CN108913759A - A kind of fluorescence PCR primer, probe and detection method for target sequence detection - Google Patents
A kind of fluorescence PCR primer, probe and detection method for target sequence detection Download PDFInfo
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Abstract
The invention discloses a kind of fluorescence PCR primer, probe and detection methods for target sequence detection.The present invention includes forward primer, reverse primer, fluorescent receptor hybridization probe and fluorogenic donor Signature probes, and the fluorescent receptor hybridization probe includes the sequence with nucleic acid array complementation to be detected.Cancel the fluorogenic donor group on universal primer, fluorogenic donor group is changed into detection probe, introduce the target sequence specific probe in a region between two primers after PCR amplification, acceptor fluorophore is modified at the nearly donor fluorophore end of target sequence specific probe, only PCR amplification be target target sequence when, fluorescent receptor hybridization probe can be just incorporated on the product of asymmetric pcr, fluorescence resonance shifts ability, false positive caused by accidentally expanding so as to avoid primer dimer or PCR, significantly improves specificity.
Description
Technical field：
The invention belongs to gene engineering technology fields, more particularly it relates to which a kind of design a segment mark on primer
Sequence is signed, target sequence detection is carried out by the solution temperature of label segment sequence using fluorescence resonance energy transfer (FRET) principle
Fluorescence PCR primer, probe and detection method.
Background technique：
Fluorescence PCR assay is the common technology for detecting target nucleic acid sequence, and currently used fluorescence PCR detecting method includes
Double probe (the Hybridization of Taqman sonde method, molecular beacon (Molecular Beacon) sonde method, fluorescent hybridization
Probe) method etc..Wherein fluorescent hybridization two probe method mainly can be with the single-stranded fluorescence hybridized of target sequence same by using two
Probe, wherein a kind of terminal modified fluorophor of a probe 3 ', the terminal modified another fluorophor of another probe 5 ', when target sequence
Being generated in the presence of column by asymmetric pcr largely can be single-stranded with this two fluorescence probe detections, when two fluorescence probe head and the tail
It is connected when hybridizing with the single stranded DNA of reverse complemental, the fluorophor that first probe 3 ' is held is held another with Article 2 probe 5 '
A fluorophor shortens on space length, causes fluorescence resonance energy transfer, and caused fluorescence signal power changes to examine
Survey the presence of target sequence.
Fluorescence probe solubility curve analytical technology is a kind of qualitative checking method based on asymmetric PCR amplification technique.Benefit
Solubility curve analysis is carried out after asymmetric PCR reaction with molecular beacon probe, Fret probe or Taqman probe, it can be because of institute
Solubility curve peak change caused by binding sequence variation, to differentiate different target sequence sites.This technology is mainly excellent
Gesture is that can use different fluorescence channels and different fluorescence probes dissolves the combination of peak temperature while detecting multiple detection sites,
And the good probe of design can differentiate different mutational sites by the variation at dissolution peak.
Fluorescence probe solubility curve technology is when same fluorescence channel is detected using a plurality of fluorescence probe at present, even if
Fluorescence probe is not in conjunction with target sequence, and accidentally hybridization can also generate back for winding mutually under cryogenic between probe itself or probe
Scape signal interferes with each other superposition between the background signal of a plurality of fluorescence probe, causes fluorescence background excessively high, to affect dissolution
The interpretation of curve.Further, since the target sequence region to be detected that fluorescence probe combines is be easy to cause due to gene mutation etc.
Solubility curve temperature change causes to judge incorrectly.
Summary of the invention：
The object of the present invention is to provide superposition will not be interfered with each other between a kind of background signal of a plurality of detection probe, also not
Meeting be influenced to cause the multiple fluorescence PCR detection primer and probe of solubility curve temperature change by target gene mutation to be detected.
Multiple fluorescence PCR detection primer of the invention and probe, including forward primer, reverse primer, fluorogenic hybridization probe
With fluorescence labels probe；
The fluorogenic hybridization probe includes the sequence with nucleic acid array complementation to be detected, and the forward primer includes just
To enriching primer, the upstream sequence reverse complemental at 3 ' ends and nucleic acid sequence to be detected of positive enriching primer, described reversely draws
Object includes the connected fluorescence labels position of sequence and reversed enriching primer, and the 3 ' of the reversed enriching primer are held and core to be detected
The downstream sequence reverse complemental of acid sequence, 5 ' ends are combined with the 3 ' ends at fluorescence labels position, and the fluorescence labels probe includes
The sequence complementary with the fluorescence labels position in reverse primer and the donor fluorophore or acceptor fluorophore for modifying the sequence；
Or the reverse primer includes reversed enriching primer, the downstream sequence at 3 ' ends and nucleic acid sequence to be detected of reversed enriching primer
Reverse complemental, the forward primer include the connected fluorescence labels position of sequence and positive enriching primer, and the forward direction is rich
Collecting the upstream sequence reverse complemental at 3 ' ends and nucleic acid sequence to be detected of primer, 5 ' ends are combined with the 3 ' ends at fluorescence labels position,
The fluorescence labels probe includes the sequence complementary with the fluorescence labels position in forward primer and the donor for modifying the sequence
Fluorophor or acceptor fluorophore.
It is preferred that when on fluorescence labels probe be donor fluorophore when, the fluorogenic hybridization probe include with it is to be detected
The sequence of nucleic acid array complementation and the acceptor fluorophore for modifying the sequence；Or working as is acceptor fluorophore on fluorescence labels probe
When, the fluorogenic hybridization probe include with the sequence of nucleic acid array complementation to be detected and modify the sequence donor fluorescent base
Group.
It is preferred that the multiple fluorescence PCR detection primer and probe, including forward primer, reverse primer, fluorescent receptor are miscellaneous
It hands over probe and fluorogenic donor Signature probes, the fluorescent receptor hybridization probe includes the sequence with nucleic acid array complementation to be detected
Column；
The forward primer includes positive universal primer and positive enriching primer, 3 ' ends of positive enriching primer with it is to be checked
The upstream sequence reverse complemental of nucleic acid sequence is surveyed, 5 ' ends are combined with 3 ' ends of positive universal primer, and the reverse primer includes
Sequence connected reversed universal primer, fluorescence labels position and reversed enriching primer, 3 ' ends of the reversed enriching primer with
The downstream sequence reverse complemental of nucleic acid sequence to be detected, 5 ' ends are combined with the 3 ' ends at fluorescence labels position, fluorescence labels position
5 ' ends are combined with 3 ' ends of reversed universal primer, and the fluorogenic donor Signature probes include and the fluorescence labels in reverse primer
The sequence of position complementation and the donor fluorophore for modifying the sequence；Or the reverse primer includes reversed universal primer and anti-
The downstream sequence reverse complemental with nucleic acid sequence to be detected is held to enriching primer, the 3 ' of reversed enriching primer, 5 ' ends lead to reversed
It is combined with 3 ' ends of primer, the forward primer includes connected positive universal primer, fluorescence labels position and the forward direction of sequence
Enriching primer, 3 ' ends of the positive enriching primer and the upstream sequence reverse complemental of nucleic acid sequence to be detected, 5 ' ends with it is glimmering
3 ' the ends at optical label position combine, and the 5 ' ends at fluorescence labels position are combined with 3 ' ends of positive universal primer, and the fluorescence supplies
Body Signature probes include the sequence complementary with the fluorescence labels position in forward primer and the donor fluorophore for modifying the sequence.
It is preferred that further including positive universal primer and reversed universal primer.
It is preferred that the fluorescent receptor hybridization probe include with the sequence of nucleic acid array complementation to be detected and modify the sequence
Acceptor fluorophore.
The sequence at the fluorescence labels position is the sequence label unrelated with nucleic acid sequence to be detected.
A second object of the present invention is to provide a kind of multi-PCR detection methods, using nucleic acid sequence to be detected as template, with
Forward primer, reverse primer carry out PCR using fluorescent receptor hybridization probe and fluorogenic donor Signature probes as probe as primer
Reaction carries out solubility curve analysis after reaction.
It is preferred that also added with individually positive universal primer and reversed universal primer in the reaction system of PCR reaction
Third object of the present invention is to provide multiple PCR detection kit, including above-mentioned forward primer, reverse primer,
Fluorescent receptor hybridization probe and fluorogenic donor Signature probes.
It is preferred that also containing positive universal primer and reversed universal primer.
As shown in Figure 1, 2, 3, the fluorogenic donor Signature probes of detection nucleic acid sequence to be detected of the invention, contain with to
The unrelated sequence label of nucleic acid sequence is detected, when the PCR reaction for detecting target sequence (nucleic acid sequence to be detected) does not carry out, fluorescence
Transmitting group (donor fluorophore) on donor Signature probes is in normal condition, and high-intensitive fluorescence signal can be detected,
And receiving on fluorescent receptor hybridization probe group (acceptor fluorophore) be because farther out with fluorogenic donor Signature probes distance, not
The fluorescence that transmitting group is launched is received, the fluorescence not excited issues.It is glimmering after the PCR for detecting target sequence, which reacts, to carry out
Light donor Signature probes and fluorescent receptor hybridization probe are in conjunction with a large amount of single stranded PCR products that PCR reaction process generates, and the two
It being closer, the fluorescence that fluorogenic donor Signature probes are issued is absorbed by the acceptor fluorophore on fluorescent receptor hybridization probe,
Fluorescence resonance transfer phenomena occurs, the fluorescence intensity of the two is made all to change.When carrying out solubility curve analysis, fluorogenic donor
Signature probes or fluorescent receptor hybridization probe are separated with the single stranded PCR products combined at a certain temperature, and the two distance becomes remote,
Fluorescence resonance transfer phenomena disappears, and the two fluorescence intensity changes again, forms the reversed dissolution peak at specific temperature.
The invention has the advantages that：
It is of the invention based on fluorescence resonance transfer phenomena and to carry out solubility curve analysis method and existing solubility curve is analyzed
Technology is compared：1, to detect, each target sequence separately designs a fluorogenic donor Signature probes and a fluorescent receptor hybridization is visited
Needle, wherein the reversed binding sequence of one (such as fluorogenic donor Signature probes) directly designs on primer, so that the probe is molten
Solution temperature is fixed, and is not influenced by area to be tested nucleotide sequence sudden change, facilitates interpretation, another probe is then directly incorporated in non-
On the target sequence that non-symmetric PCR amplification goes out, guarantee the specificity of detection；2, the sequence label on different primers can be used in design
Similar sequence realizes that a fluorescence probe hybridizes multiple sequence labels simultaneously, and it is molten to change its by the difference of number of base
Temperature is solved, the quantity of fluorescence probe is reduced, so that the change in fluorescence being wound mutually between different fluorescence probes is reduced, so that
Background background when hybrid curve is analyzed is substantially reduced, and will not influence the detection of other positive target sequences；3, when there is a plurality of fluorescence
In the presence of probe, due to fluorescent emission group (donor fluorophore) and fluorescent absorption group (acceptor fluorophore) of the invention
It is not located on same probe, the change of unbonded fluorescence probe three-dimensional structure in temperature change will not be visited as general T aqman
Needle or molecular beacon probe equally substantially change fluorescence signal intensity, and the mutual winding between fluorescence probe will not be as common
Taqman probe or molecular beacon probe equally change mutually fluorescence signal intensity, so that the background signal between probe will not be mutual
It mutually interferes, so that the interpretation of multiplex PCR is more clear.
As shown in figure 5, being detected when being incorporated into the fluorescence probe of sequence label on primer only with one, fluorescence is sent out
It penetrates group and receives group and modified respectively in the both ends of fluorescence probe, the probe for being incorporated into amplification region without the use of Article 2 comes
Guarantee specificity.Red curve (arrow 1 is signified) is 10^5copies plasmid as template, and brown (arrow 2 is signified) is NTC pairs
According to after reaction, because of the non-specific amplification of primer dimer, the positive peak as positive hole occurs in negative control.
As shown in fig. 6, yellow (arrow 1 is signified) is the NTC solubility curve figure of a fluorescence probe, blue (2 institute of arrow
Refer to) it is two same fluorescent markers but the different mixed NTC solubility curve figure of fluorescence probe of sequence, it is seen that work as fluorescence probe
When quantity increases, fluorescence superposition and mutual winding will lead to the variation of solubility curve peak type between fluorescence probe, be unfavorable for tying
Fruit interpretation.
As shown in fig. 7, brown (arrow 1 is signified) is the probe NTC solubility curve figure of a mark fluorescent donor groups, it is red
(arrow 2 is signified) is the Taqman probe NTC solubility curve figure that mark fluorescent donor groups and fluorescent receptor group are distinguished in both ends,
It can be seen that Taqman probe due to temperature change when itself secondary structure variation cause fluorogenic donor group and acceptor groups distance hair
Changing changes greatly fluorescence intensity therewith, influences interpretation.
The present invention cancels the fluorogenic donor group on universal primer, and fluorogenic donor group is changed into detection probe, and (donor is glimmering
Optical label probe) on, the target sequence specific probe for being located at the region after PCR amplification between two primers is introduced, it will be by
Body fluorophor is modified at the nearly donor fluorophore end of target sequence specific probe, and only PCR amplification is target target sequence
When, fluorescent receptor hybridization probe can be just incorporated on the product of asymmetric pcr, and fluorescence resonance shifts ability, to avoid
False positive caused by primer dimer or PCR are accidentally expanded, significantly improves specificity；It 2, will be on fluorescent receptor hybridization probe
Receptor is changed to without fluorophore, even therefore when detecting multiple target sequences using a plurality of target sequence specific probe not yet
Any fluorescence can be generated, to reduce fluorescence background value, is not influenced by dimer between target sequence specific probe；3, lead to
It crosses the directly fluorescence of detection fluorogenic donor group rather than the fluorescence of fluorescent receptor group, it is glimmering that most of fixation in the market can be compatible with
The PCR instrument of optical channel.
Detailed description of the invention：
Fig. 1 is primer and probe of the invention schematic diagram corresponding with nucleic acid sequence to be detected；
Forward and reverse enriching primer 3 ' end and template reverse complemental, 5 ' hold as universal primer binding site.Fluorescence labels
Position is located at forward or backwards between the template complementary region and universal primer combined area of enriching primer, is located in this figure reversed general
Between primer and reversed enriching primer.Fluorescent receptor hybridization probe 5 ' terminal modified fluorescent receptor group (acceptor fluorophore) is glimmering
Light donor Signature probes 3 ' terminal modified fluorogenic donor group (donor fluorophore), the fluorescence which issues can quilt
Acceptor groups on fluorescent receptor hybridization probe are absorbed, so that fluorescence intensity change.
Fig. 2 is primer and probe of the invention, and situation, schematic diagram under free state is not present in template to be measured；
When template to be measured is not present, fluorescent receptor hybridization probe, fluorogenic donor Signature probes are in free shape
Farther out, FRET effect, the fluorescence intensity that fluorophor is issued do not occur for state, fluorogenic donor group and fluorescent receptor group distance
Holding is basically unchanged.
Fig. 3 is the schematic diagram that primer and probe of the invention expands nucleic acid sequence to be detected；
Fig. 4 is melting curve figure；
Fig. 5 is melting curve figure；
Fig. 6 is melting curve figure；
Fig. 7 is melting curve figure.
Specific embodiment
It is further discussed below the present invention in the following with reference to the drawings and specific embodiments, the present invention does not address place and is suitable for existing skill
Art.It is given below specific embodiments of the present invention, but embodiment is not intended to limit this merely to the present invention is described in further detail
The claim of invention.
Embodiment 1
Using the method detection various concentration target sequence template of the embodiment of the present invention 1 to evaluate the practicality.
Target sequence template is that (it is certain section of sequence of the genomic DNA of gonococcus to the artificial plasmid comprising PCR amplification region
Column, are named as the plasmid standard of gonococcus, sequence is as shown in SEQ ID NO.1), by raw industry science skill (Shanghai) Co., Ltd.
Synthesis is 10 by concentration dilution3/ul、104/ ul various concentration.
It the use of instrument is the macro stone Slan-96p fluorescence quantitative PCR instrument in Shanghai.
Amplimer sequence is：
STD-F (forward primer):5'-TGGGTTCCCTAAGGGTTGGAGGTCCATCATCCCTCAGCCTTCATCGGTA CGCCTGCTACTTTCACGCT-3’
STD-R (reverse primer):5'-GTGCCAGCAAGATCCAATCTAGAcaatggatcggtatcactcgc-3’
GP1 (universal primer)：5'-GGGTTCCCTAAGGGTTGGA-3'
GP2 (universal primer)：5'-GTGCCAGCAAGATCCAATCTAGA-3'
Fluorescent receptor probe sequence is：
STD-RP:5 '-ggaaagtaatcagatgaaaccagttccggctgttgtcggcaagccggggt-3 ', 5 ' ends
Modify BHQ2,3 ' terminal modified C3-Spacer
Fluorogenic donor probe sequence is：
STDDP：5 '-GGTCCTTCATCGCTCAGCCTTCACCGG-3 ', 3 ' terminal modified FAM
Reaction system is：
2X PCR Mix 12.5ul (includes enzyme, Buffer, dNTP, Mg ion etc.)
Primer STD-F:100nmol；STD-R:100nmol；GP1:1pmol；GP2:10pmol
Probe STD-RP:2.5pmol；STD-DP：2.5pmol
Template 103/ul、104/ ul concentration template each 1ul, blank control ddH2O 1ul。
ddH2O polishing is to 25ul
Reaction condition：
Melting curve analysis：
Setting detection fluorescence channel is FAM
Reaction condition is：95℃2min；45℃2min；45 DEG C of -85 DEG C of melting curve analysis.
As a result as shown in figure 4, blue (arrow 1) is 103/ ul template, green (arrow 2) are 104/ ul template, yellow (arrow
3) first is NTC, it is seen that blue and green Kong Jun have a positive peak, and Tm value is 57 degree, and are expected unanimously.Yellow is without any positive peak.
2, sensitivity tests
The plasmid standard of gonococcus (NG) is done into 8 dilutions from 1 × 10 respectively7~1 × 100Copy number/μ L, with again
Fluorescent quantitative PCR (according to the reaction system and reaction condition of embodiment 1) is carried out respectively than being diluted to template, and there are Tm values
57 ± 1 degrees Celsius of inversion dissolution peak interpretation is the positive, and otherwise interpretation is feminine gender.Simultaneously using primer pair STD-F and STD-R into
Row standard PCR amplification.
Testing result shows using the sensitivity of the standard plasmid of method detection NG of the invention up to 1 × 102Copy
Number/μ L, and regular-PCR is minimum can detecte to 1 × 102Copy number/μ L illustrates method and regular-PCR method phase of the invention
Together.
3, specific test
1 detection method through the embodiment of the present invention, respectively to national standard strain sample：Gonococcus, human papilloma
Virus, chlamydia trachomatis, gonococcus, ureaplasma parvum, trichomonas vaginalis, genital tract mycoplasma, mycoplasma hominis are detected,
The result shows that other pathogens sample standard deviation is without positive dissolution peak in addition to gonococcus standard items have positive dissolution peak, it was demonstrated that this
The detection method high specificity established is invented, with other equal no cross reactions of common genital tract causal agent.
4, repetitive test
With 1 × 10 in the standard items of NG plasmid6With 1 × 104Copy number/dilution of μ L two conduct template, to this two
A dilution carries out 2 replications in different time sections, carries out 3 replications and setting 3 simultaneously to same template every time
A repeating hole carries out real-time fluorescence quantitative PCR according to method described in the embodiment of the present invention 1.Wherein：The coefficient of variation (β)=mark
Quasi- deviation (SD)/average (X), as a result repetitive test positive rate 100%.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
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gtcaggcggt gtacctgatg gtttttcaat ggatcggtat cactcgctct gccgagcaag 60
aacaaaaaga aagcatcatg cccctcattg gcgtgtttcg catatttaag ggcataattt 120
ccgaaaaagc cgccattttt gtaattcaga ccggcataat acacatccga ccccggcttg 180
ccgacaacag ccggaactgg tttcatctga ttactttcca gcgtgaaagt agcaggcgta 240
taggcggact tgctgttttg actcggaaca aattgaatgc tgccgctgaa accggaaaat 300
Claims (9)
1. a kind of multiple fluorescence PCR detection primer and probe, which is characterized in that including forward primer, reverse primer, fluorescent hybridization
Probe and fluorescence labels probe；
The fluorogenic hybridization probe includes the sequence with nucleic acid array complementation to be detected, and the forward primer includes positive rich
Collect primer, the upstream sequence reverse complemental at 3 ' ends and nucleic acid sequence to be detected of positive enriching primer, the reverse primer packet
Include the connected fluorescence labels position of sequence and reversed enriching primer, 3 ' ends of the reversed enriching primer and nucleic acid sequence to be detected
The downstream sequence reverse complemental of column, 5 ' ends are held with the 3 ' of fluorescence labels position to be combined, the fluorescence labels probe include with instead
The sequence of fluorescence labels position complementation into primer and the donor fluorophore or acceptor fluorophore for modifying the sequence；Or institute
The reverse primer stated includes reversed enriching primer, and 3 ' ends of reversed enriching primer are reversed with the downstream sequence of nucleic acid sequence to be detected
Complementation, the forward primer include the connected fluorescence labels position of sequence and positive enriching primer, and the positive enrichment is drawn
The upstream sequence reverse complemental at 3 ' ends and nucleic acid sequence to be detected of object, 5 ' ends are combined with the 3 ' ends at fluorescence labels position, described
Fluorescence labels probe include the sequence complementary with the fluorescence labels position in forward primer and the donor fluorescent for modifying the sequence
Group or acceptor fluorophore.
2. multiple fluorescence PCR detection primer according to claim 1 and probe, which is characterized in that when fluorescence labels probe
It is upper for donor fluorophore when, the fluorogenic hybridization probe includes and the sequence of nucleic acid array complementation to be detected and the modification sequence
The acceptor fluorophore of column；Or when on fluorescence labels probe be acceptor fluorophore when, the fluorogenic hybridization probe include with
The sequence of nucleic acid array complementation to be detected and the donor fluorophore for modifying the sequence.
3. multiple fluorescence PCR detection primer according to claim 1 and probe, which is characterized in that the multi-fluorescence
PCR detection primer and probe, including forward primer, reverse primer, fluorescent receptor hybridization probe and fluorogenic donor Signature probes, institute
The fluorescent receptor hybridization probe stated includes the sequence with nucleic acid array complementation to be detected；The forward primer includes positive general
Primer and positive enriching primer, the upstream sequence reverse complemental at 3 ' ends and nucleic acid sequence to be detected of positive enriching primer, 5 ' ends
It is combined with 3 ' ends of positive universal primer, the reverse primer includes the connected reversed universal primer of sequence, fluorescence labels portion
Position and reversed enriching primer, the downstream sequence reverse complemental at 3 ' ends and nucleic acid sequence to be detected of the reversed enriching primer,
5 ' ends are combined with the 3 ' ends at fluorescence labels position, and the 5 ' ends at fluorescence labels position are combined with 3 ' ends of reversed universal primer, described
Fluorogenic donor Signature probes include the sequence complementary with the fluorescence labels position in reverse primer and the donor for modifying the sequence
Fluorophor；Or the reverse primer includes reversed universal primer and reversed enriching primer, 3 ' ends of reversed enriching primer with
The downstream sequence reverse complemental of nucleic acid sequence to be detected, 5 ' ends are combined with 3 ' ends of reversed universal primer, the forward primer
Positive universal primer, fluorescence labels position and the positive enriching primer being connected including sequence, the 3 ' of the positive enriching primer
The upstream sequence reverse complemental at end and nucleic acid sequence to be detected, 5 ' ends are combined with the 3 ' ends at fluorescence labels position, fluorescence labels portion
5 ' ends of position are combined with 3 ' ends of positive universal primer, and the fluorogenic donor Signature probes include and the fluorescence in forward primer
The sequence of label position complementation and the donor fluorophore for modifying the sequence.
4. multiple fluorescence PCR detection primer according to claim 1 and probe, which is characterized in that further include positive general
Primer and reversed universal primer.
5. multiple fluorescence PCR detection primer according to claim 1 and probe, which is characterized in that the fluorescence labels
The sequence at position is the sequence label unrelated with nucleic acid sequence to be detected.
6. multiple fluorescence PCR detection primer according to claim 1 and probe, which is characterized in that the fluorescent receptor
Hybridization probe include with the sequence of nucleic acid array complementation to be detected and modify the sequence acceptor fluorophore.
7. a kind of multiple fluorescence PCR detection method, which is characterized in that using nucleic acid sequence to be detected as template, with claim 1 institute
The forward primer stated, reverse primer as primer, using fluorescent receptor hybridization probe and fluorogenic donor Signature probes as probe into
Row PCR reaction, carries out solubility curve analysis after reaction.
8. multiple fluorescence PCR detection method according to claim 7, which is characterized in that in the reaction system of PCR reaction also
Added with individual positive universal primer and reversed universal primer.
9. a kind of multiple fluorescence PCR detection reagent box, which is characterized in that including multiple described in claim 1,2,3,4,5 or 6
Fluorescent PCR detecting primer and probe.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109486912A (en) * | 2018-12-07 | 2019-03-19 | 领航基因科技（杭州）有限公司 | A kind of probe primer combination and design method for digital pcr amplification |
| CN109652521A (en) * | 2018-12-28 | 2019-04-19 | 黄劭 | A kind of hybridization probe containing manual tag |
| CN111349691A (en) * | 2018-12-21 | 2020-06-30 | 迈克生物股份有限公司 | Composition, kit and detection method for detecting EGFR gene deletion mutation |
| WO2023125560A1 (en) * | 2021-12-27 | 2023-07-06 | 迈克生物股份有限公司 | Primer probe for detection, primer probe set, and application thereof |
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| CN111349691A (en) * | 2018-12-21 | 2020-06-30 | 迈克生物股份有限公司 | Composition, kit and detection method for detecting EGFR gene deletion mutation |
| CN111349691B (en) * | 2018-12-21 | 2023-10-20 | 迈克生物股份有限公司 | Composition, kit and detection method for EGFR gene deletion mutation detection |
| CN109652521A (en) * | 2018-12-28 | 2019-04-19 | 黄劭 | A kind of hybridization probe containing manual tag |
| WO2023125560A1 (en) * | 2021-12-27 | 2023-07-06 | 迈克生物股份有限公司 | Primer probe for detection, primer probe set, and application thereof |
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Application publication date: 20181130 |