CN110184334A - Multi-PCR detection method and kit based on high-resolution fusion curve shape - Google Patents
Multi-PCR detection method and kit based on high-resolution fusion curve shape Download PDFInfo
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Abstract
The invention discloses a kind of multi-PCR detection method and kit based on high-resolution fusion curve shape.The present invention uses PCR saturated fluorescence dyestuff, and directly progress high-resolution melting, is normalized the melting curve of acquisition, differentiation or principal component analysis, single tube can be realized effectively while detecting the purpose of multiple target sequences after the completion of amplification.Identification detection is carried out according to high-resolution fusion curve shape information rather than melting temperature, significantly reduces the design difficulty of multiplex PCR assay primer, multiple pcr amplification products with small Tm difference are detected simultaneously；It avoids open pipe operation and causes the contaminated possibility of target DNA, compensate for the higher defect of single amplification reaction cost under multicolor fluorescence label or space separation in parallel, improve detection flux, reduce testing cost, there is stronger practical application value.
Description
Technical field
The invention belongs to molecular biology identification technique fields, and in particular to be examined simultaneously according to high-resolution fusion curve shape
Survey the multiple real time fluorescence PCR detection method and kit of multiple target target gene.
Background technique
Multiplex PCR (multiplex PCR) is also known as composite PCR, is a kind of PCR to grow up on the basis of Standard PCR
Technology can expand simultaneously multiple target segments by the way that two pairs or more primer is added in the same PCR system.
Relative to substance round pcr, multiplex PCR has the advantages that detection efficiency height, save the cost.
Currently, the method for detecting multiple target segments (for example, from different plant species) simultaneously includes electrophoretic analysis, sequencing
Analyze and be spatially separating method.The principle of electrophoretic analysis is to first pass through round pcr to expand segment to be detected, directly or is used
After restriction enzyme shearing, by electrophoretic analysis clip size and quantity, and then reaches and distinguish different target segments
Purpose, this method is time-consuming, laborious and can not be detected on a large scale, in addition, being less than the target of 10bp for Fragment Differential
Segment is difficult to distinguish；Sequencing analysis is relatively expensive for the detection price of batch samples, and sequencing and electrophoresis
It is operated after requiring open pipe, increases the pollution risk that sample generates aerosol；Being spatially separating rule is by spatial parallelism
Multiple single reaction detections are achieved, wherein the method for not having to open pipe operation, multicolor fluorescence detection can be real in stopped pipe
The specific detection of existing multiple target sequences, but it needs to carry out multicolor fluorescence label and requirement to instrument is also relatively high,
Need Multi-channel optical element.Probe rule is needed for every a pair of of design of primers specific probe, it is desirable that more stringent and valence
Lattice are expensive, improve testing cost.
Melting temperature analysis for single tube simultaneously detect multiple target segments provide it is a kind of simply, it is stopped pipe, at low cost
Possible ways.Different target segments since the difference of length and G/C content causes it to generate different melting temperatures,
It can be used for distinguishing multiple target segments.But have certain limitation using the method, on the one hand to design of primers require compared with
It is high, it is desirable that each primer has good specificity, and primer free dimer and non-specific amplification generate, and different targets
Segment Tm (melting temperature) value differs by more than 2 DEG C, and multipair primer is on the other hand required to be in the same reaction system, each primer tool
There is same or similar annealing temperature, and do not interfere with each other each other, so that the optimization of reaction system and reaction condition
Difficulty, and after often meeting these requirements, it is difficult to it is close to guarantee that the Tm difference between amplified production can effectively distinguish Tm
Target segment, these are all restricted the popularization of the technology.Therefore, it establishes a kind of simple and easy and can detect simultaneously more
The detection method of kind animal derived materials is always problem to be solved to improve detection efficiency.
High-resolution fusion curve (High Resolution Melting, HRM) analysis belongs to fluorescent PCR detection technique, main
Wanting principle is: the melting behavior decision of double-stranded DNA passes through in the difference of the length of DNA sequence dna, G/C content and base complement
The change of real time monitoring of DNA saturated fluorescence dyestuff signal obtains feature melting curve in fusion processes, meanwhile, by means of profession
The analysis software of property can be realized based on the test sample Genotyping of different shape melting curve or classification.For example, Chinese
Patent CN109868322A expands certain segment DNA segment of different animals source constituent using universal primer, although avoiding design
Multipair primer is still analyzed, i.e., according to the shape of high-resolution fusion curve by the differentiation etc. to high-resolution fusion curve
(profile) is not possible to distinguish corresponding to the amplification target sequence of melting temperature similitude higher (Tm is differed within 0.5 ± 0.1 DEG C)
Different animals source constituent, and due to using universal primer, the specificity of pcr amplification reaction is lower.
And existing multiple real time fluorescence PCR, for example, Chinese patent CN109868321A, for the target in goat genome
The length of the amplified production of sequence, G/C content have carried out complicated design to primer, real with the position that will pass through observation melting peakss
Now identify the target sequence of melting temperature similitude higher (Tm is differed within 0.5 ± 0.1 DEG C) simultaneously, but design of primers difficulty is larger
(increasing the ratio or length of entire amplified fragments GC%, amplified fragments Tm is increased into desired value).
Summary of the invention
The purpose of the present invention is to provide a kind of multi-PCR detection method and reagent based on high-resolution fusion curve shape
Box can be used for quickly identifying the different animals derived component in sample.
In order to achieve the above objectives, the invention adopts the following technical scheme:
It is a kind of for identifying the multiple fluorescence PCR method of different target sequences, which includes following step
It is rapid:
1) situation is formed according to the DNA in different measuring samples, determines N kind (N > 1) the target DNA sequence for needing to identify；
2) sequence fragment that there is specificity for expanding any a part in corresponding target DNA sequence is designed and synthesized
The primer of (target sequence)；
3) utilize substance PCR amplification verification step 2) in designed primer validity and specificity (confirmation is used for
The N of amplification is to primer)；
4) analyze respectively target DNA sequence and its any number of combination (any one target DNA sequence, arbitrarily
The combination of two kinds of target DNA sequences, the combination ... of any three kinds of target DNA sequences, the combination of N kind target DNA sequence), with right
Answer target DNA sequence or genomic DNA comprising target DNA sequence be template (if containing there are many target DNA sequence in template,
Can be mixed using arbitrary proportion, for example, by using equimolar ratio), it is utilized respectively by step 3) verifying for different purposes
The primer (N is to primer) of DNA sequence dna carries out multiplexed PCR amplification, and (closes immediately after the completion of amplification to amplified production
Pipe) high-resolution melting is carried out, it is carried out using the high-resolution fusion curve of normalization, differentiation or principal component analysis to acquisition special
Sign is extracted, and then will be extracted obtained characteristic information (for example, differentiation melting curve figure) and is merged, obtains standard and identify map；
5) DNA in certain measuring samples is extracted as template, carries out multiplexed PCR amplification according to step 4) and amplification is produced
Object carries out high-resolution melting and obtains high-resolution fusion curve immediately (i.e. stopped pipe), uses and acquisition to the high-resolution fusion curve
The standard identifies the same feature extracting method of map (normalization, differentiation or principal component analysis) and is analyzed, and will divide
Analysis result identifies map with the standard and compares, and according to identifying in map in the standard, through comparing, matched characteristic information is true
DNA composition situation (compositions of i.e. one or more different target DNAs) in certain fixed described measuring samples.
Preferably, in the step 3), the segment of PCR amplification is located at animal species corresponding to different target DNA sequences
In genomic DNA.
Preferably, in the step 3), by examining specificity of the determining primer in amplification goat mitochondrial DNA
The primer pair P1 of conservative region, the primer pair P2 in specific conservative region in amplification sheep mitochondrial DNA, amplification ox mitochondria
The primer pair P3 in the specific conservative region in DNA, specific conservative region in amplification buffalo mitochondrial DNA primer pair P4
In two pairs or more primers.
Preferably, the sequence of the primer pair P1 are as follows:
Upstream primer: 5'-CACCAAAATTCAACACAATACCACAT-3'
Downstream primer: 5'-AGCGTTATCTTTGTAATAGGTTTTGT-3'
The sequence of primer pair P2 are as follows:
Upstream primer: 5'-CGTAGATGTAGTATGACTTTTCCT-3'
Downstream primer: 5'-GTGAAGTTAGTTAGGAGAGTAATTATA-3'
The sequence of primer pair P3 are as follows:
Upstream primer: 5'-GTTAACAGCTAAACACCCTAGCT-3'
Downstream primer: 5'-AGGTTTGACTCCTCTTTTTACCAA-3'
The sequence of primer pair P4 are as follows:
Upstream primer: 5'-CCAAAATTTAACACAATCCCGCAA-3'
Downstream primer: 5'-CATTGGTCGTGGTTGAATTCCA-3'.
Preferably, the step 4), in step 5), the reaction system of multiplexed PCR amplification include DNA profiling, primer pair P1,
It P2, P3 and P4, PCR reaction reagent (for example, 2 × QIAGEN Multiplex PCR Master Mix), distilled water and is used for
The primer molar ratio of the saturated fluorescence dyestuff (for example, EvaGreen) of PCR real-time fluorescence amplification, P1:P2:P3:P4 is 5:7:4:
10。
Preferably, the step 4), in step 5), the response procedures of multiplexed PCR amplification are as follows: 94 DEG C of initial denaturation 5min；94
DEG C denaturation 15s, 57 DEG C of annealing 20s, 72 DEG C of extension 30s, 30 recycle；72 DEG C of extension 5min.
Preferably, the step 4), in step 5), program that high-resolution melts are as follows: closing the product of multiplexed PCR amplification
95 DEG C, while continuous fluorescence intensity are gradually heated to 0.05 DEG C/s rate since 65 DEG C under the conditions of pipe.
Preferably, the measuring samples are acquired from dairy products, lactogenesis or other biological sample.
It is a kind of for identifying the multiple fluorescence PCR kit of different target sequences, which includes for constructing multiplex PCR
The standard high-resolution of the reagent of the reaction system of amplification and the target sequence amplification product for a variety of target DNAs for comparison
Melting curve shape feature map (i.e. above-mentioned standard identification map), the standard high-resolution fusion curve shape feature map are
Be normalized by the high-resolution fusion curve of the amplified production to one of described target sequence and any amount combination,
Obtained from differentiation or principal component analysis；The reagent includes drawing for expanding the standard PCR specificity of corresponding target sequence
Object (for example, above-mentioned primer pair P1, P2, P3 and P4) and for PCR real-time fluorescence amplification saturated fluorescence dyestuff (for example,
EvaGreen)。
The beneficial effects of the present invention are embodied in:
The present invention utilizes multiplexed PCR amplification, can be to extract from the DNA of measuring samples as template, in a PCR reaction
The target sequence for expanding different target DNAs simultaneously, identifies in conjunction with segment of the high-resolution fusion curve to amplification, realizes stopped pipe
The purpose of multiple target sequences is detected simultaneously.The present invention is in the target sequence for solving to have similar Tm (Tm is differed within 0.5 ± 0.1 DEG C)
The melting peakss of amplified production, accumulation is a peak during multiplex PCR, thus can not be by analyzing the problem of Tm accurately identifies
When, it does not need to redesign primer or adjust design of primers (to consider length, the G/C content of amplified production, make new amplified production
Tm value have become apparent from difference), and verify it and expand validity and specificity.Therefore, the present invention improves detection efficiency, drop
Low testing cost.
Further, primer sequence provided by the invention improves the specificity of detection, avoids the generation of cross reaction,
Preferably realize the purpose for effectively identifying different target sequences.
Detailed description of the invention
Fig. 1 is that the target sequence of four target DNAs (ox, buffalo, goat, sheep) (is located in mitochondrial DNA) amplified production
Distinguish effect picture, in which: the normalization melting curve figure of (a) theoretical fitting；(b) obtained normalization melting curve figure is tested；
Fig. 2 is that the different target sequence amplification products of four target DNAs (ox, buffalo, goat, sheep) distinguish effect picture,
In: (a), the differentiation melting curve figure of (b) one pack system target sequence amplification product, principal component analysis (PCA) figure；(c), (d) two
Differentiation melting curve figure, principal component analysis (PCA) figure of component target sequence amplification product；(e), (f) three component and four components
Differentiation melting curve figure, principal component analysis (PCA) figure of target sequence amplification product；(g), (h) merges obtained different component
Differentiation melting curve figure, principal component analysis (PCA) figure of target sequence amplification product (15 kinds)；
Fig. 3 is the corresponding differentiation effect picture of different mixing proportion goat, cow genome group DNA, in which: (a) differentiation melts
Curve graph；(b) principal component analysis (PCA) figure；
Fig. 4 is the corresponding differentiation effect picture of different mixing proportion sheep, cow genome group DNA, in which: (a) differentiation melts
Curve graph；(b) principal component analysis (PCA) figure；
In figure: B, W, G, S respectively represent ox, buffalo, goat, sheep.
Specific embodiment
The present invention is described in further details with reference to the accompanying drawings and examples.The embodiment is only used for explaining this hair
It is bright, rather than to the limitation in the scope of the present invention.
For the identification detection of the different animals source constituent such as goat, sheep, ox, buffalo in dairy products, obtain first
The genomic DNA of goat, sheep, ox, buffalo: fresh cow milk sample picks up from the Xi'an Weiyang District cattle farm Cao Tan；Fresh sheep cream picks up from
Xi'an Weiyang District market；From Taobao, lactogenesis is saved backup in -20 DEG C for fresh buffalo's milk and the purchase of sheep cream；Each lactogenesis obtains
Time is in June, 2018.(Tiangeng is raw using Beads enrichment method genome DNA extracting reagent kit for extracting genome DNA in lactogenesis
Object Science and Technology Ltd.).
(1) the target sequence primer design of target DNA to be detected
According to the complete mtdna sequence of ox, buffalo, goat, sheep on NCBI, drawn using software design specificity
Object.Primer is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd, and primer sequence is, for example, that (the design of primers deadline is 2018
August):
Goat primer sequence (primer pair P1, reference sequences GU295658):
Upstream primer: 5'-CACCAAAATTCAACACAATACCACAT-3'
Downstream primer: 5'-AGCGTTATCTTTGTAATAGGTTTTGT-3'
Sheep primer sequence (primer pair P2, reference sequences AF010406):
Upstream primer: 5'-CGTAGATGTAGTATGACTTTTCCT-3'
Downstream primer: 5'-GTGAAGTTAGTTAGGAGAGTAATTATA-3'
Ox primer sequence (primer pair P3, reference sequences AF492351):
Upstream primer: 5'-GTTAACAGCTAAACACCCTAGCT-3'
Downstream primer: 5'-AGGTTTGACTCCTCTTTTTACCAA-3'
Buffalo primer sequence (primer pair P4, reference sequences NC-006295):
Upstream primer: 5'-CCAAAATTTAACACAATCCCGCAA-3'
Downstream primer: 5'-CATTGGTCGTGGTTGAATTCCA-3'.
For target sequence to be detected, it is fitted high-resolution fusion curve using u-Melt software, predicts that different target DNAs are same
When detect and identify distinguish a possibility that.Shown in fitting result such as Fig. 1 (a), show that each amplification target sequence has oneself in figure
The high-resolution fusion curve of specific characteristic, and the smallest goat of melting temperature difference and buffalo DNA (Tm differs 0.5 ± 0.1 DEG C),
It cannot be distinguished according to both melting temperatures, and can significantly be distinguished according to high-resolution fusion curve, explanation can pass through
The shape feature of melting curve identifies four (ox, buffalo, goat, sheep) different target DNAs.
Substance PCR reaction verifying:
Expand use PCR reaction system are as follows: 1 μ L (20ng/ μ L) template (ox, buffalo, goat or sheep genome
DNA), 10 μM of upstream primer and downstream primer each 0.25 μ L, 5 μ L 2 × Taq PCR Master Mix, 0.5 20 × Eva of μ L
Green dyestuff adds water to complement to 10 μ L.
PCR response procedures are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 15s, 57 DEG C of annealing 20s, 72 DEG C of extension 30s are carried out
30 circulations；Last 72 DEG C re-extend 5min.Fluorescence is acquired in annealing stage, for obtaining amplification curve.
High-resolution melt program are as follows: amplified production under the conditions of stopped pipe, melting temperature from 65 DEG C with 0.05 DEG C/s gradually
95 DEG C are warming up to, fluorescence signal is collected since 65 DEG C.
Verified, referring to Fig. 1 (b), the melting curve for testing acquisition is similar with the curve of theoretical fitting, therefore confirms benefit
With high-resolution fusion curve to ox, buffalo, goat, 4 species DNA discriminatory analysis of sheep feasibility.
(2) multi-PRC reaction
1. design of primers
The primer expanded using P1, P2, P3 and P4 as Quadruple- PCR.
2.PCR reaction
Expand the PCR reaction system used are as follows: 5 μ L 2 × QIAGEN Multiplex PCR Master Mix, 0.5 μ L
20 × Eva Green dyestuff, 1 μ L (20ng/ μ L) DNA profiling, it is P1:P2:P3:P4=5:7:4 that primer, which matches (molar ratio):
10, add water to complement to 10 μ L.Goat in end reaction system, sheep, ox and buffalo upstream and downstream primer concentration be respectively 0.25 μ
M, 0.35 μM, 0.4 μM and 1 μM.
PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 15s, 57 DEG C of annealing 20s, 72 DEG C of extension 30s are carried out
30 circulations；Last 72 DEG C re-extend 5min.Fluorescence is acquired in annealing stage, for obtaining amplification curve.
High-resolution melt program are as follows: amplified production under the conditions of stopped pipe, melting temperature from 65 DEG C with 0.05 DEG C/s gradually
95 DEG C are warming up to, fluorescence signal is collected since 65 DEG C.
3. detecting multiple target DNAs simultaneously
The detection of 3.1 15 kinds of difference DNA components
It is (single to be divided into template with 4 species (goat, sheep, ox and buffalo), 15 kinds of different genes group DNA group that may be present
4 kinds of component, 6 kinds of bi-component, three 4 kinds of components, a kind of four component), PCR amplification is carried out respectively, after the completion of amplification, is carried out immediately high
It differentiates and melts, after melting, melted to obtain to distinguish effect and more significantly analyze as a result, analyzing the differentiation that experiment obtains
Curve and PCA analysis chart, as a result as shown in Fig. 2 (a)-Fig. 2 (h), in differentiation melting curve analysis figure, each melting curve position
Between distinguish obvious and stablize, Tm value differs closer goat and buffalo is also distinguished significantly.Different purpose in PCA analysis chart
DNA is drawn in corresponding independent cluster, and it is obvious mutually to distinguish effect.Result above is proved by carrying out to amplified production
High-resolution fusion curve analysis (for example, difference analysis, PCA are analyzed), can effectively identify different target DNA and its mixing
Object.
The detection of 3.2 two kinds of target DNA different mixing proportion samples
(1) goat/ox sample of different proportion is distinguished:
Using cow genome group DNA and goat genomic DNA as control group, by the genomic DNA of ox and goat
(20ng/ μ L) is mixed according to the ratio (V/V) of 0:100,50:50,40:60,30:70,10:90,5:95,1:99,100:0 and is made
For template, 8 groups of amplification experiments are done, every group of experiment does three multiple holes (repetition) respectively, it is molten to carry out high-resolution after amplification respectively
Solution, obtain corresponding differentiation melting curve and principal component analysis figure (https: //www.metaboanalyst.ca//
Faces/ModuleView.xhtml), Fig. 3 (a) is shown, generates different special linearity curves respectively under different mixing proportion, and
Obvious, Fig. 3 (b) display is distinguished between each curve, although containing the different animal derived materials (genomes of ox and goat
DNA), but due to mixed proportion difference, different groups can be divided into, and three repetitions under each mixed proportion divided into it is main at
Divide identical one kind.
(2) sheep/ox sample of different proportion is distinguished:
Using cow genome group DNA and ovine genome DNA as control group, by the genomic DNA of sheep and ox
(20ng/ μ L) is mixed according to the ratio (V/V) of 0:100,50:50,60:40,70:30,90:10,95:5,99:1,100:0 and is made
For template, 8 groups of amplification experiments are done, every group of experiment does three multiple holes (repetition) respectively, it is molten to carry out high-resolution after amplification respectively
Solution, obtain corresponding differentiation melting curve and principal component analysis figure (https: //www.metaboanalyst.ca//
Faces/ModuleView.xhtml), Fig. 4 (a) is shown, generates different special linearity curves respectively under different mixing proportion, and
Obvious, Fig. 4 (b) display is distinguished between each curve, although containing the different animal derived materials (genomes of ox and sheep
DNA), but due to mixed proportion difference, different groups can be divided into, and three repetitions under each mixed proportion divided into it is main at
Divide identical one kind.
(3) Blind Test of analog sample
By carrying out Blind Test to analog sample, to assess the detection of multiplex PCR combination high-resolution fusion curve analysis method
Performance.Analysis shows that its recognition accuracy is 100%, show that this method has good reproducibility.
(4) the quadruple fluorescent PCR kit and application method of different target DNAs are detected simultaneously
(1) kit forms
Have in the kit: 2 × QIAGEN Multiplex PCR Master Mix, specific primer (P1, P2, P3,
P4), 20 × Eva Green and distilled water；Target target DNA (identifies map for constructing standard).
(2) kit application method
By 2 × QIAGEN Multiplex PCR Master Mix, 5 μ L, 20 × EvaGreen, 0.5 μ L, 10 μM of mesh
The corresponding specific primer of gene and distilled water mixing, obtain quadruple fluorescent PCR amplification reaction solution.According to experiment when use
Sample needs to be added template DNA, and end reaction system is 10 μ L.According to the PCR reaction condition of foundation, pass through Quadruple- PCR
Amplification (simultaneously can expand multiple target sequences in the same reaction system), molten in conjunction with the high-resolution to amplified production
Solution curve analyzes (according to the shape of melting curve), can judge target DNA whether is contained in sample to be tested.
In short, the invention discloses a kind of multi-PCR detection method and kit based on high-resolution fusion curve shape.
The present invention uses PCR saturated fluorescence dyestuff, and high-resolution melting is directly carried out after the completion of amplification, is returned to the melting curve of acquisition
One change, differentiation or principal component analysis, can be realized single tube effectively while detecting the purpose of multiple target sequences.It is molten according to high-resolution
Solution curve shape information rather than melting temperature carry out identification detection, significantly reduce the design difficulty of multiplex PCR assay primer,
Multiple pcr amplification products with small Tm difference are detected simultaneously；Avoiding open pipe operation causes target DNA dirty
A possibility that dye, compensates for the higher defect of single amplification reaction cost under multicolor fluorescence label or space separation in parallel,
Detection flux is improved, testing cost is reduced, there is stronger practical application value.
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Claims (7)
1. a kind of for identifying the multiple fluorescence PCR method of different target sequences, it is characterised in that: the multiple fluorescence PCR method packet
Include following steps:
1) situation is formed according to the DNA in different measuring samples, determines the different target DNA sequences for needing to identify；
2) designing and synthesizing has drawing for specific sequence fragment for expanding any a part in corresponding target DNA sequence
Object；
3) utilize substance PCR amplification verification step 2) in designed primer validity and specificity；
4) target DNA sequence and its any number of combination are analyzed respectively, to correspond to target DNA sequence or comprising purpose
The genomic DNA of DNA sequence dna be template, be utilized respectively by step 3) verifying the primer for different target DNA sequences into
Row multiplexed PCR amplification, and high-resolution melting is carried out to amplified production after the completion of amplification, using normalization, differentiation or master
Constituent analysis carries out feature extraction to the high-resolution fusion curve of acquisition, then will extract obtained characteristic information and merges, obtains
Standard identifies map；
5) extract DNA in certain measuring samples as template, according to step 4) carry out multiplexed PCR amplification and to amplified production into
Row high-resolution melts and obtains high-resolution fusion curve, uses to the high-resolution fusion curve and obtains the standard identification map
Same feature extracting method is analyzed, and analysis result is identified map with the standard and is compared, and is reflected according in the standard
The matched characteristic information of institute is compared in other map determines DNA composition situation in certain described measuring samples.
2. a kind of for identifying the multiple fluorescence PCR method of different target sequences according to claim 1, it is characterised in that: institute
It states in step 3), the segment of PCR amplification is located in the genomic DNA of corresponding animal species.
3. according to claim 1 or claim 2 a kind of for identifying the multiple fluorescence PCR method of different target sequences, it is characterised in that:
In the step 3), by the primer for examining specific conservative region of the determining primer in amplification goat mitochondrial DNA
To P1, amplification sheep mitochondrial DNA in specific conservative region primer pair P2, amplification ox mitochondrial DNA in specificity
The primer pair P3 of conservative region, specific conservative region in amplification buffalo mitochondrial DNA primer pair P4 in two pairs or more
Primer.
4. a kind of for identifying the multiple fluorescence PCR method of different target sequences according to claim 3, it is characterised in that: institute
State the sequence of primer pair P1 are as follows:
Upstream primer: 5'-CACCAAAATTCAACACAATACCACAT-3'
Downstream primer: 5'-AGCGTTATCTTTGTAATAGGTTTTGT-3'
The sequence of primer pair P2 are as follows:
Upstream primer: 5'-CGTAGATGTAGTATGACTTTTCCT-3'
Downstream primer: 5'-GTGAAGTTAGTTAGGAGAGTAATTATA-3'
The sequence of primer pair P3 are as follows:
Upstream primer: 5'-GTTAACAGCTAAACACCCTAGCT-3'
Downstream primer: 5'-AGGTTTGACTCCTCTTTTTACCAA-3'
The sequence of primer pair P4 are as follows:
Upstream primer: 5'-CCAAAATTTAACACAATCCCGCAA-3'
Downstream primer: 5'-CATTGGTCGTGGTTGAATTCCA-3'.
5. a kind of for identifying the multiple fluorescence PCR method of different target sequences according to claim 1, it is characterised in that: institute
State step 4), in step 5), the program that high-resolution melts are as follows: open the product of multiplexed PCR amplification from 65 DEG C under the conditions of stopped pipe
Begin to be gradually heated to 95 DEG C, while continuous fluorescence intensity with 0.05 DEG C/s rate.
6. a kind of for identifying the multiple fluorescence PCR method of different target sequences according to claim 1, it is characterised in that: institute
Measuring samples acquisition is stated from dairy products, lactogenesis or other biological sample.
7. a kind of for identifying the multiple fluorescence PCR kit of different target sequences, it is characterised in that: the kit includes being used for structure
Build the reagent of the reaction system of multiplexed PCR amplification and the target sequence amplification product for a variety of target DNAs for comparison
Standard high-resolution fusion curve shape feature map, the standard high-resolution fusion curve shape feature map is by described
One of target sequence and any amount combination amplified production high-resolution fusion curve be normalized, differentiation or
Obtained from principal component analysis；The reagent includes PCR primer for expanding corresponding target sequence and for PCR real-time fluorescence
The saturated fluorescence dyestuff of amplification.
Priority Applications (1)
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| CN111690753A (en) * | 2020-06-19 | 2020-09-22 | 陕西科技大学 | Single-tube detection kit and detection method for identifying 15 animal-derived components based on high-resolution melting curve shape |
| CN114606298A (en) * | 2020-12-08 | 2022-06-10 | 厦门致善生物科技股份有限公司 | Method for detecting length of one or more nucleic acid molecule amplification products in sample |
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