CN106191214B - PCR detection method for multicolor fluorescence melting curve - Google Patents
PCR detection method for multicolor fluorescence melting curve Download PDFInfo
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Abstract
The invention relates to the technical field of Polymerase Chain Reaction (PCR), in particular to a PCR detection method of a multicolor fluorescence melting curve, which adopts a bar code probe capable of being specifically hybridized with a target gene to be detected and a fluorescence detection probe capable of being specifically combined with the bar code probe to simultaneously detect a plurality of target genes in a sample to be detected, generates a specific bar code sequence corresponding to each target gene to be detected in the DNA amplification process, forms DNA products with different lengths and marked with the specificities of different fluorescent groups and quenching groups by the bar code sequence through DNA amplification, and realizes the PCR multiple detection based on melting curve analysis through final multicolor fluorescence melting curve analysis.
Description
Technical field
The present invention relates to polymerase chain reaction,PCR (PCR), more particularly to a kind of detection side multicolor fluorescence melting curve PCR
Method.
Background technique
Polymerase chain reaction,PCR (PCR) is a kind of technology for carrying out gene magnification in vitro using hot resistant DNA polymerase, in base
Because the multiple fields such as clone/medical diagnosis on disease/forensic identification are widely used.Regular-PCR technology needs after amplification, to product
Electrophoretic analysis is carried out, analytic process is cumbersome time-consuming.With TaqManTMThe inventions of hydrolysis probes and double-stranded DNA fluorescent dyestuff
It uses, PCR product can carry out analysis detection by the method for real-time fluorescence, and this new round pcr is referred to as real-time fluorescence
Quantitative PCR.Compared with regular-PCR technology, real-time fluorescence quantitative PCR has apparent advantage, Real-Time Fluorescent Quantitative PCR Technique
A possibility that DNA cloning carries out simultaneously with result detection, eliminates the electrophoretic analysis after reacting, while reducing pollution.
In the prior art, using TaqManTMThe principle of the real-time fluorescence quantitative PCR of hydrolysis probes is: in upstream primer
Downstream increases the TaqMan of an identification target sequenceTMHydrolysis probes, the 5 ' of probe and 3 ' ends mark luminophore respectively and quench
Go out group, quenching group by fluorescence resonance energy transfer (fluorescence resonance energy transfer,
FRET the fluorescent energy for) absorbing luminophore, is quenched the fluorescence signal of luminophore, during PCR amplification, archaeal dna polymerase
It holds from 5 ' toward 3 ' ends and extends together with upstream primer, extend to TaqManTMWhen at hydrolysis probes, the circumscribed core in the 5 ' of archaeal dna polymerase
Sour enzyme plays a role, and the hydrolysis probes since 5 ' ends, luminophore and quenching group separate, and luminophore is excited concurrently
Penetrate fluorescence.By the TaqMan that the fluorophor of a plurality of label different wave length in same reaction system, is arrangedTMHydrolysis probes,
Multiplex PCR detection can be achieved.But it is based on TaqManTMThe defect of the real-time fluorescence quantitative PCR detection method of hydrolysis probes exists
In: a kind of fluorophor of color can only one TaqMan of labelTMHydrolysis probes detect a testing gene, therefore, multiple
Detectability is limited.
In the prior art, it is using the principle of the real-time fluorescence quantitative PCR of double-stranded DNA fluorescent dyestuff: double-stranded DNA fluorescent dye
Material cannot be excited before being not embedded into DNA, once in insertion double-stranded DNA, so that it may it is excited and emits the glimmering of specific wavelength
Light, in the excessive situation of dyestuff, the power of the fluorescence signal detected is proportional with the amount of reactant double center chain DNA, therefore
It can be by the power of instrument record fluorescence signal come the concentration of DNA in real-time detection PCR product.On the basis of the technology
On, it is also been developed melting curve analysis method (Melting Curve analysis), melting curve analysis method is to utilize difference
There is DNA sequence dna the feature of different Tm values (temperature when double-spiral structure degradation half of DNA) to pass through after PCR reaction
The temperature of reactant is gradually increased, being denaturalized the double-stranded DNA with different Tm values successively becomes single stranded DNA, still, due to fluorescence
Dyestuff cannot be excited in conjunction with single stranded DNA, and therefore, at the corresponding temperature of the specific Tm value of double-stranded DNA, fluorescence intensity is significantly
Weaken, melting curve analysis method can be to different length DNA in PCR product or equal length difference sequence using this principle
DNA is analyzed.
With the raising to multiplex PCR detection demand, new technology is needed to upgrade original technology.But due to
What fluorophor itself emitted is not the fluorescence of single wavelength, and PCR instrument has the fluorescence discrimination that different fluorophors emit
Limit, therefore, existing PCR instrument can only detect the fluorescence signal of 4 ~ 6 kinds of fluorophors transmitting, this is but also using TaqManTMWater
The Multiple detection ability for solving the real-time fluorescence quantitative PCR of sonde method is restricted.And apply the real-time glimmering of double-stranded DNA fluorescent dyestuff
Fluorescent Quantitative PCR, since dyestuff does not have the recognition capability of distinguished sequence, can only be used in same reaction system a kind of double
Chain DNA fluorescent dye.
Summary of the invention
A kind of at low cost, Multiple detection ability is provided it is an object of the invention to avoid shortcoming in the prior art
Stronger multicolor fluorescence melting curve PCR detection method and the kit that testing gene is detected using this method.
The purpose of the present invention is achieved through the following technical solutions:
A kind of multicolor fluorescence melting curve PCR detection method is provided, comprising the following steps:
A, a pair of sequences is synthesized according to the specific sequence of target gene to be measured；
B, bar code detecting probe is synthesized, 3 ' ends of bar code detecting probe are the probe that target gene to be measured is located between two primers
Sequence, 5 ' ends are the bar code sequence with certain length of label quenching group；
C, synthesize fluorescent detection probe, fluorescent detection probe is the sequence with loop-stem structure, 3 ' ends for institute
The bar code recognition sequence of bar code sequence specific binding, 5 ' terminal sequences and 3 ' terminal sequence complementary pairings are stated, centre is any
One section it is uncorrelated to target gene to be measured and with the incoherent sequence of bar code sequence, 3 ' end and 5 ' end marks luminophore respectively
And quenching group；
D, PCR amplification:
1) in each round PCR reaction, after 92-97 DEG C of denaturing step, in 50-65 DEG C of annealing steps, probe sequence
It is identified with target gene to be measured, upstream primer and downstream primer and target gene to be measured identify；
2) in 58-75 DEG C of extension step, under archaeal dna polymerase effect, upstream primer and downstream primer start to extend, tool
There is the archaeal dna polymerase hydrolysis probes sequence of 5 ' -3 ' exonuclease activities, is released the bar code sequence of bar code detecting probe；
3) enter in next round PCR reaction, in 92-97 DEG C of denaturing step, the loop-stem structure of fluorescent detection probe is opened,
In 50-65 DEG C of annealing steps, the bar code recognition sequence of the bar code sequence and fluorescent detection probe that are discharged in last round of PCR cycle
Column specific binding；
4) enter 58-75 DEG C of extension step, the bar code sequence in last round of PCR cycle is using fluorescent detection probe as template
Synthetic dsdna product；
E, multicolor fluorescence melting curve tests and analyzes PCR product:
After PCR reaction terminates, using fluorescent detection probe as the double stranded DNA product of templated synthesis, with the liter of temperature
Height starts unwinding, and luminophore separates together with DNA chain with quenching group, the fluorescence for the different wave length that luminophore issues
It is detected by PCR instrument, PCR product is analyzed according to multicolor fluorescence melt curve analysis.
Melting curve analysis method is the feature for having different Tm values using different DNA sequence dnas, after PCR reaction, is passed through
The temperature of reactant is gradually increased, being denaturalized the double-stranded DNA with different Tm values successively becomes single stranded DNA, according to this principle
Different length DNA in PCR product or equal length difference sequence DNA can be analyzed.
In above-mentioned technical proposal, bar code detecting probe 3 ' hold probe sequence completely with target gene complementary pairing to be measured, no
The bar code detecting probe of complete complementary pairing cannot be by the archaeal dna polymerase hydrolysis release bar shaped with 5 ' -3 ' exonuclease activities
Code sequence.
In above-mentioned technical proposal, the fluorescence probe system includes multiple bar code detecting probes and multiple fluorescent detection probes,
The sequence length of the loop-stem structure of each fluorescent detection probe is different, so that different fluorescent detection probes is with different
Tm value forms the DNA product that the different length of specificity of different fluorophors and quenching group is marked after PCR amplification,
And by final multicolor fluorescence melting curve analysis, different Tm value DNA products are generated in multiple fluorescence channels and specifically melt song
Line peak, to realize the PCR Multiple detection based on melting curve analysis.
In above-mentioned technical proposal, the type of quenching group is any one in Dabcyl, BHQ-1, QYS-7, BHQ-2,
Fluorophor be Pacific Blue, Oregon Green, Bodipy FL-X, FAM, TET, Bodipy R6G-X, JOE, HEX,
Cy3, Rhodamine Red-X, TAMRA, any one in Cy3.5, Texas Red-X, ROX.
It is described the present invention also provides the kit using multicolor fluorescence melting curve PCR detection method detection testing gene
Kit includes:
A, bar code detecting probe, 3 ' ends are the probe sequence that target gene to be measured is located between two primers, and 5 ' ends are mark
Remember the bar code sequence with certain length of quenching group；
B, with the fluorescent detection probe of loop-stem structure, 3 ' ends are the bar shaped specifically bound with the bar code sequence
Code identification sequence, 5 ' terminal sequences and 3 ' terminal sequence complementary pairings, it is intermediate be any one section it is uncorrelated to target gene to be measured and with
The incoherent sequence of bar code sequence, 3 ' ends and 5 ' ends mark luminophore and quenching group respectively；
C, hot resistant DNA polymerase；
D, the pair of primers synthesized according to testing gene；
E, dATP, dTTP, dCTP and dGTP；
F, PCR buffer.
In above-mentioned technical proposal, the hot resistant DNA polymerase is poly- for the heat-resistant dna with 5 ' -3 ' exonuclease activities
Synthase.
In above-mentioned technical proposal, the PCR buffer be containing magnesium ion, Triton X-100, ammonium sulfate, potassium chloride,
The mixed solution of Tris-HCl.
Beneficial effects of the present invention:
A kind of multicolor fluorescence melting curve PCR detection method of the invention, using can be with target gene to be measured specificity
The bar code detecting probe of hybridization and the fluorescent detection probe that can be specifically bound with bar code detecting probe, to multiple in sample to be tested
It is corresponding with each target gene to be measured special by generating during DNA cloning when target gene is detected simultaneously
Bar code sequence, then the specificity that different fluorophors and quenching group are marked is formed by DNA cloning by bar code sequence
Different length DNA product, the fluorescence of each wavelength detected using PCR instrument can distinguish multiple and different Tm values
Melt curve analysis peak, and by final multicolor fluorescence melting curve analysis, different Tm value DNA products are generated in multiple fluorescence channels
Special melting curve peak comes whether interpretation detects target gene, to realize the PCR Multiple detection based on melting curve analysis.With
The prior art is compared, and the Multiple detection ability of detection method of the invention is stronger, can detect more target gene simultaneously,
And cost is relatively low, and this method is used to detect the kit of testing gene, suitable for the popularization in fields such as each medical treatment, scientific researches
Using.
Detailed description of the invention
Using attached drawing, the present invention will be further described, but the content in attached drawing does not constitute any limitation of the invention.
Fig. 1 is a kind of schematic illustration of multicolor fluorescence melting curve PCR detection method of the invention.
Fig. 2 is a kind of the glimmering of the FAM fluorophor of the embodiment of multicolor fluorescence melting curve PCR detection method of the invention
Light melting curve figure.
Fig. 3 is a kind of the glimmering of the HEX fluorophor of the embodiment of multicolor fluorescence melting curve PCR detection method of the invention
Light melting curve figure.
Appended drawing reference:
1 --- bar code detecting probe, 11 --- probe sequence, 12 --- bar code sequence；
2 --- fluorescent detection probe, 21 --- bar code recognition sequences；
R --- fluorophor；
Q --- quenching group；
Pol --- the archaeal dna polymerase with 5'-3' exonuclease activity.
Specific embodiment
With the following Examples and attached drawing the invention will be further described.
The present embodiment is congratulated by Multiple detection staphylococcus aureus FemA gene, single increasing listeria spp Hly gene, will
Salmonella IpaH gene and salmonella InvA gene, are illustrated detection method of the invention.
1, the design of target gene specific primer sequence and probe sequence to be measured:
1) the FemA gene order 13: DQ352463 of the common strain of staphylococcus aureus is recalled from GeneBank,
DQ352462, DQ352461, DQ352460, DQ352459, DQ352458, DQ352457, DQ352456, DQ352467,
DQ352466, DQ352465, DQ352464, DQ352455 carry out sequence alignment using ClustalW software, choose highly conserved
Section carry out design of primers as target, then by sequence inputting GeneBank, carry out BLAST comparison, determine the sequence
It is low with other species gene homologys in addition to staphylococcus aureus, it is soft that ABI primer Express 3.0 is reapplied later
Part design primer sequence and probe sequence guarantee that the Tm value of primer is 55 ± 3 DEG C, and the Tm value of probe sequence is 60-63 DEG C, amplification
Product length is 80-300 bp, finally carries out BLAST comparison to primer sequence and probe sequence, it is ensured that the spy of primer and probe
It is anisotropic.Primer sequence and probe sequence are as follows:
Upstream primer sequence: 5 '-gccatacagtcatttcacgca(SEQ ID NO:1)；
Downstream primer sequence: 5 '-acagcagtaagtaagcaagctg(SEQ ID NO:2)；
Probe sequence: 5 '-aactgttggccactatgagttaa；
2) the Hly gene order five: MLU25452 of the common strain of Listeria monocytogenes is recalled from GeneBank,
JF712527, JF712528, JF712529, JQ015301, referring to FemA gene primer and probe design process design primer and
Probe.Primer and probe sequence are as follows:
Upstream primer sequence: 5 '-gtgccgccaagaaaaggtta(SEQ ID NO:3)；
Downstream primer sequence: 5 '-tttcacgagagcacctggat(SEQ ID NO:4)；
Probe sequence: 5 '-ccaagttgtgaatgcaatttcgagcc(SEQ ID NO:5)；
3) the IpaH gene order of Shigella not homophyletic is recalled from GeneBank, wherein shigella flexneri not homophyletic
IpaH gene order four: M76445, DQ448042, DQ448040, FJ227542；Shigella dysenteriae not homophyletic IpaH gene
One: DQ132807；Shigella bogdii not homophyletic IpaH gene one: AKNA01000029；Shigella sonnei not homophyletic
IpaH gene six: DQ448041, DQ448039, KP116110, KP116109, KP116108, JQ638640, referring to FemA base
Because of primer and probe design process design primer and probe.Primer and probe sequence are as follows:
Upstream primer sequence: 5 '-aggacattgcccgggataaa(SEQ ID NO:6)；
Downstream primer sequence: 5 '-gacacgccatagaaacgcat(SEQ ID NO:7)；
Probe sequence: 5 '-agagaaacttcagctctccactgcc(SEQ ID NO:8)；
4) the InvA gene order nine: DQ644630 of common salmonella not homophyletic is recalled from GeneBank,
DQ644631, DQ644632, DQ644633, M90846, DQ644629, DQ644625, DQ644627, DQ644628, reference
FemA gene primer and probe design process design primer and probe.Primer and probe sequence are as follows:
Upstream primer sequence: 5 '-caacgtttcctgcggtactg(SEQ ID NO:9)；
Downstream primer sequence: 5 '-taacgaattgcccgaacgtg(SEQ ID NO:10)；
Probe sequence: 5 '-ccacgctctttcgtctggcattatc(SEQ ID NO:11).
2, the design of bar code detecting probe:
The nucleotide sequence that four length are 26 nt is designed, " bar code sequence " is named as, then respectively at this
The probe sequence with target gene specific bond to be measured designed in 3 ' end connection above-mentioned steps 1 of four bar code sequences, from
And designing four bar code detecting probes and numbering respectively is 1,2,3,4；The probe sequence point at the 3 ' ends of this four bar code detecting probes
Staphylococcus aureus FemA gene, single increasing listeria spp Hly gene, Shigella IpaH gene and sramana can not be identified
Salmonella InvA gene, 5 ' end label quenching groups, the type of quenching group is in Dabcyl, BHQ-1, QYS-7, BHQ-2
Any one.
The bar code detecting probe sequence of number 1 corresponding with testing gene staphylococcus aureus FemA gene and list respectively
Increase bar code detecting probe sequence, the number 3 corresponding with Shigella IpaH gene of the corresponding number 2 of listeria spp Hly gene
Bar code detecting probe sequence and number 4 corresponding with salmonella InvA gene bar code detecting probe sequence difference it is as follows:
1:5 '-quenching group-AGAGTCATGACTGTATGAGAGCACTC-aactgttggccactatgagttaa(SEQ
ID NO:12)；
2:5 '-quenching group-AGAATACAGACAGACATGTCTGACAC-ccaagttgtgaatgcaatttcgagcc
(SEQ ID NO:13)；
3:5 '-quenching group-AGTAGATCTGCATCTATAGAGTCGTC-agagaaacttcagctctccactgcc
(SEQ ID NO:14)；
4:5 '-quenching group-AGGATTACACTCACTGACATCTCCAC-ccacgctctttcgtctggcattatc
(SEQ ID NO:15).
, fluorescent detection probe design:
The 11 nt nucleotide sequences at 5 ' ends of the bar code sequence designed using in above-mentioned steps 2 are examined as corresponding fluorescence
5 ' ends of probing needle, using the inverted repeats of bar code sequence as 3 ' ends, centre has loop-stem structure and length for one section
Not equal nucleotide sequence, so that different fluorescent detection probes has different Tm values.5 ' end labels of fluorescent detection probe
Quenching group, the type of quenching group are any one in Dabcyl, BHQ-1, QYS-7, BHQ-2；Fluorescent detection probe
5 ' end mark fluorescent groups, fluorophor be Pacific Blue, Oregon Green, Bodipy FL-X, FAM, TET,
It is Bodipy R6G-X, JOE, HEX, any in Cy3, Rhodamine Red-X, TAMRA, Cy3.5, Texas Red-X, ROX
It is a kind of.
(1) fluorescent detection probe sequence (SEQ ID NO:16) corresponding with FemA gene:
5 '-quenching group-AGAGTCATGAC-ctgcacgct-GAGTGCTCTCATACAGTCATGACTCT-FAM；
(2) fluorescent detection probe sequence (SEQ ID NO:17) corresponding with Hly gene:
5 '-quenching group-AGAATACAGAC-ctgtgcactcgcacgcggctaac-
GTGTCAGACATGTCTGTCTGTATTCT-FAM；
(3) fluorescent detection probe sequence (SEQ ID NO:18) corresponding with IpaH gene:
5 '-quenching group-AGTAGATCTGC-ctgcacgct-GACGACTCTATAGATGCAGATCTACT-HEX；
(4) fluorescent detection probe sequence (SEQ ID NO:19) corresponding with InvA gene:
5 '-quenching group-AGGATTACACT-ctgtgcactcgcacgcggctaac-
GTGGAGATGTCAGTGAGTGTAATCCT-HEX。
4, the preparation and processing of sample:
The staphylococcus aureus being incubated overnight, single increasing listeria spp, Shigella, 200 μ l of salmonella are taken respectively
Into centrifuge tube, 10 times of gradient dilutions of physiological saline are used after mixing, dilute 10000 times；Bacterium solution after taking 1 ml to dilute is to 1.5ml
In centrifuge tube, 14000g is centrifuged 1 minute, abandoning supernatant, 100 μ l DNA extracting solutions of addition (10mM Tris-HCl (pH 7.5),
1% Triton X-100,2% Chelex-100,0.1mM EDTA), after mixing well, 95 DEG C of heating are cracked 15 minutes,
20000g is centrifuged 2 minutes, is extracted -20 DEG C of DNA solution of gained and is stored for future use.
5, PCR amplification and melting curve analysis:
PCR reaction solution contains: 1-3 mmol/l MgSO4,20 ~ 60 mmol/l (NH4)2SO4, 5 ~ 40 mmol/l KCl, 10
~ 30 Tris-HCl(pH8.1 ~ 8.8 mmol/l), 0.01 ~ 0.1% Triton X-100(v/v), 0.1 ~ 0.4 mmol/l
DNTPs, single primer 2 .5 ~ 15 μm ol/l, 0.01 ~ 0.2U/ of hot resistant DNA polymerase μ with 5 ' -3 ' exonuclease activities
L, UNG enzyme 0.01 ~ 0.05U/ μ l, 2.5 ~ 5 μm of ol/l of bar code detecting probe, 2.5 ~ 5 μm of ol/l of fluorescent detection probe, reaction solution are overall
20 ~ 50 μ l of product, 1 ~ 10 μ l of nucleic acid template is added in every person-portion, or 1 ~ 10 μ l sterile water is added as blank control.
This detection carries out in 96 instrument of CFX of Bio-Rad company, and response procedures are:
(1) 50 DEG C of 5min --- 95 DEG C of 10min；
(2) 95 DEG C of 15s --- 54 DEG C of 15s --- 72 DEG C of 20s, 50 circulations；
60 DEG C ~ 95 DEG C of (3) 95 DEG C of 1min --- 35 DEG C of 1min ---, wherein 60 DEG C ~ 95 DEG C of heatings with 0.4 DEG C/5s
Speed carries out melting curve analysis, and acquires FAM and HEX fluorescence signal.
6, result interpretation:
Above-mentioned steps " in 1,2,3 ", fluorescence detection corresponding with staphylococcus aureus and single increasing listeria spp respectively
FAM fluorophor is marked in probe, and HEX fluorescence is marked in fluorescent detection probe corresponding with Shigella, salmonella respectively
Group, melting curve is as shown in Figures 2 and 3, and since the length of four fluorescent detection probes is different, corresponding Tm value tag is not
Together, blank control detects signal without melting curve, is sentenced according to the appearance situation at melting curve peak special under corresponding fluorescence channel
It is disconnected whether to detect four kinds of microorganisms to be checked.The corresponding fluorophor of four kinds of tested microorganisms and Tm value relationship are as shown in table 1:
The corresponding fluorophor of 1. 4 kinds of tested microorganisms of table and Tm value relationship
As shown in Figures 2 and 3, method of the invention is to the staphylococcus aureus in sample to be tested, single increasing Listeria
Bacterium, Shigella, salmonella specific gene carried out Multiple detection, can be bent by melting under multiple fluorescence channels
Line peak comes whether interpretation detects target gene, to judge whether to detect object bacteria.The fluorescent detection probe that the present invention uses is only
It needs two kinds of fluorophors of label that can detect four kinds of target gene simultaneously, compares TaqManTMThe multiple inspection of hydrolysis probes method
Survey ability is stronger, and cost is relatively low.
Finally it should be noted that the above examples are only used to illustrate the technical scheme of the present invention rather than protects to the present invention
The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed
Solution, can with modification or equivalent replacement of the technical solution of the present invention are made, without departing from technical solution of the present invention essence and
Range.
<110> Steve Jia Chang Yu；Ai Jiesi Biotechnology Co., Ltd；Austria strong biotechnology (Guangzhou) has
Limit company
<120>a kind of multicolor fluorescence melting curve PCR detection method
<160> 19
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 1
gccatacagt catttcacgc a 21
<210> 2
<211> 22
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 2
acagcagtaa gtaagcaagc tg 22
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 3
gtgccgccaa gaaaaggtta 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 4
tttcacgaga gcacctggat 20
<210> 5
<211> 26
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 5
ccaagttgtg aatgcaattt cgagcc 26
<210> 6
<211> 20
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 6
aggacattgc ccgggataaa 20
<210> 7
<211> 20
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 7
gacacgccat agaaacgcat 20
<210> 8
<211> 25
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 8
agagaaactt cagctctcca ctgcc 25
<210> 9
<211> 20
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 9
caacgtttcc tgcggtactg 20
<210> 10
<211> 20
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 10
taacgaattg cccgaacgtg 20
<210> 11
<211> 25
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 11
ccacgctctt tcgtctggca ttatc 25
<210> 12
<211> 49
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 12
agagtcatga ctgtatgaga gcactcaact gttggccact atgagttaa 49
<210> 13
<211> 52
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 13
agaatacaga cagacatgtc tgacacccaa gttgtgaatg caatttcgag cc 52
<210> 14
<211> 51
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 14
agtagatctg catctataga gtcgtcagag aaacttcagc tctccactgc c 51
<210> 15
<211> 51
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 15
aggattacac tcactgacat ctccacccac gctctttcgt ctggcattat c 51
<210> 16
<211> 46
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 16
agagtcatga cctgcacgct gagtgctctc atacagtcat gactct 46
<210> 17
<211> 60
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 17
agaatacaga cctgtgcact cgcacgcggc taacgtgtca gacatgtctg tctgtattct 60
<210> 18
<211> 46
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 18
agtagatctg cctgcacgct gacgactcta tagatgcaga tctact 46
<210> 19
<211> 60
<212> DNA
<213>artificial sequence
<400> SEQ ID NO: 19
aggattacac tctgtgcact cgcacgcggc taacgtggag atgtcagtga gtgtaatcct 60
Claims (7)
1. a kind of multicolor fluorescence melting curve PCR detection method, it is characterised in that: the following steps are included:
A, pair of primers is synthesized according to the specific sequence of target gene to be measured；
B, bar code detecting probe is synthesized, 3 ' ends of bar code detecting probe are the probe sequence that target gene to be measured is located between two primers
Column, 5 ' ends are the bar code sequence with certain length of label quenching group；
C, fluorescent detection probe is synthesized, fluorescent detection probe is the sequence with loop-stem structure, and 3 ' ends are and the item
The bar code recognition sequence that shape code sequence-specific combines, 5 ' terminal sequences and 3 ' terminal sequence complementary pairings, centre are any one section
It is uncorrelated to target gene to be measured and with the incoherent sequence of bar code sequence, label and is quenched luminophore respectively for 3 ' ends and 5 ' ends
Go out group；
D, PCR amplification:
1) in each round PCR reaction, after denaturing step, in annealing steps, probe sequence and target gene to be measured are identified,
Upstream primer and downstream primer and target gene to be measured identify；
2) in extending step, under archaeal dna polymerase effect, upstream primer and downstream primer start to extend, circumscribed with 5 ' -3 '
The archaeal dna polymerase hydrolysis probes sequence of nuclease, is released the bar code sequence of bar code detecting probe；
3) enter in next round PCR reaction, in denaturing step, the loop-stem structure of fluorescent detection probe is opened, in annealing steps,
The bar code sequence discharged in last round of PCR cycle is in conjunction with the bar code recognition sequence-specific of fluorescent detection probe；
4) enter and extend step, bar code sequence is using fluorescent detection probe as templated synthesis double stranded DNA product；
E, multicolor fluorescence melting curve tests and analyzes PCR product:
After PCR reaction terminates, using fluorescent detection probe as the double stranded DNA product of templated synthesis, open as the temperature rises
Beginning unwinding, luminophore separate together with DNA chain with quenching group, and the fluorescence for the different wave length that luminophore issues is glimmering
The detection of light PCR instrument analyzes PCR product according to multicolor fluorescence melt curve analysis.
2. a kind of multicolor fluorescence melting curve PCR detection method according to claim 1, it is characterised in that: bar code is visited
Completely with target gene complementary pairing to be measured, the bar code detecting probe of not fully complementary pairing cannot be had the probe sequence that needle 3 ' is held
There is the archaeal dna polymerase hydrolysis release bar code sequence of 5 ' -3 ' exonuclease activities.
3. a kind of multicolor fluorescence melting curve PCR detection method according to claim 1, it is characterised in that: the fluorescence
Probe system includes multiple bar code detecting probes and multiple fluorescent detection probes, the sequence of the loop-stem structure of each fluorescent detection probe
Length is different.
4. a kind of multicolor fluorescence melting curve PCR detection method according to claim 1,2 or 3, it is characterised in that: described
Quenching group be Dabcyl, BHQ-1, QYS-7, BHQ-2 in any one, the fluorophor be Pacific Blue,
Oregon Green、Bodipy FL-X、FAM、TET、Bodipy R6G-X、JOE、HEX、Cy3、Rhodamine Red-X、
Any one in TAMRA, Cy3.5, Texas Red-X, ROX.
5. detecting testing gene using multicolor fluorescence melting curve PCR detection method described in Claims 1-4 any one
Kit, it is characterised in that: the kit includes:
A, bar code detecting probe, 3 ' ends are the probe sequence that target gene to be measured is located between two primers, and 5 ' ends are quenched for label
Go out the bar code sequence with certain length of group；
B, with the fluorescent detection probe of loop-stem structure, 3 ' ends are known for the bar code specifically bound with the bar code sequence
Other sequence, 5 ' terminal sequences and 3 ' terminal sequence complementary pairings, centre are any one section of and and bar shaped uncorrelated to target gene to be measured
The code incoherent sequence of sequence, 3 ' ends and 5 ' ends mark luminophore and quenching group respectively；
C, hot resistant DNA polymerase；
D, the pair of primers synthesized according to testing gene；
E, dATP, dTTP, dCTP and dGTP；
F, PCR buffer.
6. the kit according to claim 5 using multicolor fluorescence melting curve PCR detection method detection testing gene,
It is characterized by: the hot resistant DNA polymerase is the hot resistant DNA polymerase with 5 ' -3 ' exonuclease activities.
7. the reagent of the detection testing gene according to claim 5 using multicolor fluorescence melting curve PCR detection method
Box, it is characterised in that: the PCR buffer be containing magnesium ion, Triton X-100, ammonium sulfate, potassium chloride, Tris-HCl it is mixed
Close solution.
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| CN108823287B (en) * | 2017-04-28 | 2019-04-12 | 厦门大学 | A method of detection target nucleic acid sequence |
| CN108531566A (en) * | 2018-04-17 | 2018-09-14 | 厦门基科生物科技有限公司 | DNA detection methods and composite coding probe based on the hybridization of composite coding probe |
| CN108796047A (en) * | 2018-05-31 | 2018-11-13 | 江洪 | Primer 5 ' holds the fluorescent PCR of reverse complemental |
| CN111100908B (en) * | 2018-10-26 | 2022-07-12 | 厦门大学 | Method and kit for detecting deletion of nucleotide fragment |
| CN111926063B (en) * | 2020-08-21 | 2021-03-23 | 中国人民解放军陆军军医大学第一附属医院 | DNA molecule detection method using 3D bar code |
| 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 |
| CN114317699B (en) * | 2021-12-23 | 2023-01-10 | 郑州华之源医学检验实验室有限公司 | Melting curve positive and negative peak shape analysis-based multiplex PCR detection method and application |
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| CN101899499A (en) * | 2009-05-26 | 2010-12-01 | 厦门大学 | Method for detecting human beta-globin gene mutation |
| CN102016075A (en) * | 2008-05-06 | 2011-04-13 | 凯杰有限公司 | Simultaneous detection of multiple nucleic acid sequences in a reaction |
| CN103276094A (en) * | 2013-06-08 | 2013-09-04 | 瀚吉康生物科技（北京）有限公司 | Multi-PCR detection method and kit |
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| CN102016075A (en) * | 2008-05-06 | 2011-04-13 | 凯杰有限公司 | Simultaneous detection of multiple nucleic acid sequences in a reaction |
| CN101899499A (en) * | 2009-05-26 | 2010-12-01 | 厦门大学 | Method for detecting human beta-globin gene mutation |
| CN103276094A (en) * | 2013-06-08 | 2013-09-04 | 瀚吉康生物科技（北京）有限公司 | Multi-PCR detection method and kit |
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