CN106191214A - A kind of multicolor fluorescence melting curve PCR detection method - Google Patents
A kind of multicolor fluorescence melting curve PCR detection method Download PDFInfo
- Publication number
- CN106191214A CN106191214A CN201510215155.1A CN201510215155A CN106191214A CN 106191214 A CN106191214 A CN 106191214A CN 201510215155 A CN201510215155 A CN 201510215155A CN 106191214 A CN106191214 A CN 106191214A
- Authority
- CN
- China
- Prior art keywords
- sequence
- bar code
- pcr
- probe
- melting curve
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to polymerase chain reaction,PCR (PCR) technical field, particularly relate to a kind of multicolor fluorescence melting curve PCR detection method, this detection method use can with the bar code detecting probe of target gene specific hybrid to be measured and can be specific binding with bar code detecting probe fluorescent detection probe, multiple target genes in testing sample are detected simultaneously, by during DNA cloning, generate the special bar code sequence corresponding with each target gene to be measured, form the DNA product of the specific different length that marked different fluorophor and quenching group again through DNA cloning by bar code sequence, and by final multicolor fluorescence melting curve analysis, thus realize PCR Multiple detection based on melting curve analysis.
Description
Technical field
The present invention relates to polymerase chain reaction,PCR (PCR), particularly relate to a kind of multicolor fluorescence melting curve PCR detection method.
Background technology
Polymerase chain reaction,PCR (PCR) is a kind of technology utilizing hot resistant DNA polymerase to carry out gene amplification in vitro, is widely used in multiple fields such as gene clone/medical diagnosis on disease/forensic identifications.Regular-PCR technology needs after amplification, and product carries out electrophoretic analysis, analyzes process loaded down with trivial details time-consuming.Along with TaqManTMThe invention of hydrolysis probes, and the use of double-stranded DNA fluorescent dyestuff, PCR primer can be analyzed 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 obvious advantage, and the DNA cloning of Real-Time Fluorescent Quantitative PCR Technique is carried out with result detection simultaneously, eliminates reacted electrophoretic analysis, reduces the probability of pollution simultaneously.
In prior art, apply TaqManTMThe principle of the real-time fluorescence quantitative PCR of hydrolysis probes is: increase a TaqMan identifying target sequence in the downstream of forward primerTMHydrolysis probes, the 5 ' of probe and 3 ' ends labelling luminophore and quenching group respectively, quenching group passes through FRET (fluorescence resonance energy transfer) (fluorescence resonance energy
Transfer, FRET) absorb luminophore fluorescent energy, the fluorescence signal of cancellation luminophore, in PCR amplification procedure, archaeal dna polymerase together with forward primer from 5 ' end toward 3 ' end extend, extend to TaqManTMTime at hydrolysis probes, 5 ' exonucleases of archaeal dna polymerase play a role, and start hydrolysis probes from 5 ' ends, and luminophore and quenching group separate, and luminophore is excited and launches fluorescence.By in same reaction system, the TaqMan of the fluorophor of a plurality of labelling different wave length is setTMHydrolysis probes, can realize multiplex PCR detection.But, based on TaqManTMThe defect of the real-time fluorescence quantitative PCR detection method of hydrolysis probes is: the fluorophor of a kind of color can only one TaqMan of labellingTMHydrolysis probes, detects a testing gene, and therefore, its Multiple detection is limited in one's ability.
In prior art, the principle of the real-time fluorescence quantitative PCR of application double-stranded DNA fluorescent dyestuff is: double-stranded DNA fluorescent dyestuff is before being not embedded into DNA, can not be excited, once embed in double-stranded DNA, just can be excited and launch the fluorescence of specific wavelength, in the case of dyestuff excess, the power of the fluorescence signal detected is proportional with the amount of reactant double center chain DNA, therefore can detect the concentration of DNA in PCR primer in real time by the power of instrument record fluorescence signal.On the basis of this technology, also been developed melting curve analysis method (Melting Curve analysis), melting curve analysis method be utilize different DNA sequence have different Tm value (double-spiral structure of DNA degrade half time temperature) feature, after PCR reacts, by gradually stepping up the temperature of reactant, the double-stranded DNA degeneration successively with different Tm value is made to become single stranded DNA, but, it is excited owing to fluorescent dye can not be combined with single stranded DNA, therefore, at a temperature of double-stranded DNA specific T m value correspondence, fluorescence intensity significantly weakens, melting curve analysis method utilizes this principle can be analyzed different length DNA in PCR primer or equal length difference sequence DNA.
Along with the raising to multiplex PCR detection demand, need new technique that original technology is upgraded.But, launch due to fluorophor itself is not the fluorescence of single wavelength, and the phosphor region indexing that different fluorophors are launched by PCR instrument is limited, and therefore, existing PCR instrument can only detect the fluorescence signal that 4 ~ 6 kinds of fluorophors are launched, and this also makes to apply TaqManTMThe Multiple detection ability of the real-time fluorescence quantitative PCR of hydrolysis probes method is restricted.And apply the real-time fluorescence quantitative PCR of double-stranded DNA fluorescent dyestuff, do not possess the identification ability of distinguished sequence due to dyestuff, therefore, same reaction system can only use a kind of double-stranded DNA fluorescent dyestuff.
Summary of the invention
It is an object of the invention to avoid weak point of the prior art and the higher multicolor fluorescence melting curve PCR detection method of a kind of low cost, Multiple detection ability is provided and uses the test kit of the method detection testing gene.
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, comprises the following steps:
A, synthesize pair of sequences according to the specific sequence of target gene to be measured；
B, synthesis bar code detecting probe, 3 ' ends of bar code detecting probe are the target gene to be measured probe sequence between two primers, and 5 ' ends are the bar code sequence with certain length of labelling quenching group；
C, synthesis fluorescent detection probe, fluorescent detection probe is a sequence with loop-stem structure, its 3 ' end is the bar code recognition sequence specific binding with described bar code sequence, 5 ' terminal sequences and 3 ' terminal sequence complementary pairings, centre is any one section of uncorrelated with target gene to be measured and incoherent with bar code sequence sequence, 3 ' ends and 5 ' ends labelling luminophore and quenching group respectively；
D, PCR expand:
1) each take turns in PCR reaction, after 92-97 DEG C of denaturing step, at 50-65 DEG C of annealing steps, probe sequence and target gene identification to be measured, forward primer and downstream primer and target gene identification to be measured；
2) extending in step at 58-75 DEG C, under archaeal dna polymerase effect, forward primer and downstream primer start to extend, and have 5 '-3 ' the archaeal dna polymerase hydrolysis probes sequence of exonuclease activity, make the bar code sequence of bar code detecting probe be released；
3) entering in the reaction of next round PCR, in 92-97 DEG C of denaturing step, the loop-stem structure of fluorescent detection probe is opened, and in 50-65 DEG C of annealing steps, in last round of PCR cycle, the bar code sequence of release is combined with the bar code recognition sequence-specific of fluorescent detection probe；
4) entering 58-75 DEG C and extend step, the bar code sequence in last round of PCR cycle is with fluorescent detection probe for templated synthesis double stranded DNA product；
E, multicolor fluorescence melting curve detection analysis PCR primer:
After PCR reaction terminates, double stranded DNA product with fluorescent detection probe as templated synthesis, along with the rising of temperature starts to unwind, luminophore separates together with DNA with quenching group, the fluorescence of the different wave length that luminophore sends is detected by PCR instrument, analyzes PCR primer according to multicolor fluorescence melt curve analysis.
Melting curve analysis method is the feature utilizing different DNA sequence to have different Tm value, after PCR reacts, by gradually stepping up the temperature of reactant, make the double-stranded DNA degeneration successively with different Tm value become single stranded DNA, different length DNA in PCR primer or equal length difference sequence DNA can be analyzed according to this principle.
In technique scheme, the probe sequence that bar code detecting probe 3 ' is held completely with target gene complementary pairing to be measured, the bar code detecting probe of not fully complementary pairing can not be had 5 '-3 ' the archaeal dna polymerase hydrolysis release bar code sequence of exonuclease activity.
In technique scheme, described fluorescent probe system includes multiple bar code detecting probe and multiple fluorescent detection probe, the sequence length of the loop-stem structure of each fluorescent detection probe is different, so that different fluorescent detection probes has different Tm values, the DNA product of the specific different length that marked different fluorophor and quenching group is formed after PCR expands, and by final multicolor fluorescence melting curve analysis, produce different Tm value DNA product special melting curve peak at multiple fluorescence channels, thus realize PCR Multiple detection based on melting curve analysis.
In technique scheme, the kind of quenching group is any one in Dabcyl, BHQ-1, QYS-7, BHQ-2, and fluorophor is Pacific Blue, Oregon
Any one in Green, Bodipy FL-X, FAM, TET, Bodipy R6G-X, JOE, HEX, Cy3, Rhodamine Red-X, TAMRA, Cy3.5, Texas Red-X, ROX.
The present invention also provides for using the test kit of multicolor fluorescence melting curve PCR detection method detection testing gene, and described test kit includes:
A, bar code detecting probe, its 3 ' end is the target gene to be measured probe sequence between two primers, and 5 ' ends are the bar code sequence with certain length of labelling quenching group；
B, there is the fluorescent detection probe of loop-stem structure, its 3 ' end is the bar code recognition sequence specific binding with described bar code sequence, 5 ' terminal sequences and 3 ' terminal sequence complementary pairings, centre is any one section of uncorrelated with target gene to be measured and incoherent with bar code sequence sequence, 3 ' ends and 5 ' ends labelling 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 technique scheme, described hot resistant DNA polymerase is to have 5 '-3 ' hot resistant DNA polymerase of exonuclease activity.
In technique scheme, described PCR buffer is containing magnesium ion, Triton X-100, ammonium sulfate, potassium chloride, the mixed solution of Tris-HCl.
Beneficial effects of the present invention:
nullA kind of multicolor fluorescence melting curve PCR detection method of the present invention，Employing can with the bar code detecting probe of target gene specific hybrid to be measured and can be specific binding with bar code detecting probe fluorescent detection probe，When multiple target genes in testing sample are detected simultaneously，By during DNA cloning，Generate the special bar code sequence corresponding with each target gene to be measured，Form the DNA product of the specific different length that marked different fluorophor and quenching group again through DNA cloning by bar code sequence，The fluorescence of wavelength can distinguish the melt curve analysis peak of multiple different Tm value to utilize PCR instrument each of to detect，And by final multicolor fluorescence melting curve analysis，Produce different Tm value DNA product special melting curve peak at multiple fluorescence channels and come whether interpretation detects target gene，Thus realize PCR Multiple detection based on melting curve analysis.Compared with prior art, the Multiple detection ability of the detection method of the present invention is higher, it is possible to detect more genes of interest simultaneously, and cost is relatively low, the method is for detecting the test kit of testing gene, it is adaptable in the popularization and application in the fields such as each medical treatment, scientific research.
Accompanying drawing explanation
The present invention will be further described to utilize accompanying drawing, but the content in accompanying drawing does not constitute any limitation of the invention.
Fig. 1 is the principle schematic of a kind of multicolor fluorescence melting curve PCR detection method of the present invention.
Fig. 2 is the fluorescence melting curve figure of the FAM fluorophor of the embodiment of a kind of multicolor fluorescence melting curve PCR detection method of the present invention.
Fig. 3 is the fluorescence melting curve figure of the HEX fluorophor of the embodiment of a kind of multicolor fluorescence melting curve PCR detection method of the present invention.
Reference:
1 bar code detecting probe, 11 probe sequences, 12 bar code sequences；
2 fluorescent detection probes, 21 bar code recognition sequences；
R fluorophor；
Q quenching group；
Pol has the archaeal dna polymerase of 5'-3' exonuclease activity.
Detailed description of the invention
With the following Examples and accompanying drawing the invention will be further described.
The detection method of the present invention, by Multiple detection staphylococcus aureus FemA gene, single increasing listeria spp Hly gene, shigella IpaH gene and Salmonella InvA gene, is illustrated by the present embodiment.
1, target gene specific primer sequence to be measured and the design of probe sequence:
1) from GeneBank, recall the FemA gene order 13: DQ352463 of the common strain of staphylococcus aureus, DQ352462, DQ352461, DQ352460, DQ352459, DQ352458, DQ352457, DQ352456, DQ352467, DQ352466, DQ352465, DQ352464, DQ352455, application ClustalW software carries out sequence alignment, the section choosing high conservative carries out design of primers as target, then by this sequence inputting GeneBank, carry out BLAST comparison, determine that this sequence is low with other species gene homology in addition to staphylococcus aureus, reapply ABI afterwards
Primer Express 3.0 software design primer sequence and probe sequence, the Tm value of guarantee primer is 55 ± 3 DEG C, and the Tm value of probe sequence is 60-63 DEG C, amplified production a length of 80-300 bp, finally primer sequence and probe sequence are carried out BLAST comparison, it is ensured that primer and the specificity of probe.Primer sequence and probe sequence are as follows:
Forward primer sequence: 5 '-gccatacagtcatttcacgca(SEQ ID NO:1)；
Downstream primer sequence: 5 '-acagcagtaagtaagcaagctg(SEQ ID NO:2)；
Probe sequence: 5 '-aactgttggccactatgagttaa；
2) from GeneBank, the Hly gene order five of the common strain of Listeria monocytogenes is recalled: MLU25452, JF712527, JF712528, JF712529, JQ015301, reference FemA gene primer and probe design process design primer and probe.Primer and probe sequence are as follows:
Forward 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) from GeneBank, the IpaH gene order of shigella not homophyletic, wherein shigella flexneri not homophyletic IpaH gene order four are recalled: 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, design primer and probe with reference to FemA gene primer and probe design process.Primer and probe sequence are as follows:
Forward 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) from GeneBank, recall the InvA gene order nine: DQ644630 of common Salmonella not homophyletic, DQ644631, DQ644632, DQ644633, M90846, DQ644629, DQ644625, DQ644627, DQ644628, design primer and probe with reference to FemA gene primer and probe design process.Primer and probe sequence are as follows:
Forward 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:
Design the nucleotide sequence of four a length of 26 nt, by its named " bar code sequence ", 3 ' the ends at these four bar code sequences connect the probe sequence with target gene specific bond to be measured of design in above-mentioned steps 1 the most respectively, thus design four bar code detecting probes difference numbered 1,2,3,4；The probe sequence that the 3 ' of these four bar code detecting probes are held is able to identify staphylococcus aureus FemA gene, single increasing listeria spp Hly gene, shigella IpaH gene and Salmonella InvA gene, its 5 ' end labelling quenching group, the kind of quenching group is any one in Dabcyl, BHQ-1, QYS-7, BHQ-2.
The bar code detecting probe sequence of the numbering 3 that the bar code detecting probe sequence of the numbering 1 answered with testing gene staphylococcus aureus FemA gene pairs respectively and single bar code detecting probe sequence increasing the numbering 2 that listeria spp Hly gene pairs is answered are answered with shigella IpaH gene pairs and the bar code detecting probe sequence of numbering 4 answered with Salmonella InvA gene pairs are as follows:
1:5 '-quenching group-AGAGTCATGACTGTATGAGAGCACTC-aactgttggccactatgagttaa(SEQ ID NO:12)；
2:5 '-quenching group-AGAATACAGACAGACATGTCTGACAC-ccaagttgtgaatgcaatttcgagcc(SE Q ID NO:13)；
3:5 '-quenching group-AGTAGATCTGCATCTATAGAGTCGTC-agagaaacttcagctctccactgcc(SEQ ID NO:14)；
4:5 '-quenching group-AGGATTACACTCACTGACATCTCCAC-ccacgctctttcgtctggcattatc(SEQ ID NO:15).
, the design of fluorescent detection probe:
In above-mentioned steps 2,5 ' the 11 nt nucleotide sequences held of the bar code sequence of design are as 5 ' ends of corresponding fluorescent detection probe, using the inverted repeat of bar code sequence as 3 ' ends, centre is one section of nucleotide sequence not having loop-stem structure and length not etc. so that different fluorescent detection probes has different Tm values.5 ' end labelling quenching groups of fluorescent detection probe, the kind of quenching group is any one in Dabcyl, BHQ-1, QYS-7, BHQ-2；5 ' end mark fluorescent groups of fluorescent detection probe, fluorophor is Pacific Blue, Oregon
Any one in Green, Bodipy FL-X, FAM, TET, Bodipy R6G-X, JOE, HEX, Cy3, Rhodamine Red-X, TAMRA, Cy3.5, Texas Red-X, ROX.
(1) the fluorescent detection probe sequence (SEQ ID NO:16) answered with FemA gene pairs:
5 '-quenching group-AGAGTCATGAC-ctgcacgct-GAGTGCTCTCATACAGTCATGACTCT-FAM；
(2) the fluorescent detection probe sequence (SEQ ID NO:17) answered with Hly gene pairs:
5 '-quenching group-AGAATACAGAC-ctgtgcactcgcacgcggctaac-GTGTCAGACATGTCTGTCTG TATTCT-FAM；
(3) the fluorescent detection probe sequence (SEQ ID NO:18) answered with IpaH gene pairs:
5 '-quenching group-AGTAGATCTGC-ctgcacgct-GACGACTCTATAGATGCAGATCTACT-HEX；
(4) the fluorescent detection probe sequence (SEQ ID NO:19) answered with InvA gene pairs:
5 '-quenching group-AGGATTACACT-ctgtgcactcgcacgcggctaac-GTGGAGATGTCAGTGAGTGT AATCCT-HEX.
4、The preparation of sample and process:
Take respectively in the staphylococcus aureus of incubated overnight, single increasing listeria spp, shigella, Salmonella 200 μ l to centrifuge tube, with 10 times of gradient dilutions of normal saline after mixing, dilute 10000 times；Taking the bacterium solution after 1 ml dilution in 1.5ml centrifuge tube, 14000g is centrifuged 1 minute, abandons supernatant, adds 100 μ l DNA extracting solution (10mM Tris-HCl (pH 7.5), 1% Triton X-100,2% Chelex-100,0.1mM
EDTA), fully after mixing, 95 DEG C add thermal cracking 15 minutes, and 20000g is centrifuged 2 minutes, extract gained DNA solution-20 DEG C and store for future use.
5、PCR Amplification and melting curve analysis:
PCR reactant liquor contains: 1-3 mmol/l MgSO4,20 ~ 60 mmol/l (NH4)2SO4, 5 ~ 40 mmol/l KCl, 10 ~ 30
Mmol/l Tris-HCl(pH8.1 ~ 8.8), 0.01 ~ 0.1% Triton X-100(v/v), 0.1 ~ 0.4 mmol/l dNTPs, wall scroll primer 2 .5 ~ 15
μm ol/l, have 5 '-3 ' hot resistant DNA polymerase 0.01 ~ 0.2U/ μ l of exonuclease activity, UNG enzyme 0.01 ~ 0.05U/ μ l, bar code detecting probe 2.5 ~ 5 μm ol/l, fluorescent detection probe 2.5 ~ 5 μm ol/l, reactant liquor cumulative volume 20 ~ 50 μ l, every person-portion adds nucleic acid masterplate 1 ~ 10 μ l, or adds 1 ~ 10 μ l sterilized water as blank.
This detection is carried out in CFX 96 instrument of Bio-Rad company, and response procedures is:
95 DEG C of 10min of (1) 50 DEG C of 5min；
(2) 95 DEG C of 15s 72 DEG C of 20s of 54 DEG C of 15s, 50 circulations；
35 DEG C of 1min of (3) 95 DEG C of 1min 60 DEG C ~ 95 DEG C, wherein 60 DEG C ~ 95 DEG C carry out melting curve analysis with the programming rate of 0.4 DEG C/5s, and gather FAM and HEX fluorescence signal.
6, result interpretation:
In above-mentioned steps " 1,2,3 ", marked FAM fluorophor with staphylococcus aureus and single fluorescent detection probe corresponding to listeria spp that increase respectively, fluorescent detection probe corresponding with shigella, Salmonella respectively marked HEX fluorophor,Melting curve such as figure 2 And figure 3 Shown in,Owing to the length of four fluorescent detection probes is different, corresponding Tm value tag is different, and blank detects signal without melting curve, judges whether to detect four kinds of microorganisms to be checked according to the appearance situation at melting curve peak special under corresponding fluorescence channel.Fluorophor and Tm value relation that four kinds of tested microorganism are corresponding are as shown in table 1:
Fluorophor that 1. 4 kinds of tested microorganism of table are corresponding and Tm value relation
As shown in Figures 2 and 3, the method of the present invention has carried out Multiple detection to the staphylococcus aureus in testing sample, single increasing listeria spp, shigella, the specific gene of Salmonella, can come whether interpretation detects target gene by the melting curve peak under multiple fluorescence channels, thus judge whether to detect object bacteria.The fluorescent detection probe that the present invention uses has only to two kinds of fluorophors of labelling and just can detect four kinds of genes of interest simultaneously, compares TaqManTMThe Multiple detection ability of hydrolysis probes method is higher, and cost is relatively low.
Finally should be noted that; above example is merely to illustrate technical scheme rather than limiting the scope of the invention; although the present invention being explained in detail with reference to preferred embodiment; it will be understood by those within the art that; technical scheme can be modified or equivalent, without deviating from the spirit and scope of technical solution of the present invention.
<110> Steve Jia Chang
Yu；Ai Jiesi bio tech ltd；Strong biotechnology (Guangzhou) company limited difficult to understand
<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 multicolor fluorescence melting curve PCR detection method, it is characterised in that: comprise the following steps:
A, synthesize pair of primers according to the specific sequence of target gene to be measured；
B, synthesis bar code detecting probe, 3 ' ends of bar code detecting probe are the target gene to be measured probe sequence between two primers, and 5 ' ends are the bar code sequence with certain length of labelling quenching group；
C, synthesis fluorescent detection probe, fluorescent detection probe is a sequence with loop-stem structure, its 3 ' end is the bar code recognition sequence specific binding with described bar code sequence, 5 ' terminal sequences and 3 ' terminal sequence complementary pairings, centre is any one section of uncorrelated with target gene to be measured and incoherent with bar code sequence sequence, 3 ' ends and 5 ' ends labelling luminophore and quenching group respectively；
D, PCR expand:
1) each take turns in PCR reaction, after denaturing step, at annealing steps, probe sequence and target gene identification to be measured, forward primer and downstream primer and target gene identification to be measured；
2) extending in step, under archaeal dna polymerase effect, forward primer and downstream primer start to extend, and have 5 '-3 ' the archaeal dna polymerase hydrolysis probes sequence of exonuclease activity, make the bar code sequence of bar code detecting probe be released；
3) entering in the reaction of next round PCR, in denaturing step, the loop-stem structure of fluorescent detection probe is opened, and in annealing steps, in last round of PCR cycle, the bar code sequence of release is combined with the bar code recognition sequence-specific of fluorescent detection probe；
4) entering extension step, bar code sequence is with fluorescent detection probe for templated synthesis double stranded DNA product；
E, multicolor fluorescence melting curve detection analysis PCR primer:
After PCR reaction terminates, double stranded DNA product with fluorescent detection probe as templated synthesis, along with the rising of temperature starts to unwind, luminophore separates together with DNA with quenching group, the fluorescence of the different wave length that luminophore sends is detected by fluorescent PCR instrument, analyzes PCR primer according to multicolor fluorescence melt curve analysis.
A kind of multicolor fluorescence melting curve PCR detection method the most according to claim 1, it is characterized in that: the probe sequence that bar code detecting probe 3 ' is held completely with target gene complementary pairing to be measured, the bar code detecting probe of not fully complementary pairing can not be had 5 '-3 ' the archaeal dna polymerase hydrolysis release bar code sequence of exonuclease activity.
A kind of multicolor fluorescence melting curve PCR detection method the most according to claim 1, it is characterised in that: described fluorescent probe system includes multiple bar code detecting probe and multiple fluorescent detection probe, and the sequence length of the loop-stem structure of each fluorescent detection probe is different.
4. according to a kind of multicolor fluorescence melting curve PCR detection method described in claim 1,2 or 3, it is characterized in that: described quenching group is any one in Dabcyl, BHQ-1, QYS-7, BHQ-2, described fluorophor is Pacific Blue, Oregon Green, any one in Bodipy FL-X, FAM, TET, Bodipy R6G-X, JOE, HEX, Cy3, Rhodamine Red-X, TAMRA, Cy3.5, Texas Red-X, ROX.
5. use the test kit of power multicolor fluorescence melting curve PCR detection method detection testing gene described in 1 to 4 any one, it is characterised in that: described test kit includes:
A, bar code detecting probe, its 3 ' end is the target gene to be measured probe sequence between two primers, and 5 ' ends are the bar code sequence with certain length of labelling quenching group；
B, there is the fluorescent detection probe of loop-stem structure, its 3 ' end is the bar code recognition sequence specific binding with described bar code sequence, 5 ' terminal sequences and 3 ' terminal sequence complementary pairings, centre is any one section of uncorrelated with target gene to be measured and incoherent with bar code sequence sequence, 3 ' ends and 5 ' ends labelling 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.
The test kit of employing multicolor fluorescence melting curve PCR detection method the most according to claim 5 detection testing gene, it is characterised in that: described hot resistant DNA polymerase is to have 5 '-3 ' hot resistant DNA polymerase of exonuclease activity.
The test kit of the detection testing gene of employing multicolor fluorescence melting curve PCR detection method the most according to claim 5, it is characterised in that: described PCR buffer is containing magnesium ion, Triton X-100, ammonium sulfate, potassium chloride, the mixed solution of Tris-HCl.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510215155.1A CN106191214B (en) | 2015-04-30 | 2015-04-30 | PCR detection method for multicolor fluorescence melting curve |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510215155.1A CN106191214B (en) | 2015-04-30 | 2015-04-30 | PCR detection method for multicolor fluorescence melting curve |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106191214A true CN106191214A (en) | 2016-12-07 |
| CN106191214B CN106191214B (en) | 2019-05-10 |
Family
ID=57458238
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510215155.1A Active CN106191214B (en) | 2015-04-30 | 2015-04-30 | PCR detection method for multicolor fluorescence melting curve |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106191214B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108823287A (en) * | 2017-04-28 | 2018-11-16 | 厦门大学 | A method of detection target nucleic acid sequence |
| WO2019228542A1 (en) * | 2018-05-31 | 2019-12-05 | Jiang Hong | Primer 5' end reverse complementary fluorescent pcr and test kit |
| CN111100908A (en) * | 2018-10-26 | 2020-05-05 | 厦门大学 | Method and kit for detecting deletion of nucleotide fragment |
| CN111926063A (en) * | 2020-08-21 | 2020-11-13 | 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 |
| CN114317699A (en) * | 2021-12-23 | 2022-04-12 | 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 |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2015
- 2015-04-30 CN CN201510215155.1A patent/CN106191214B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108823287A (en) * | 2017-04-28 | 2018-11-16 | 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 |
| WO2019228542A1 (en) * | 2018-05-31 | 2019-12-05 | Jiang Hong | Primer 5' end reverse complementary fluorescent pcr and test kit |
| CN111100908A (en) * | 2018-10-26 | 2020-05-05 | 厦门大学 | Method and kit for detecting deletion of nucleotide fragment |
| CN111100908B (en) * | 2018-10-26 | 2022-07-12 | 厦门大学 | Method and kit for detecting deletion of nucleotide fragment |
| CN111926063A (en) * | 2020-08-21 | 2020-11-13 | 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 |
| WO2022120914A1 (en) * | 2020-12-08 | 2022-06-16 | 厦门致善生物科技股份有限公司 | Method for measuring length of amplification product of one or more nucleic acid molecules in sample |
| CN114317699A (en) * | 2021-12-23 | 2022-04-12 | 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 |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106191214B (en) | 2019-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106191214A (en) | A kind of multicolor fluorescence melting curve PCR detection method | |
| CN111073989B (en) | Rapid constant-temperature detection method and application of shigella nucleic acid | |
| CN101880711B (en) | Nucleic acid screening method of staphylococcus aureus, salmonella, shigella and listeria monocytogenes | |
| CN109468384B (en) | Composite amplification detection kit for simultaneously detecting 45Y loci | |
| CN105803074B (en) | primer type nucleic acid fluorescent probe displaced by bidirectional strand | |
| Zhuang et al. | Small RNA expression profiling by high‐throughput sequencing: Implications of enzymatic manipulation | |
| CN104726549A (en) | Novel method for isothermal amplification detection of double-stranded nucleic acid based on nicking enzyme | |
| CN106995841A (en) | A kind of genetically engineered soybean detection multiple PCR reagent kit and detection method | |
| CN105002285A (en) | Liquid-stage chip constant-temperature detection method for tiny RNA | |
| CN104480202B (en) | A kind of Fructus Luffae reference gene and application thereof | |
| CN107893109A (en) | A kind of low abundance gene mutation enrichment method based on removal wild-type sequence | |
| CN101886122A (en) | Method for detecting chlamydia pneumoniae by loop-mediated isothermal amplification and detection kit | |
| CN108342511A (en) | Detect primer sets and its application of cell enlargement irido virus | |
| CN108300775B (en) | Annular dumbbell-shaped probe and application thereof | |
| CN102162012A (en) | Qualitative PCR detection method for transgenic rice kefeng No. 6 | |
| CN113481284A (en) | Colorimetric nucleic acid detection kit based on constant temperature amplification and detection method | |
| US20200377943A1 (en) | Flexible and high-throughput sequencing of targeted genomic regions | |
| CN104404162A (en) | Real-time fluorescence PCR method for detecting multiple genes or different targets with primer associated universal probe | |
| CN108823288B (en) | Detection module and method | |
| CN109852670A (en) | A kind of high specific nucleic acid detection reagent and its application method | |
| CN110295252A (en) | Improve reaction solution, primer pair, probe and the kit of the detection dog parainfluenza of specificity | |
| CN105018604A (en) | Kit for detecting drug resistance gene polymorphism at a room temperature by probe | |
| CN116083541A (en) | Method for enriching low-abundance mononucleotide variant | |
| CN114622041A (en) | Primer and TaqMan probe for detecting canine torque teno virus and application thereof | |
| CN103725777A (en) | Real-time fluorescence PCR (Polymerase Chain Reaction) method for rapidly detecting transgenic soybean MON89788 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20171218 Address after: Second layer 204-205 unit, No. 5, spiral Road, Guangzhou International Biological Island, Guangzhou, Guangdong, 510000, China Applicant after: Bio Tech (Guangzhou) Co., Ltd. Address before: 361, Ruth Town, San Francisco, Calif. Applicant before: Yu Jiachang Applicant before: Ai Jiesi bio tech ltd Applicant before: Bio Tech (Guangzhou) Co., Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |