CN107083327A - Determine kit, method and the application of the two of the gene copy numbers of DEFA1 3 - Google Patents
Determine kit, method and the application of the two of the gene copy numbers of DEFA1 3 Download PDFInfo
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- CN107083327A CN107083327A CN201710519565.4A CN201710519565A CN107083327A CN 107083327 A CN107083327 A CN 107083327A CN 201710519565 A CN201710519565 A CN 201710519565A CN 107083327 A CN107083327 A CN 107083327A
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- 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
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- C12Q1/6851—Quantitative amplification
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Abstract
The invention provides a kind of kit, method and the application of the two of the measure gene copy numbers of DEFA1 3, it is related to technical field of biological, the kit for the measure gene copy numbers of DEFA1 3 that the present invention is provided, including droplet digital pcr detection reagent and droplet card, wherein, droplet digital pcr detection reagent includes premixed liquid, and premixed liquid includes the primer and probe of the genes of specific detection DEFA1 3.The kit that the present invention is provided has the advantages that high sensitivity, high accuracy, high precision, high repeatability.The assay method for the gene copy numbers of DEFA1 3 that the present invention is provided, applies the kit for the measure gene copy numbers of DEFA1 3 that the present invention is provided, easy with operating method, quick, sensitive, accurate advantage.
Description
Technical field
The present invention relates to technical field of biological, more particularly, to a kind of reagent of measure DEFA1-3 gene copy numbers
Box, method and the application of the two.
Background technology
People is 2 times of body biologies (diploid genome), and most gene only has two copies, but also has some bases
Because having multiple copies.The gene of multicopy has the variation of copy number, such as DEFB1-3 genes, in height on the different person
Plus with rope people its copy number variation between 2-12, the bright seminar in direction finds Chinese's DEFA1-3 gene copy numbers
Variation is between 2-16.It has now been found that the variation of DEFA1-3 gene copy numbers may be related to human disease's development prognosis, especially
It is infectious diseases.
Method at present through commonly used measurement gene copy number has three kinds i.e. qPCR, paralog ratio test
And multiplex ligation-dependent probe amplification (MLPA) (PRT).They are in specific design
It is upper different, but the principle all used is exactly the gene dosage that DEFA1-3 is measured to be copied with one or more
Gene dosage of the number without variation is compared, and DEFA1-3 copy numbers are inferred with this.Early stage has more above-mentioned three kinds of the research of scholar
Method, it is most accurate in these three methods, most reliable method to find MLPA, but its shortcoming is exactly that method is complicated, is taken
Long, the expense for measuring a sample is higher.MLPA principle be using hybridization probe method amplification DEFA1-3 and other
30 genetic fragments, then calculate DEFA1-3 by comparing DEFA1-3 fragments with the amount of other genetic fragments product after amplification
Copy number.
Therefore, develop a kind of easy, the time-consuming short, cost of method low, and accurately and reliably determine DEFA1-3 gene copies
Several methods is particularly important.
In view of this, it is special to propose the present invention.
The content of the invention
First purpose of the present invention is to provide a kind of kit of measure DEFA1-3 gene copy numbers, of the invention
Second purpose is to provide application of the mentioned reagent box in DEFA1-3 gene copy numbers are determined, the 3rd mesh of the invention
Be a kind of assay method of DEFA1-3 gene copy numbers is provided, fourth object of the present invention is to provide said determination
Application of the method in DEFA1-3 gene copy numbers are determined, DEFA1-3 copy number sides are determined to alleviate present in prior art
Method is complicated, and time-consuming, costly technical problem.
The invention provides a kind of kit of measure DEFA1-3 gene copy numbers, the kit includes：
Droplet digital pcr detection reagent and droplet card；
The droplet digital pcr detection reagent includes premixed liquid, and the premixed liquid includes specific detection DEFA1-3 genes
Primer and probe；
The upstream and downstream primer of the specific detection DEFA1-3 genes is respectively：
DEFA1-3-F：5'-CCGTCCTTCCCTCTAGACTTAGC-3'(SEQ ID NO.1)；
DEFA1-3-R：5'-GAGCAGATTGCAGCGGACAT-3'(SEQ ID NO.2)；
The probe of the specific detection DEFA1-3 genes is：FAM-ACTGCTAACTCCATACTC-MGB(SEQ ID
NO.3)。
Further, the premixed liquid also includes 2 × ddPCR Supermix for probes and RNase Free
dH2O。
Further, 2 × ddPCR Supermix for probes, DEFA1-3-F, DEFA1-3- in the premixed liquid
R, probe and RNase Free dH2O volume ratio is 55-65：10-20：10-20：1-10：20-30.
Further, the droplet digital pcr detection reagent also includes RNase Free dH2O, droplet occur oil, 2 ×
Control liquid, positive control, negative control, 10 × CutSmart buffer and restriction enzyme.
Further, the positive control includes the sample that known copy number is 2,4,6,8,10,12,14,16.
Further, RNase Free dH in the droplet digital pcr detection reagent2Oil, 2 occur for O, premixed liquid, droplet
× control liquid, positive control, negative control, the volume ratio of 10 × CutSmart buffer and restriction enzyme are 250-
350：200-250：800-1200：500-700：80-120：8-16：250-350：0.5-2.
Present invention also offers application of the above-mentioned kit in DEFA1-3 gene copy numbers are determined.
Present invention also offers a kind of assay method of DEFA1-3 gene copy numbers, using above-mentioned measure DEFA1-3 bases
Because of the kit of copy number.
Further, the assay method comprises the following steps：
Step (a)：Handle sample DNA；
Step (b)：Droplet digital pcr reaction solution is prepared, the droplet digital pcr reaction solution includes sample to be tested reaction solution
With blank control reaction solution, the sample to be tested reaction solution includes the sample DNA after being handled in the step (a)；
Step (c)：Sample to be tested reaction solution and blank control reaction solution in the step (b) is separately added into described micro-
Drop occurs in the hole in the middle of card；
Step (d)：Occur to add droplet generation oil in the hole of card bottom in the droplet, and seal；
Step (e)：The droplet is placed in droplet generation instrument and generates droplet, the droplet is created on described micro-
Drop occurs in the hole on card top；
Step (f)：The droplet is taken, performing PCR of going forward side by side reaction；
Step (g)：The droplet for completing the PCR reactions is tested and analyzed, the gene copy number is obtained.
In addition, the application present invention also offers above-mentioned assay method in DEFA1-3 gene copy numbers are determined.
The kit for the measure DEFA1-3 gene copy numbers that the present invention is provided, including droplet digital pcr detection reagent and
Droplet card, wherein, droplet digital pcr detection reagent includes premixed liquid, and premixed liquid includes specific detection DEFA1-3 genes
Primer and probe.The kit that the present invention is provided have high sensitivity, high accuracy, high precision, high repeatability it is excellent
Point.The assay method for the DEFA1-3 gene copy numbers that the present invention is provided, applies the measure DEFA1-3 genes that the present invention is provided
The kit of copy number, quick, sensitive, accurate advantage easy with operating method.
Embodiment
Technical scheme is clearly and completely described below in conjunction with embodiment, it is clear that described reality
It is a part of embodiment of the invention to apply example, rather than whole embodiments.Based on the embodiment in the present invention, the common skill in this area
The every other embodiment that art personnel are obtained under the premise of creative work is not made, belongs to the model that the present invention is protected
Enclose.
At present, the method through commonly used measurement gene copy number has three kinds i.e. qPCR, PRT and MLPA, wherein, MLPA is
It is most accurate in these three methods, most reliable method, but its shortcoming is exactly that method is complicated, time-consuming, measures a sample
Expense it is higher.
Therefore, in view of the above-mentioned problems, the invention provides a kind of kit of measure DEFA1-3 gene copy numbers, including：
Droplet digital pcr detection reagent and droplet card；
Droplet digital pcr detection reagent includes premixed liquid, the primer of premixed liquid including specific detection DEFA1-3 genes and
Probe；
Wherein, the upstream and downstream primer of specific detection DEFA1-3 genes is respectively：
DEFA1-3-F：5'-CCGTCCTTCCCTCTAGACTTAGC-3'(SEQ ID NO.1)；
DEFA1-3-R：5'-GAGCAGATTGCAGCGGACAT-3'(SEQ ID NO.2)；
The probe of specific detection DEFA1-3 genes is：FAM-ACTGCTAACTCCATACTC-MGB(SEQ ID
NO.3)。
Droplet digital pcr (droplet digital PCR), this method is that the PCR reaction solutions that will be prepared are led to with oil
The method for crossing high pressure becomes the minute water oil mixing drop of drops up to ten thousand, and PCR reactions occur in this tiny droplets, machine after reaction terminates
Device can read fluorescent positive drop number, then the Poisson distribution method in program can be used to measure nucleotide sequence it is dense
Degree.Gene copy number is determined using this method, can alleviate can not reach asking for resolving effect by traditional quantitative PCR method
Topic, the research to hereditary disease, cancer, infectious diseases provides a kind of brand-new technical thought and means.
In the present invention, DEFA1-3 genes behaviour DEFA1-3 genes.
In the present invention, premixed liquid also includes 2 × ddPCR Supermix for probes and RNase Free dH2O。
In the present invention, 2 × ddPCR Supermix for probes in premixed liquid, DEFA1-3-F, DEFA1-3-R,
Probe and RNase Free dH2O volume ratio is 55-65：10-20：10-20：1-10：20-30.
Wherein, 2 × ddPCR Supermix for probes, DEFA1-3-F, DEFA1-3-R, probe and RNase
Free dH2O volume ratio such as can be, but be not limited to 55：10：10：1：20、60：15：15：5：25 or 65：20：20：
10：30.
In one preferred embodiment, 2 × ddPCR Supermix for probes, DEFA1-3-F, DEFA1-
3-R, probe and RNase Free dH2O volume ratio is 60：15：15：5：25.
Wherein, the concentration of DEFA1-3-F, DEFA1-3-R and probe is 10 μM.
In the present invention, droplet digital pcr detection reagent also includes RNase Free dH2Oil, 2 × matter occur for O, droplet
Draining, positive control, negative control, 10 × CutSmart buffer and restriction enzyme.
Wherein, droplet occurs oil and occurs oil for sonde method ddPCR droplets, and 2 × control liquid is 2 × ddPCR sonde method Quality Controls
Liquid, positive control includes the sample that known copy number is 2,4,6,8,10,12,14,16, and negative control is RNase Free
dH2O, restriction enzyme is Msel enzymes.
The positive template for selecting known copy number to be 2,4,6,8,10,12,14,16, uses 0.01M pH8.0 Tris-
Freezen protective after the dilution of edta buffer liquid, will examine qualified positive control preparation quantitative separating.
Wherein, the whether qualified method of inspection positive control is：Using real-time fluorescence quantitative PCR, target gene is utilized
DEFA1-3 and reference gene ALB quantitative fluorescent PCR examine the copy number of positive control to be 2,4,6,8,10,12,14,16, glimmering
Fluorescent Quantitative PCR reaction condition is 95 DEG C, 3min；95℃ 30s；54 DEG C of 30s, 72 DEG C of 30s, 40 circulations；72℃ 1min.
In the present invention, RNase Free dH in droplet digital pcr detection reagent2O, premixed liquid, droplet occur oil, 2 ×
Control liquid, positive control, negative control, the volume ratio of 10 × CutSmart buffer and restriction enzyme are 250-
350：200-250：800-1200：500-700：80-120：8-16：250-350：0.5-2.
Wherein, RNase Free dH2Oil, 2 × control liquid, positive control, negative control, 10 occur for O, premixed liquid, droplet
The volume ratio of × CutSmart buffer and restriction enzyme such as can be, but be not limited to 250：200：800：500：
80：8：250：0.5、300：225：1000：600：100：12：300：1 or 350：250：1200：700：120：16：350：2.
In one preferred embodiment, RNase Free dH2Oil, 2 × control liquid, sun occur for O, premixed liquid, droplet
Property control, negative control, the volume ratio of 10 × CutSmart buffer and restriction enzyme be 300：225：1000：
600：100：12：300：1.
In the present invention, each reagent in the kit of DEFA1-3 gene copy numbers is determined with suitable external packing box bag
Dress, labelling sign title, lot number, date of manufacture, the term of validity.
The kit that the present invention is provided has the advantages that high sensitivity, high accuracy, high precision, high repeatability.
Present invention also offers application of the above-mentioned kit in DEFA1-3 gene copy numbers are determined.
Present invention also offers a kind of assay method of DEFA1-3 gene copy numbers, using above-mentioned measure DEFA1-3 bases
Because of the kit of copy number.
In the present invention, assay method comprises the following steps：
Step (a)：Handle sample DNA；
Wherein, single DNA sample process system composition is：ddH2O 20.3μL；10×CutSmart buffer 2.5μ
L；The μ L of Msel enzymes 0.2；The μ L (50ng or so) of sample DNA 2.After the completion of configuration, 37 DEG C, after 60min, 65 DEG C, 20minPCR instruments
Middle carry out endonuclease reaction.
Step (b)：Droplet digital pcr reaction solution is prepared, droplet digital pcr reaction solution includes sample to be tested reaction solution and sky
White control reaction liquid, sample to be tested reaction solution includes the sample DNA after being handled in step (a)；
Wherein, the volume of sample to be tested reaction solution and blank control reaction solution is respectively 20 μ L, sample to be tested reaction solution bag
Include 18 μ L premixed liquid and processing after sample DNA；
Step (c)：Sample to be tested reaction solution and blank control reaction solution in step (b) is separately added into droplet card
In 8 middle holes, supplied during less than 8 samples with 20 μ L 1 × ddPCR sonde method control liquids；
Step (d)：Occur each 70 μ L droplets that add in 8 holes of card bottom in droplet and occur oil, and lid rubber cushion is sealed；
Step (e)：Droplet is placed in droplet generation instrument and generates droplet, droplet is created on droplet and occurs card top
Hole in；
Step (f)：The droplet for taking 40 μ L to generate, is added in 96 orifice plates, sealer, performing PCR of going forward side by side reaction；
Wherein, PCR reaction conditions are：
Pre-degeneration：96 DEG C of 10min of temperature, 1 circulation；
Denaturation：98 DEG C of 30s of temperature, annealing：58 DEG C of 45s of temperature, totally 40 circulations；
Terminate reaction：98 DEG C of 10min of temperature, 1 circulation；
Wherein, PCR reacts instrument warming and cooling rate less than 2.5 DEG C/sec；
Step (g)：96 orifice plates for completing PCR reactions are put into plate holder and assembled, droplet reading is put into afterwards
Take in instrument, open QuantaSoft softwares, droplet is tested and analyzed, testing gene copy number is obtained.
The assay method for the DEFA1-3 gene copy numbers that the present invention is provided, applies the measure DEFA1-3 that the present invention is provided
The kit of gene copy number, quick, sensitive, accurate advantage easy with operating method.
In addition, the application present invention also offers above-mentioned assay method in DEFA1-3 gene copy numbers are determined.
In order to contribute to it is clearer understand present disclosure, be described in detail as follows in conjunction with specific embodiment.
The design and screening of the primer of embodiment 1 and probe
According to (the numbering of target gene DEFA1-3 corresponding sequences in GenBank：MGI:MGI:99588), selection length is 50
~150bp conservative fragments, using the Software for Design specific probes of Primer Express 2.0 and primer, final choice is sensitive
Degree highest, the probe of the target gene of high specificity and primer are tested.
The upstream and downstream primer of resulting specific detection DEFA1-3 genes is respectively：
DEFA1-3-F：5'-CCGTCCTTCCCTCTAGACTTAGC-3'(SEQ ID NO.1)；
DEFA1-3-R：5'-GAGCAGATTGCAGCGGACAT-3'(SEQ ID NO.2)；
The probe of specific detection DEFA1-3 genes is：FAM-ACTGCTAACTCCATACTC-MGB
(SEQ ID NO.3)。
The selection of the kit of embodiment 2 and the pretreatment of reagent
The kit for the measure DEFA1-3 gene copy numbers that the selection present invention is provided, including droplet digital pcr detection reagent
And droplet card.
Wherein, droplet digital pcr detection reagent includes RNase Free dH2O, 1,1.2mL；DdPCR sonde methods are premixed
Liquid, one, 1.1mL；Oil, one, 4mL occur for sonde method ddPCR droplets；2 × ddPCR sonde method control liquids, 2.4mL；It is positive
Control, it is known that each one of the sample that copy number is 2,4,6,8,10,12,14,16, every 40 μ L；Negative control (RNase Free
dH2O), one, 48 μ L；10×CutSmart buffer 1.2mL；Msel enzymes one, 40 units (4 μ L).
The positive template for selecting known copy number to be 2,4,6,8,10,12,14,16, uses 0.01M pH8.0 Tris-
Edta buffer liquid is diluted after 100 times, in -80 DEG C of freezen protectives, qualified positive control preparation will be examined quantitatively to divide by 400 μ L
Dress.
Its 1.1mL of ddPCR sonde method premixed liquids composition is：600 μ L 2 × ddPCR Supermix for probes,
Upstream and downstream primer is respectively 150 μ L, and specific probe is 50 μ L, and the concentration of primer and probe is 10 μM, RNase Free
dH2O 250μL。
The extraction and processing of the sample to be tested of embodiment 3
15 urgent patients of random selection and 5 Healthy Peoples extract 2mL whole bloods from peripheral vein and are put into EDTA anticoagulant tubes
In.Sample total DNA is extracted using DNA extraction kit (QIAGEN, Valencia, CA), and is numbered.
Each STb gene of extraction is configured to system for handling and reacted, wherein, system for handling is as follows：
| Sample | Volume (μ L) |
| ddH2O | 20.3 |
| 10×CutSmart buffer | 2.5 |
| Msel enzymes | 0.2 |
| Sample DNA | 2 |
Reaction condition is as follows：
| Temperature (DEG C) | Time (min) |
| 37 | 60 |
| 65 | 20 |
The measure of the sample to be tested DEFA1-3 gene copy numbers of embodiment 4
The kit and the embodiment of the present invention 3 of the measure DEFA1-3 gene copy numbers provided using the embodiment of the present invention 2 are carried
The sample to be tested DNA of confession, carries out the measure of DEFA1-3 gene copy numbers, and step is as follows：
Step (A)：DdPCR reaction solutions are prepared, the μ L of cumulative volume 20 add following reactants into amplification pipe：DdPCR probes
The μ L of method premixed liquid 18, the treated μ L of DNA profiling 2 that the embodiment of the present invention 3 is provided.
Blank control：With RNase Free dH2O replaces expanding under DNA profiling, similarity condition；
Step (B)：Droplet is taken to be placed in Kato fixed, 20 μ L reaction solutions are added into droplet occurs a row in the middle of card
8 holes in, supplied during less than 8 samples with 20 μ L 1 × ddPCR sonde method control liquids；
Step (C)：Oil occurs for each 70 μ L droplets that add in minimum 8 holes of next row of card occur for droplet, and lid rubber cushion is close
Envelope；
Step (D)：Above Kato is lightly steadily positioned in droplet generation instrument, starts to generate droplet；
Step (E)：Droplet, which is created on droplet, to be occurred in one round of card the top, slow to draw 40 μ L, then squeezes into 96 orifice plates
In the hole of relevant position；
Step (F)：Seal and enter performing PCR reaction after film in 96 hole PCR instruments, warming and cooling rate is less than 2.5 DEG C/sec；
Wherein, PCR conditions are as follows：
Step (G)：96 orifice plates for completing PCR are put into plate holder and assembled, are lightly steadily put into afterwards
Droplet is read in instrument, sequentially draws the droplet of each sample and with carrier fluid stream one by one by double-colored detector, there is fluorescence signal
Droplet is the positive, and the droplet of unstressed configuration signal is feminine gender, opens QuantaSoft softwares and is tested and analyzed, software records are each
The ratio of positive droplet in sample, and automatically analyze data.
Amplification known copy number is a (value is 2,4,6,8,10,12,14,16), and positive control DEFA1-3 signals are m,
RPP30 signals are n, and sample DEFA1-3 signal is m ', and RPP30 signals are n, then the copy number of sample is (m '/m) × a.
The DEFA1-3 number of copies carried according to patient, it can be determined that the neurological susceptibility that patient's severe sepsis occurs.When
When DEFA1-3 gene copy numbers are more than 8, severe sepsis neurological susceptibility substantially increases.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, it will be understood by those within the art that：Its according to
The technical scheme described in foregoing embodiments can so be modified, or which part or all technical characteristic are entered
Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme.
SEQUENCE LISTING
<110>Zhejiang University
<120>Determine kit, method and the application of the two of DEFA1-3 gene copy numbers
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence
<400> 1
ccgtccttcc ctctagactt agc 23
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
gagcagattg cagcggacat 20
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence
<400> 3
actgctaact ccatactc 18
Claims (10)
1. a kind of kit of measure DEFA1-3 gene copy numbers, it is characterised in that the kit includes：
Droplet digital pcr detection reagent and droplet card；
The droplet digital pcr detection reagent includes premixed liquid, and the premixed liquid includes drawing for specific detection DEFA1-3 genes
Thing and probe；
The upstream and downstream primer of the specific detection DEFA1-3 genes is respectively：
DEFA1-3-F：5'-CCGTCCTTCCCTCTAGACTTAGC-3'(SEQ ID NO.1)；
DEFA1-3-R：5'-GAGCAGATTGCAGCGGACAT-3'(SEQ ID NO.2)；
The probe of the specific detection DEFA1-3 genes is：FAM-ACTGCTAACTCCATACTC-MGB(SEQ ID
NO.3)。
2. kit according to claim 1, it is characterised in that the premixed liquid also includes 2 × ddPCR Supermix
For probes and RNase Free dH2O。
3. kit according to claim 2, it is characterised in that 2 × ddPCRSupermix in the premixed liquid
Probes, DEFA1-3-F, DEFA1-3-R, probe and RNase Free dH2O volume ratio is 55-65：10-20：10-20：
1-10：20-30.
4. kit according to claim 1, it is characterised in that the droplet digital pcr detection reagent also includes RNase
Free dH2Oil, 2 × control liquid, positive control, negative control, 10 × CutSmart buffer and limitation occur for O, droplet
Property restriction endonuclease.
5. kit according to claim 4, it is characterised in that it is 2,4,6 that the positive control, which includes known copy number,
8,10,12,14,16 sample.
6. kit according to claim 5, it is characterised in that RNase in the droplet digital pcr detection reagent
Free dH2O, premixed liquid, droplet occur oil, 2 × control liquid, positive control, negative control, 10 × CutSmart buffer with
And the volume ratio of restriction enzyme is 250-350：200-250：800-1200：500-700：80-120：8-16：250-350：
0.5-2。
7. application of the kit in DEFA1-3 gene copy numbers are determined as described in claim any one of 1-6.
8. a kind of assay method of DEFA1-3 gene copy numbers, it is characterised in that described in application claim any one of 1-6
Determine the kit of DEFA1-3 gene copy numbers.
9. assay method according to claim 8, it is characterised in that the assay method comprises the following steps：
Step (a)：Handle sample DNA；
Step (b)：Droplet digital pcr reaction solution is prepared, the droplet digital pcr reaction solution includes sample to be tested reaction solution and sky
White control reaction liquid, the sample to be tested reaction solution includes the sample DNA after being handled in the step (a)；
Step (c)：Sample to be tested reaction solution and blank control reaction solution in the step (b) is separately added into the droplet hair
In hole in the middle of raw card；
Step (d)：Occur to add droplet generation oil in the hole of card bottom in the droplet, and seal；
Step (e)：The droplet is placed in droplet generation instrument and generates droplet, the droplet is created on the droplet hair
In the hole on raw card top；
Step (f)：The droplet is taken, performing PCR of going forward side by side reaction；
Step (g)：The droplet for completing the PCR reactions is tested and analyzed, the gene copy number is obtained.
10. application of the assay method as claimed in claim 8 or 9 in DEFA1-3 gene copy numbers are determined.
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| CN201710519565.4A CN107083327A (en) | 2017-06-29 | 2017-06-29 | Determine kit, method and the application of the two of the gene copy numbers of DEFA1 3 |
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| CN106676189A (en) * | 2017-02-23 | 2017-05-17 | 珠海出入境检验检疫局检验检疫技术中心 | Quantitative detection method of bovine-derived and porcine-derived components based on droplet digital PCR (polymerase chain reaction) as well as primer, probe and kit |
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