 minutes; Observe supernatant, if unlimpid, increase centrifugation time, if limpid with the slow sucking-off supernatant liquor of pipettor in new 1.5ml EP pipe, recording volume, adds the pre-cold isopropanol of equal-volume (20 ℃ of precoolings), turns upside down gently for several times to white cotton-shaped DNA and separates out; 12000g, 4 ℃ centrifugal 3 minutes, manage as seen end white precipitate, abandon supernatant; Add 500ul precooling 75% ethanol (20 ℃ of precoolings), the washing precipitation of softly turning upside down; 12000g, 4 ℃ centrifugal 2 minutes, abandon supernatant; On clean experiment table, place new filter paper, back-off EP pipe, blots liquid, air-dry DNA precipitation; Range estimation precipitation size, adds 50-100ul DNA Rehydration Solution, and 65 ℃ of water-baths are placed 15 minutes, takes out the vortex abundant dissolving DNA precipitation of vibrating every 5-6 minute; After dissolving completely, with Nano-Space ultraviolet spectrophotometer, measure nucleic acid concentration and purity, nucleic acid concentration is greater than 20ng/ul and is considered as qualifiedly, as concentration is not enough, can add ethanol again precipitate DNA and dissolve.At tube wall and pipe, cover SD sample number again, preserve DNA sample standby to-80 ℃ of refrigerators.
Embodiment 4 polymerase chain reactions and separation and purification
(1) triple PCR reaction
At reagent, prepare district preparation 50ul PCR reaction system (except DNA profiling), each component and addition are as following table:
| Component | Volume | Final concentration |
| 2×PCR?Mix | 25ul | 1× |
| SNP1 upstream primer | 1ul | 0.2uM |
| SNP1 downstream primer | 1ul | 0.2uM |
| SNP2 upstream primer | 1ul | 0.2uM |
| SNP2 downstream primer | 1ul | 0.2uM |
| SNP3 upstream primer | 1ul | 0.2uM |
| SNP3 downstream primer | 1ul | 0.2uM |
| Water | 20ul | ? |
At sample, prepare district to of short duration centrifugal rear the taking-ups 1ul(～30ng of EP pipe of Whole Blood Genomic DNA is housed) to reaction system, and on PCR reaction tubes mark sample number into spectrum and each combination number, PCR reaction system vortex mixes rear of short duration centrifugal; In reaction zone, carry out pcr amplification reaction, according to following loop parameter, amplification instrument be set:
| Step number | Temperature | Treatment time | Cycle number |
| 1 | 95℃ | 3min | ? |
| 2 | 95℃ | 15s | ? |
| 3 | 60℃ | 30s | ? |
| 4 | 72℃ | 20s | Go?to?step2,for?50cycle |
| 5 | 72℃ | 5min | ? |
| 6 | 4℃ | Holding | ? |
After setting program, selective reaction system 50ul, clicks " start " and starts instrument operation.
(2) biotin labeling strand pcr amplification product purifying
Before purifying, reagent and instrument are prepared: before Sample Purification on Single, guarantee that all solution reaches room temperature, open precise temperature control heating container, regulate temperature to reach 80 ℃.
Strand sample separation purification process: the annealing buffer that adds in advance 40 μ l to contain SNP1, SNP2 and each 0.3 μ M sequencing primer of SNP3 in PSQ 96 plates, generally adds each 10pM sequencing primer 2ul; Use Vertex to mix Sepharose beads; The Sepharoe beads total amount (every sample 2.5 μ l) that needs are used is transferred in an Eppendorf pipe; In Sepharose bead, add binding buffer, make average each sample approximately have the volume of 40 μ l, mixture is mixed; Above mixture is added in PCR product (50 μ l reaction volume) to every sample 40 μ l; PCR product is mixed to 10 minutes at normal temperatures, make the abundant combination of beads and vitamin H, for compared with long segment, can proper extension mixing time; In Vacuum prep workstation, in four sample panel, add successively 180ml high purity water, 70% ethanol, washing buffer and 120ml Denaturation buffer; Open the pump of vacuum prep workstation, vacuum prep tool is cleaned 30 seconds in high purity water; Then vacuum prep tool is moved on in PCR plate, capture sepharose beads(and please at beads, be combined with PCR product in latter three minutes and complete this operation, do not allow Beads sink to again and manage at the end); Pick up PCR plate, check whether most of beads has been attracted on vacuum prep tool; Vacuum prep tool is put into 70% ethanol 5 seconds; Then move on in denatureation buffer 5 seconds; Move on to again in washing buffer and clean 5-10 second; Suction nozzle is placed on to the top of the corresponding plate hole that contains sequencing primer, does not contact liquid level, turn off pump; Vacuum prep tool is put into the PSQ96 plate that contains sequencing primer, shake, discharge sepharose beads(sequencing primer and also can finally add); PSQ 96 plates that are placed with sample are placed in accurate temperature control box to 80 ℃ of heating 2 minutes, take out cool to room temperature, can carry out Pyrosequencing reaction.
After separation and purification, clean: do not open vacuum pump and valve, use high purity water to clean vacuum prep tool, the Beads not coming off is on a small quantity eluted; After changing high purity water, open again vacuum pump and valve, with about 250ml high purity water, clean tool; Turn off vacuum pump and valve, vacuum prep tool is sidelong, room temperature is dried; Clean the plastic channel of all splendid attire reagent solutions, naturally dry, powered-down, hides plant and instrument and prevents dust.
Embodiment 5 tetra-sodium sequencing reactions
Tetra-sodium sequenator is opened preheating in advance for 90 minutes, sets working procedure, calculates each using amount of reagent of this experiment and is added into corresponding agent bin; The sample preparing and agent bin are put into instrument correspondence position, click " Run " working procedure; After detection completes, close software process status window, preserve sequencing result.
Tetra-sodium sequencing result is analyzed: in " SNP Runs " folder, open above-mentioned operating file, select " SNP mode ", click " Analyze All " all detection samples are carried out to gene type assay.
According to embodiment 4-5 operation three times, as shown in Figure 1, this figure can clear interpretation rs1780623, rs11536972 and the genotype in rs2232618 site for A, B, tri-analytical resultss of C altogether.
Finally explanation is, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (9)
1. the method that detects LBP gene label list nucleic acid pleomorphism site, is characterized in that: comprise the following steps:
1) design of Auele Specific Primer
According to confirmed LBP gene label list nucleic acid pleomorphism site, design specificity amplification primer and sequencing primer, wherein, in described specificity amplification primer one with biotin labeling;
2) pcr amplification
The DNA of sample to be measured of take is template, with the specificity amplification primer described in step 1), carries out PCR reaction, obtains pcr amplification thing;
3) tetra-sodium order-checking
By step 2) gained pcr amplification thing adopts alkaline denaturation to make it be separated into strand, get wherein by after biotin labeled strand pcr amplification product purifying, carry out hybridization with sequencing primer described in step 1), and analytical results is to judge whether the target list nucleic acid of described sample to be measured possesses polymorphism.
2. the method for detection according to claim 1 LBP gene label list nucleic acid pleomorphism site, is characterized in that: described LBP gene label list nucleic acid pleomorphism site comprises: one or more in rs1780623, rs11536972 and rs2232618.
3. the method for detection according to claim 2 LBP gene label list nucleic acid pleomorphism site, it is characterized in that: the nucleotide sequence of the upstream amplimer in described rs1780623 site is as shown in SEQ ID NO:1, the nucleotide sequence of downstream amplimer is as shown in SEQ ID NO:2, and sequencing primer is as shown in SEQ ID NO:3; The nucleotide sequence of the upstream amplimer in described rs11536972 site is as shown in SEQ ID NO:4, and the nucleotide sequence of downstream amplimer is as shown in SEQ ID NO:5, and sequencing primer is as shown in SEQ ID NO:6; The nucleotide sequence of the upstream amplimer in described rs2232618 site is as shown in SEQ ID NO:7, and the nucleotide sequence of downstream amplimer is as shown in SEQ ID NO:8, and sequencing primer is as shown in SEQ ID NO:9.
4. the method for detection LBP gene label list nucleic acid pleomorphism site according to claim 2, is characterized in that: described rs1780623 site, rs11536972 site and rs2232618 Sites Combination are carried out triple PCR amplification.
5. for detection of the test kit of LBP gene label list nucleic acid pleomorphism site, it is characterized in that: described test kit comprises the specificity amplification primer of LBP gene label list nucleic acid pleomorphism site, the tetra-sodium sequencing primer of LBP gene label list nucleic acid pleomorphism site, and PCR reaction solution, PCR product screening liquid, tetra-sodium sequencing reaction liquid.
6. the test kit for detection of LBP gene label list nucleic acid pleomorphism site according to claim 5, it is characterized in that: described specificity amplification primer is as shown in SEQ ID NO:1-2, and/or as shown in SEQ ID NO:4-5, and/or as shown in SEQ ID NO:7-8.
7. the test kit for detection of LBP gene label list nucleic acid pleomorphism site according to claim 5, is characterized in that: described sequencing primer is as shown in SEQ ID NO:3, and/or as shown in SEQ ID NO:6, and/or as shown in SEQ ID NO:9.
8. the test kit for detection of LBP gene label list nucleic acid pleomorphism site according to claim 5, is characterized in that: described test kit also comprises the vitamin H of amplimer described in mark.
9. for detection of the Auele Specific Primer of LBP gene label list nucleic acid pleomorphism site, it is characterized in that: described Auele Specific Primer is as shown in SEQ ID NO:1-2, and/or as shown in SEQ ID NO:4-5, and/or as shown in SEQ ID NO:7-8.
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| CN109507323A (en) * | 2018-12-07 | 2019-03-22 | 上海浩港生物技术有限公司 | A kind of kit with LBP content in mass spectrograph detection human blood |
| CN109633176A (en) * | 2019-01-11 | 2019-04-16 | 广东医科大学附属医院 | A kind of nephrosis gene therapy diagnostic kit |
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| CN104407156A (en) * | 2014-12-05 | 2015-03-11 | 重庆中元生物技术有限公司 | Latex enhanced immunoturbidimetry kit for detecting lipopolysaccharide binding protein (LBP) |
| CN109507323A (en) * | 2018-12-07 | 2019-03-22 | 上海浩港生物技术有限公司 | A kind of kit with LBP content in mass spectrograph detection human blood |
| CN109633176A (en) * | 2019-01-11 | 2019-04-16 | 广东医科大学附属医院 | A kind of nephrosis gene therapy diagnostic kit |
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