CN108531566A - DNA detection methods and composite coding probe based on the hybridization of composite coding probe - Google Patents
DNA detection methods and composite coding probe based on the hybridization of composite coding probe Download PDFInfo
- Publication number
- CN108531566A CN108531566A CN201810343607.8A CN201810343607A CN108531566A CN 108531566 A CN108531566 A CN 108531566A CN 201810343607 A CN201810343607 A CN 201810343607A CN 108531566 A CN108531566 A CN 108531566A
- Authority
- CN
- China
- Prior art keywords
- probe
- sub
- composite coding
- detection methods
- hybridization
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the DNA detection methods hybridized based on composite coding probe and composite coding probe, the composite coding probe is the nucleic acid sequence that a double hairpin motif is formed by by the first sub- probe and the second sub- probe portion pairing, it includes：The ring-shaped areas loop, stem end region and modification group area；Base sequence on the first sub- probe and the second sub- probe positioned at stem end region is mutually paired, and complementary series is constituted with the base sequence of the different zones of the positive-sense strand of target gene to be measured and antisense strand respectively positioned at the base sequence of the ring-shaped areas loop on the first sub- probe and the second sub- probe；The quantity in the modification group area is two, which includes matched fluorophor and quenching group.Detection method provided by the invention has the characteristics that quick and easy, at low cost, non-instrument dependence, Multiple detection ability, can the target spot number of multiplex real-time identification be significantly more than the detectable fluorescence channel number of current instrument.
Description
Technical field
The present invention relates to DNA detection fields more particularly to it is a kind of based on composite coding probe hybridization DNA detection methods and
Composite coding probe.
Background technology
Fluorescent quantitative PCR (Real-time PCR, FQ-PCR) technology advantage most outstanding is real
Existing DNA cloning is synchronous with detection to be carried out.The technology have the characteristics that it is easy to operate, rapidly and efficiently, hypersensitivity, extensively use
It is quickly to detect in many research fields such as molecule diagnosis, molecular biology research, the animal and plant quarantine and food safety detections
One of modernism.
The enrichment process of FQ-PCR amplified productions can both be indicated by non-specific fluorescence dye method, can also use spy
Anisotropic fluorescent Resonance energy transfer (FRET) probe technique detects to realize.Non-specific fluorescence dye method although cost is relatively low,
But false positive issue caused by being interfered due to PCR non-specific amplification products can not be overcome, and since fluorescent dye shines wave
Long single limitation is greatly limited in practical applications so cannot achieve the detection of multiple gene target spot.
FRET probes are in the nature an oligonucleotides, both ends one reporter fluorescence group of label and a quenching fluorescence respectively
After group, during specific amplification products generate, since reporter fluorescence group and quenching fluorescence group detach, so that
Fluorescence monitoring system can receive fluorescence signal, realizes the accumulation of fluorescence signal and PCR product forms fully synchronized, solve
The false positive issue that non-specific amplification is brought.Current at least 5 kinds of fluorophors are marked for FRET probe modifications, therefore by
FRET probe techniques realize the Genotyping application that FQ-PCR is relied in multiple gene target spot.
At present, include mainly using the technology of FRET probes in the market：Hydrolysis probes (the quotient that PE companies of the U.S. develop
The name of an article：TaqMan, United States Patent (USP) No.6,485,203), Epoch companies increase joint efficiency on the basis of hydrolysis probes
MGB probes (United States Patent (USP) No.7,205,105), and, molecular beacon probe (United States Patent (USP) No.5,225,517).
Hydrolysis probes have the characteristics that high sensitivity, favorable reproducibility, but due to the limitation of detection zone itself, in principle
Specific recognition can not be carried out to particular target point.And molecular beacon probe is based on its unique space structure, the target with height
Point detection specificity, but it there are annealing slack and then causes its detection sensitivity limited during PCR.Meanwhile FRET
The quantity that probe technique is also detected fluorescence channel by existing PCR detecting instruments is limited, and current PCR detecting instruments can be examined simultaneously
5 kinds of fluorophors are surveyed, are theoretically at best able to detect 5 kinds of target sequences simultaneously.It would therefore be highly desirable to which people go to solve by technological innovation
The low problem of FQ-PCR measurement capacities.
Chinese invention patent application (No.CN106121214A)：A kind of PCR detection method of multicolor fluorescence melting curve,
Which disclose a kind of monochromatic or double-colored multiple FQ-PCR technologies, are in the nature a kind of detection of PCR terminals multiple target point analysis
Mode.Such technology cannot achieve amplification it is synchronous with detection while, also by the interference of " false positive " melting peakss, most
High detection target sequence number is also limited by analysis temperature range.Target sequence is enriched with multiple PCR technique (TEM-PCR), by nest
Nucleic acid chip technology and streaming detection technique of fluorescence are effectively combined one by formula PCR by streaming fluorescent technique (xMAP) again
It rises, largely solves the Genotyping application difficult point of multiple gene target spot dependence.But the technology has to particular instrument
Dependence, testing process are cumbersome, easily cause the shortcomings such as amplified production pollution.Chinese invention patent application
(No.CN1936019A)：For the probe coding method of multiplex real-time nucleic acid amplification detection, the scheme of the patent disclosure overcomes
The shortcomings that above-mentioned technology, but its probe synthetic modification, there are technology unstability, testing cost also accordingly increases.
Invention content
It is an object of the invention to overcome the shortcomings of that measurement capacity is small in existing DNA detection techniques, and provide a kind of fast
Fast simple, at low cost, non-instrument dependence, the stronger DNA detection methods of Multiple detection ability pass through and provide a kind of compound volume
Code probe to realize the detection method can the target spot number of multiplex real-time identification be significantly more than the detectable fluorescence of current instrument
Number of active lanes.
To achieve the above object, the first aspect of the present invention provides a kind of DNA inspections hybridized based on composite coding probe
Survey method, includes the following steps：
A. one or more composite coding probe is synthesized according to the specific sequence of target gene to be measured；
The composite coding probe is to be formed by a pair of hair fastener knot by the first sub- probe and the second sub- probe portion pairing
The nucleic acid sequence of structure, it includes：The ring-shaped areas loop, stem end region and modification group area；
Base sequence on the first sub- probe and the second sub- probe positioned at stem end region is mutually paired, first son
On probe and the second sub- probe positioned at the ring-shaped areas loop base sequence respectively with the positive-sense strand and antisense strand of target gene to be measured
Different zones base sequence constitute complementary series；
The quantity in the modification group area is two, which includes matched fluorophor and quenching group；
B. sense primer and downstream primer are synthesized according to the specific sequence of target gene to be measured；
C. reaction system is built using the probe of above-mentioned acquisition and primer, PCR amplification is carried out to target gene to be measured；
D. after pcr amplification reaction, the fluorescence signal of PCR product is collected to form corresponding amplification curve, is treated
Target gene is surveyed to be analyzed.
Fig. 1 shows the structure of composite coding probe of the present invention, and with reference to figure 1, the composite coding probe 1 is visited by the first son
Needle 11 and 12 portion paired of the second sub- probe form, further, it includes：The ring-shaped areas loop 2, stem end region 3 and modification base
Area 4 of group.
Base sequence on first sub- probe 11 and the second sub- probe 12 positioned at stem end region 3 is mutually paired, in realization
State the portion paired of the first sub- probe and the second sub- probe；And positioned at the ring-shaped areas loop 1 base sequence respectively with target to be measured
The positive-sense strand of gene and the base sequence of the different zones of antisense strand constitute complementary series, this makes the first sub- probe and the second son
Probe is in following pcr amplification reactions, specific sequence of the sequence energy greater probability of the ring-shaped areas the loop ground with target gene to be measured
Row carry out pairing combination, and otherwise, the first sub- probe 11 and the second sub- probe 12 are located at the base sequence meeting of the ring-shaped areas loop 1 certainly
Row pairing, then can reduce the probability that probe is combined with target gene to be measured so that detection can not carry out.The modification group area 4
Quantity be two, which includes matched fluorophor and quenching group.The fluorophor and quenching group
The mechanism of action it is similar to the group luminescence mechanism of existing fluorescence probe, i.e., when fluorophor and quenching group close to each other,
Fluorescence resonance energy transfer (FRET) can occur, the signal that fluorophor is sent out is quenched group absorptions；And fluorophor with
When quenching group detaches, fluorophor sends out the fluorescence of respective wavelength, and is detected by PCR instrument.
In this way, according to target gene to be measured different specific sequence that may be present, can synthesize one or more compound
Oded p, PCR reaction systems are built using the composite coding probe, and PCR amplification is carried out to target gene to be measured：
(1) in the denaturing step of each round pcr amplification reaction, not only there is the unwinding of DNA double chain, the compound volumes
The stem end region of code probe has also carried out corresponding unwinding；
(2) pass through after denaturing step, the first sub- probe and the second sub- probe after annealing steps, unwinding are located at loop
The base sequence of ring-shaped area identifies corresponding specific sequence on target gene to be measured, and sense primer and downstream primer are also identified and waited for
Survey corresponding specific sequence on target gene；
(3) in extending step, by the effect of archaeal dna polymerase in system, sense primer and downstream primer start to extend,
Form double stranded DNA product；Meanwhile fluorophor and quenching group are separated from each other, and send out the fluorescence of corresponding wavelength；
(4) it subsequently enters in next round pcr amplification reaction, and until cycle terminates.After pcr amplification reaction, receive
Collect fluorescence signal caused by PCR product to form corresponding amplification curve, and target gene to be measured is analyzed.
In the above process, due to the first, second sub- probe portion pairing, and include two groups of matched fluorophors and
Quenching group, since fluorophor and quenching group distance are closer, will not send out fluorescence letter in the state that unwinding does not occur
Number, thus the composite coding probe structure of the formation is stablized；And in extending step, due to the fluorophor of two modification groups
The fluorescence of corresponding wavelength can be sent out, therefore the specific signals of the composite coding probe are that two fluorophors are sent out
The combination of fluorescence signal, in the case of the PCR detecting instruments with identical quantity fluorescence channel detectability, expand
The Species differences of specific signals, and the Species differences do not put forward higher requirements PCR detecting instruments, so as to
According to the combination for the different fluorescence signals that composite coding probe has, analysis is detected to target gene to be measured, is overcome
Fluorescence probe in the prior art can only carry 1 fluorophor and 1 quenching group, thus the type of specific signals is pair
The quantity for the fluorescence channel that should can be detected in PCR detecting instruments improves measurement capacity, instrument dependence in the same circumstances
Low, Multiple detection ability is good, and detection efficiency is high.
In a certain embodiment：The quantity of the stem end region is two, and the both ends of the ring-shaped areas loop are separately connected institute
Two stem end regions are stated, the other end of two stem end regions is separately connected described two modification group areas so that two modifications
Group area is located at the both ends of composite coding probe.
In a certain embodiment：Described two modification groups are respectively the first modification group and the second modification group；First
Included two fluorophor of modification group and the second modification group is located at the ends 5' and the ends 3' on same sub- probe.
In a certain embodiment：Described two modification groups are respectively the first modification group and the second modification group；First
Included two fluorophor of modification group and the second modification group is located at the ends 5' or 3' on different sub- probes
End.
The above-mentioned different embodiments about two fluorophor positions correspond respectively to the feelings shown by Fig. 2 a and Fig. 2 b
Condition.
In a certain embodiment：First fluorophor and the second fluorophor are respectively adopted in group as described below
It is a kind of comprising but be not limited to：ALEXA350、FAM、HEX、TET、JOE、VIC、ROX、Texas Red、Cy5、Cy5.5、
TAMRA；One kind in group as described below is respectively adopted in first quenching group and the second quenching group comprising but it is unlimited
In：Dabcyl、BHQ-1、BHQ-2、QYS-7.
In a certain embodiment：The first sub- probe and the second sub- probe are the fluorescence probe that can be used for FQ-PCR, can
Using one kind in following probe comprising but be not limited to：Hydrolysis probes, MGB probes, molecular beacon probe, displacement probe, scorpion
Sub- primer, Amplifier primers, to which composite coding probe can be based on improving on the basis of existing fluorescence probe, design cost
It is low.
In a certain embodiment：The length of the ring-shaped areas loop be include 15-40 base-pair；The stem end region
Length be include 6-20 base-pair, to ensure double hairpin motif stablize.
In a certain embodiment：On first sub- probe and the second sub- probe positioned at the stem end region base sequence with
The specific sequence of target gene to be measured is uncorrelated, to prevent the special of the base sequence of stem end region and target gene to be measured
Property sequence is matched, interference detection results.
In a certain embodiment：PCR amplification in the step c, denaturation temperature is 95 DEG C, annealing temperature is 60 DEG C,
Elongating temperature is 72 DEG C.
Based on above spirit, to achieve the above object, the second aspect of the present invention provides a kind of composite coding
Probe is suitable for the DNA detection methods based on PCR, and the composite coding probe is in the above technical solution
Composite coding probe.
Description of the drawings
Fig. 1 is the structural schematic diagram of composite coding probe in an embodiment of the present invention, wherein the circle with Filling pattern
Point represents fluorophor, and the dot without Filling pattern represents quenching group；
Fig. 2 a are the schematic diagram of two fluorophor present positions in an embodiment of the present invention, wherein with Filling pattern
Dot represents fluorophor, and the dot without Filling pattern represents quenching group；
Fig. 2 b are the schematic diagram of two fluorophor present positions in another embodiment of the present invention, wherein having Filling pattern
Dot represent fluorophor, the dot without Filling pattern represents quenching group；
Fig. 3 a show the gradient dilution amplification curve of sample to be tested in embodiment one；
Fig. 3 b show the Ct values and DNA starting copy numbers of the gradient dilution amplification curve of sample to be tested in embodiment one
Logarithmic relationship curve；
Fig. 4 a-4d, which are respectively illustrated, contains only that there are four types of any one in hypotype HPV viruse in sample to be tested in embodiment two
Corresponding amplification curve when kind.
Specific implementation mode
Below in conjunction with drawings and the specific embodiments, the present invention is further illustrated.
Embodiment one
The present embodiment selects human body ALDH2 genes (Gene ID:217) it is target gene to be measured, it is corresponding according to the present invention
Spirit, the particular sequence for designing corresponding primer and composite coding probe is as follows, wherein the first son of composition composite coding probe
Probe and the second sub- probe are indicated (similarly hereinafter) with probe P1 and probe P2 respectively：
Sense primer：5'-GCCCAGTCACCCTTTGGT-3'
Downstream primer：5'-GACCCTCAAGCCCCAACA-3'
Probe P1：5'-ROX-AATCGCGGGGTTTTGTCGGGGAGTGGCCGGGAGTTTGCGCTGACGA-FAM-3'
Probe P2：5'-BHQ1-TCGTCAGCGCAAAGGACCTGCTGGGGGCTCAGGAAACCCCGCGATT-BHQ1-3'
Select dosage for 1 × 108copies/μL、1×107copies/μL、1×106copies/μL、1×
105copies/μL、1×104copies/μL、1×103The ALDH2 gene plasmids of copies/ μ L (being 10 times of gradient dilutions)
Standard items are as reaction template.ALDH2 gene plasmid standard items containing 2 μ L in the PCR reaction systems of 10 μ L, 10mmol/L
Tris-HCl, pH=8.3,50mmol/L KCl, 0.5U Taq enzyme, 2.5mmol/L dNTP, 3.5mmol/L Mg2+, 0.45
μm ol/L probes P1,0.45 μm of ol/L probes P2,0.3 μm of ol/L sense primer, 0.3 μm of ol/L downstream primer.Pcr amplification reaction
Program be：95 DEG C 3 minutes；95 DEG C 10 seconds, 60 DEG C 15 seconds, 72 DEG C 15 seconds, 45 cycle；Fluorescence is acquired in 60 DEG C of annealing stages
Signal.
In the present embodiment, the ends 5' and 3' of probe P1 are marked with two different fluorophors, respectively FAM groups
With ROX groups, and the ends 5' and 3' of probe P2 are labeled identical BHQ1 quenching groups.In its natural state, probe P1 and
Probe P2 forms probe complex, fluorophor and quenching group is close to each other does not generate fluorescence signal.In pcr amplification reaction
Annealing stage, probe P1 and P2 are combined with PCR product, i.e., the fluorophor of probe P1 and P2 and quenching group separate, and produce
Raw two different fluorescence signals.With the increase of PCR cycle number, fluorescence signal in system with the accumulation of PCR product and
It gradually increases to form two typical amplification curves.Since probe P1 carries two different fluorophors and with same simultaneously
Fluorescence signal mechanism of production, therefore two amplification curves are identical.The gradient dilution amplification curve of the present embodiment is shown in Fig. 3 a.
As shown in Figure 3a, the preferable gradient of amplification curve 1a embodiments of 10 times of gradient dilution samples, and 2a (negative control ddH2O, as
There is no the presence of template) amplification without specific product, therefore can't detect nonspecific signal.With reference to Fig. 3 b, it illustrates wait for test sample
The logarithmic relationship curve of the Ct values and DNA starting copy numbers of this gradient dilution amplification curve, it can be seen that 1 × 108copies
The Ct values of the reaction tube of template consumption are that 20.25 (Ct values are meant that：Fluorescence signal in each reaction tube reaches the domain of setting
The recurring number undergone when value), 1 × 107The Ct values of the reaction tube of copies template consumptions are 23.66,1 × 106Copies templates
The Ct values of the reaction tube of dosage are 27.45,1 × 105The Ct values of the reaction tube of copies template consumptions be 31.07,1 ×
104The Ct values of the reaction tube of copies template consumptions are 33.23,1 × 103The Ct values of the reaction tube of copies template consumptions are
36.82, thus, the above result shows that being formed by composite coding probe in FQ- by probe P1, P2 used by the present embodiment
In round pcr have feasibility, the PCR reaction systems have higher reaction efficiency, the Ct values of gradient sample amplification curve with
The logarithm of its DNA starting copy number has good linear relationship (R2=0.996) the stronger quantitative detection energy of the technology, is embodied
Power.
Embodiment two
The present embodiment with the HPV viruse (HPV18, HPV33, HPV52, HPV58) of four kinds of high-risk hypotypes be research object,
It is as follows for above four kinds of hypotype design primers and probe：
The sequence of primer used is：
HPV18-F2：5'-AATTATTTGTTACTGTGGTAGATACCAC-3'
HPV18-R2：5'-ACTGAAAAATAAACTGCAAATCATATTCC-3'
HPV33-F2：5'-AGGTATTTGTTACTGTGGTAGATACC-3'
HPV33-R2：5'-GCATAGTTGAAAAACAAACTGTAGATC-3'
HPV52-F2：5'-ATCAGTTGTTTGTCACAGTTGTG-3'
HPV52-R2：5'-CATCAGCTGTTAATGTAATTTTGCAC-3'
HPV58-F2：5'-AGTTATTTGTTACCGTGGTTGATAC-3'
HPV58-R2：5'-AAGCTGAAAAACAAACTGTAAGTCA-3'
The sequence of composite coding probe used is：
HPV18-P1：5'-HEX-AATCGCAAAGTTTCTTCTACACAGTCTCCTGTACCTGGGC-
-TTTACTGCGTTT-ROX-3'
HPV18-P2：5'-BHQ1-AAACGCAGTAAACCAAATTTAAGCAGTATAGCAGACATG-
-AAACTTTGCGATT-BHQ1-3'
HPV33-P1：5'-FAM-AATCGCAAAGTTTTGACTTTATGCACACAAGTAACTAGTG-
-ACAGTTTACTGCGTTTG-FAM-3'
HPV33-P2：5'-BHQ1-CAAACGCAGTAAAAATTTTAAAGAATATATAAGACATGTT-
-GAAGAAACTTTGCGATT-BHQ1-3'
HPV52-P1：5'-HEX-AATCGCAAAGTTTGTGCTGAGGTTAAAAAGGAAAGCACA-
-TTTACTGCGTTT-CY5-3'
HPV52-P2：5'-BHQ1-AAACGCAGTAAATTTAAGGAATACCTTCGTCATGGCGAA-
-AACTTTGCGATT-BHQ1-3'
HPV58-P1：5'-CY5-AATCGCAAAGTTTGCACTGAAGTAAATAAGGAAGGTACTT-
-TACTGCGTTTG-FAM-3'
HPV58-P2：5'-BHQ1-CAAACGCAGTAAAAAGGAATATGTACGTCATGTTGAAGA-
-AACTTTGCGATT-BHQ1-3'
Template containing 2 μ L in the PCR reaction systems of 20 μ L, 10mmol/L Tris-HCl, pH=8.3,
50mmol/L KCl, 0.5U Taq enzymes, 2.5mmol/L dNTP, 3.5mmol/L Mg2+, the final concentration of each probe are all
The final concentration of 0.45 μm of ol/L, each primer are all 0.3 μm of ol/L.Pcr amplification reaction program is：95 DEG C 3 minutes；95 DEG C 10 seconds,
60 DEG C 15 seconds, 72 DEG C 15 seconds, 45 cycle；Fluorescence signal is acquired in 60 DEG C of annealing stages.
Above the established multi-PRC reaction system can detect to contain only the HPV of any one in four kinds of hypotype HPV
The sample of hypotype, the curve that corresponding amplification curve goes out as shown in figures 4a-4d respectively include HPV18, HPV33, HPV52
With the testing result of the sample of HPV58, it can be seen that cross reaction is not present between four kinds of different HPV high-risk subtype virus,
The present embodiment only needs one-time detection experiment that can accurately be detected to sample to be tested, embodies the higher detection effect of the technology
Rate.
Embodiment three
The present embodiment is with TP53 genes (the Gene ID of people:7157) SNP site (rs12951053) in is research
Object carries out Genotyping with the method for the present invention, and designed primer and probe sequence is as follows：
Primers F 5H-A1：5'-GTGGATGGGTAGTAGTATGGA-3'
Primers F 5H-C2-2A：5'-GTGGATGGGTAGTAGTATGaC-3', wherein label underscore and lowercase
Base is the base mismatch being artificially introduced
Primers F 5H-R：5'-ATCCTCACCATCATCACACTG-3'
Probe F5H-P1：5'-HEX-AATCGCAAAGTTTGGTGGGCCCAGGGGTCAGAGGCAA-
-GCTTTACTGCGTTT-FAM-3'
Probe F5H-P2：5'-BHQ1-AAACGCAGTAAAGCAGGGTGGCAAGTGGCTCCTGACC-
-TAAACTTTGCGATT-BHQ1-3'
Sequencing primer：
F：5'-GGGAGCAGTAAGGAGATTC-3'
R：5'-TCTGCTTGCCTCTGACCC-3'
The reaction system formed comprising two kinds of compositing formulas of reaction solution A and reaction solution B in the present embodiment, is respectively used to examine
Whether the SNP site for surveying sample to be tested contains A types gene and c-type gene.
Reaction solution A：10 μ L PCR reaction systems include 2 μ L templates, 10mmol/L Tris-HCl, pH=8.3,
50mmol/L KCl, 0.5U Taq enzymes, 2.5mmol/L dNTP, 3.5mmol/L Mg2+, 0.45 μm of ol/L probe F5H-P1,
0.45 μm of ol/L probes F5H-P2,0.3 μm of ol/L primers Fs 5H-A1,0.3 μm of ol/L primers Fs 5H-R.
Reaction solution B：10 μ L PCR reaction systems include 2 μ L templates, 10mmol/L Tris-HCl, pH=8.3,
50mmol/L KCl, 0.5U Taq enzymes, 2.5mmol/L dNTP, 3.5mmol/L Mg2+, 0.45 μm of ol/L probe F5H-P1,
0.45 μm of ol/L probes F5H-P2,0.3 μm of ol/L primers Fs 5H-C2-2A, 0.3 μm of ol/L primers Fs 5H-R.
Pcr amplification reaction program is：95 DEG C 3 minutes；95 DEG C 10 seconds, 60 DEG C 15 seconds, 72 DEG C 15 seconds, 45 cycle；60℃
Annealing stage acquires fluorescence signal.
23 peripheral blood samples provided by First Hospital of Xiamen are be provided, above-mentioned two kinds of reactants of A, B are utilized
System carries out Genotyping detection to above 23 samples respectively and counts respective testing result, is combined to obtain waiting for test sample
The genotype results of this SNP site.
In 23 samples to be tested, the SNP site testing result of TP53 genes is that have 12 in rs12951053 be AA types,
10 are AC types, and 1 is CC types, shown in table 1 specific as follows.Inventor is also to second of pcr amplification product of 23 samples point
Sanger sequencings have not been carried out, and acquired results are identical with table 1.
Table 1
Thus, in the present embodiment, composite coding probe provided by the invention and corresponding DNA detection methods can be efficient
Ground is carried out at the same time detection to the SNP site of multiple samples simultaneously, and obtains accurate believable result.And with Sanger PCR sequencing PCR phases
Than, the present embodiment substantially increases the flux of sequencing, can effectively be detected to the SNP site of multiple samples simultaneously, and
The step of reducing meso sample processing, improves conventional efficient, and testing cost is lower.
The foregoing is merely the preferred embodiment of the present invention, it is not intended to limit its scope of the claims, it is every to utilize the present invention
Equivalent structure transformation made by specification and accompanying drawing content is directly or indirectly used in other related technical areas, similarly
It is included within the scope of the present invention.
Claims (10)
1. the DNA detection methods based on the hybridization of composite coding probe, which is characterized in that include the following steps：
A. composite coding probe is synthesized according to the specific sequence of target gene to be measured；
The composite coding probe is to be formed by a double hairpin motif by the first sub- probe and the second sub- probe portion pairing
Nucleic acid sequence, it includes：The ring-shaped areas loop, stem end region and modification group area；
Base sequence on the first sub- probe and the second sub- probe positioned at stem end region is mutually paired, the first sub- probe
With on the second sub- probe be located at the ring-shaped areas loop base sequence respectively with the positive-sense strand of target gene to be measured and antisense strand not
Base sequence with region constitutes complementary series；
The quantity in the modification group area is two, which includes matched fluorophor and quenching group；
B. sense primer and downstream primer are synthesized according to the specific sequence of target gene to be measured；
C. reaction system is built using the probe of above-mentioned acquisition and primer, PCR amplification is carried out to target gene to be measured；
D. after pcr amplification reaction, the fluorescence signal of PCR product is collected to form corresponding amplification curve, to target to be measured
Mark gene is analyzed.
2. the DNA detection methods as described in claim 1 based on the hybridization of composite coding probe, it is characterised in that：
The quantity of the stem end region is two, and the both ends of the ring-shaped areas loop are separately connected described two stem end regions, this two
The other end of a stem end region is separately connected described two modification group areas so that Liang Ge modification groups area is located at composite coding spy
The both ends of needle.
3. the DNA detection methods as claimed in claim 2 based on the hybridization of composite coding probe, it is characterised in that：
Described two modification groups are respectively the first modification group and the second modification group；
Included two fluorophor of first modification group and the second modification group is located at the 5' on same sub- probe
End and the ends 3'.
4. the DNA detection methods as claimed in claim 2 based on the hybridization of composite coding probe, it is characterised in that：
Described two modification groups are respectively the first modification group and the second modification group；
Included two fluorophor of first modification group and the second modification group is located on different sub- probes
The ends 5' or the ends 3'.
5. the DNA detection methods based on the hybridization of composite coding probe as described in any one of claim 3 or 4, feature exist
In：
One kind in group as described below is respectively adopted in first fluorophor and the second fluorophor comprising but it is unlimited
In：ALEXA350、FAM、HEX、TET、JOE、VIC、ROX、Texas Red、Cy5、Cy5.5、TAMRA；
One kind in group as described below is respectively adopted in first quenching group and the second quenching group comprising but it is unlimited
In：Dabcyl、BHQ-1、BHQ-2、QYS-7.
6. the DNA detection methods as claimed in claim 2 based on the hybridization of composite coding probe, it is characterised in that：
The first sub- probe and the second sub- probe are the fluorescence probe that can be used for FQ-PCR, and one in following probe can be used
Kind comprising but be not limited to：Hydrolysis probes, MGB probes, molecular beacon probe, displacement probe, scorpions, Amplifier draw
Object.
7. the DNA detection methods as claimed in claim 2 based on the hybridization of composite coding probe, it is characterised in that：
The length of the ring-shaped areas loop be include 15-40 base-pair；The length of the stem end region be include 6-20 alkali
Base pair.
8. the DNA detection methods as claimed in claim 2 based on the hybridization of composite coding probe, it is characterised in that：
Positioned at the special of base sequence and the target gene to be measured of the stem end region on first sub- probe and the second sub- probe
Property sequence is uncorrelated.
9. the DNA detection methods as claimed in claim 2 based on the hybridization of composite coding probe, it is characterised in that：
PCR amplification in the step c, denaturation temperature is 95 DEG C, annealing temperature is 60 DEG C, elongating temperature is 72 DEG C.
10. a kind of composite coding probe is suitable for the DNA detection methods based on PCR, it is characterised in that：It is described
Composite coding probe is the composite coding probe described in any one of the claims 1-8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810343607.8A CN108531566A (en) | 2018-04-17 | 2018-04-17 | DNA detection methods and composite coding probe based on the hybridization of composite coding probe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810343607.8A CN108531566A (en) | 2018-04-17 | 2018-04-17 | DNA detection methods and composite coding probe based on the hybridization of composite coding probe |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108531566A true CN108531566A (en) | 2018-09-14 |
Family
ID=63480434
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810343607.8A Pending CN108531566A (en) | 2018-04-17 | 2018-04-17 | DNA detection methods and composite coding probe based on the hybridization of composite coding probe |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108531566A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406475A (en) * | 2018-12-13 | 2019-03-01 | 四川大学 | Double labelling quick response nucleic acid aptamer probe and its method for detecting aflatoxin B1 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1936019A (en) * | 2005-10-18 | 2007-03-28 | 厦门大学 | Probe coding method for multiplex real-time nucleic acid amplification detection |
| CN104109712A (en) * | 2004-02-18 | 2014-10-22 | 克罗莫塞尔公司 | Methods and materials using signaling probes |
| CN104404142A (en) * | 2014-11-11 | 2015-03-11 | 中国科学院上海微***与信息技术研究所 | Fluorescent probe for fluorescent quantitative PCR reactions |
| CN106191214A (en) * | 2015-04-30 | 2016-12-07 | 余家昌 | A kind of multicolor fluorescence melting curve PCR detection method |
-
2018
- 2018-04-17 CN CN201810343607.8A patent/CN108531566A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104109712A (en) * | 2004-02-18 | 2014-10-22 | 克罗莫塞尔公司 | Methods and materials using signaling probes |
| CN1936019A (en) * | 2005-10-18 | 2007-03-28 | 厦门大学 | Probe coding method for multiplex real-time nucleic acid amplification detection |
| CN104404142A (en) * | 2014-11-11 | 2015-03-11 | 中国科学院上海微***与信息技术研究所 | Fluorescent probe for fluorescent quantitative PCR reactions |
| CN106191214A (en) * | 2015-04-30 | 2016-12-07 | 余家昌 | A kind of multicolor fluorescence melting curve PCR detection method |
Non-Patent Citations (2)
| Title |
|---|
| BRYAN A. BAKER ET AL.: "Hybridization kinetics between immobilized double-stranded DNA probes and targets containing embedded recognition segments", 《NUCLEIC ACIDS RESEARCH》 * |
| 邓炜 等: "基于聚乙烯亚胺还原的金纳米颗粒为基底构建的"signal on"型高灵敏ECL传感器铅离子检测", 《化学传感器》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406475A (en) * | 2018-12-13 | 2019-03-01 | 四川大学 | Double labelling quick response nucleic acid aptamer probe and its method for detecting aflatoxin B1 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shen et al. | Recent advances and perspectives of nucleic acid detection for coronavirus | |
| CN105821138B (en) | A kind of method that double loop-stem structure DNA profiling detection nucleic acid are built based on coupled reaction | |
| CN103540660B (en) | Based on the loop-mediated isothermal amplification method of TaqMan probe and special LAMP primer thereof and test kit | |
| CN107988325B (en) | RAA constant temperature fluorescence detection method and reagent for shrimp liver Enterocytozoon (EHP) | |
| CN102827929B (en) | Method for detecting nucleic acid | |
| CN106834456B (en) | Y-STR multiplex amplification detection kit marked by adopting fluorescence marking method and use method thereof | |
| CA2768478A1 (en) | Detection of short rna sequences | |
| CN105803074A (en) | Primer-type nucleic acid fluorescent probe subjected to two-way strand displacement | |
| CN110144384A (en) | The fluorescence chemical sensor and its detection method of a kind of test side intragranular oxidative damage and application | |
| CN107988326A (en) | Prawn Acute Hepatic pancreatic necrosis（AHPND）RAA constant temperature fluorescence detection method and reagent | |
| CN106399577B (en) | A kind of real-time fluorescence PCR detection method for the double target nucleic acids of interest present detections of single channel | |
| CN101899521A (en) | Loop-mediated isothermal amplification (LAMP) detection method of Angiostrongylus cantonensis | |
| CN113186313A (en) | Salmonella detection primer group, method and kit based on RPA-LbCas12a-TTECDS system | |
| Liu et al. | Ratiometric fluorescence method for ctDNA analysis based on the construction of a DNA four-way junction | |
| CN112725531A (en) | Hepatitis B virus rapid detection system combining MCDA with biosensor | |
| CN114381550A (en) | Multi-target nucleic acid detection kit and detection method for HPV typing | |
| CN112391498B (en) | Application of pregnancy test paper in on-site instant detection of hepatitis B virus drug-resistant mutant gene | |
| CN108531566A (en) | DNA detection methods and composite coding probe based on the hybridization of composite coding probe | |
| ZHANG et al. | Highly sensitive fluorescent aptasensor for thrombin detection based on competition triggered rolling circle amplification | |
| Zhang et al. | A one-step dipstick assay for the on-site detection of nucleic acid | |
| CN107893120B (en) | Primer group for detecting motion gene SNP, application and product thereof, and detection method and application for detecting motion gene SNP | |
| CN106319079A (en) | Method for detecting 22q11.2 copy number deletion | |
| CN104450954B (en) | Detect the fluorescent PCR kit and its method of 13 kinds of hypotypes of human papilloma virus | |
| Xu et al. | Long-stem shaped multifunctional molecular beacon for highly sensitive nucleic acids determination via intramolecular and intermolecular interactions based strand displacement amplification | |
| CN104988235B (en) | One kind cutting a gram enzyme mediated isothermality amplification small molecule beacon quantitative measurement technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180914 |
|
| RJ01 | Rejection of invention patent application after publication |