CN104404162A - Real-time fluorescence PCR method for detecting multiple genes or different targets with primer associated universal probe - Google Patents

Real-time fluorescence PCR method for detecting multiple genes or different targets with primer associated universal probe Download PDF

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Publication number
CN104404162A
CN104404162A CN201410772831.0A CN201410772831A CN104404162A CN 104404162 A CN104404162 A CN 104404162A CN 201410772831 A CN201410772831 A CN 201410772831A CN 104404162 A CN104404162 A CN 104404162A
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primer
probe
general
sequence
target
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高利飞
白宪鹤
孟浪
李桂林
付光宇
吴学炜
苗拥军
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Autobio Diagnostics Co Ltd
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Autobio Diagnostics Co Ltd
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    • 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

Abstract

The invention discloses a real-time fluorescence PCR method for detecting multiple genes or different targets with a primer associated universal probe. The method comprises the following steps: firstly, relying on a composite sequence primer with a special structure, and introducing a combined position of a universal probe and a universal amplification primer at the tail end of an amplified product in the process of initial amplification so as to realize different targets; using the same probe for real-time fluorescence detection. The method has the advantage that the production process is simple and easy to control, the absolute usage amount of the probe is decreased, and the reagent cost is reduced. In a single detection, different items can share one probe, so that the production process is simplified and is convenient to control. In multiple detections, different targets realize specific real-time fluorescence detection by using the same probe without different probes, so that the absolute usage amount of the probe is directly reduced, the problems of poor detection curves and low sensitivity caused by a high multi-probe fluorescent background are solved, and besides, the reagent cost is also reduced by using the same probe.

Description

Primer association general probe is adopted to detect the real time fluorescent PCR method of multiple gene or different target
Technical field
The present invention relates to real-time fluorescent PCR amplification detection method, especially relate to a kind of employing primer correlating method, realize the real time fluorescent PCR method that general probe detects multiple gene or different target.
Background technology
Polymerase chain reaction (Polymerase Chain Reaction, PCR) be the isothermal DNA amplification that the mid-80 grows up, it has special, responsive, the outstanding advantages such as productive rate is high, quick, easy, reproducible, easy automatization, in vitro the goal gene that will study or a certain DNA fragmentation can be expanded to 100,000 and even 1,000,000 times within a few hours at one, see United States Patent (USP) the 4th, 683,195 and the 4,683, No. 202.The ultimate principle of round pcr is similar to the natural reproduction process of DNA, and its specificity depends on the Oligonucleolide primers with the complementation of target sequence two ends.PCR is by sex change--annealing--, and extension three primitive reaction steps are formed: the 1. sex change of template DNA: template DNA is after being heated to about 93 DEG C certain hours, make template DNA double-strand or dissociate through the double-stranded DNA that pcr amplification is formed, make it to become strand, so that it is combined with primer, for lower whorl reaction is prepared; 2. the annealing (renaturation) of template DNA and primer: template DNA becomes after strand through heat denatured, and temperature is down to about 55 DEG C, and the complementary sequence of primer and template DNA strand matches and combines; 3. the extension of primer: DNA profiling--primer binding substances is under the effect of Taq archaeal dna polymerase, take dNTP as reaction raw materials, target sequence is template, by base pairing and semiconservative replication principle, synthesize a semiconservative replication chain that the is new and complementation of template DNA chain, recirculation sex change--annealing--extends three processes, just can obtain more " semiconservative replication chain ", and this new chain can become again the template of circulation next time.Often complete a circulation and need 2 ~ 4 minutes, within 2 ~ 3 hours, just can will wait that expanding goal gene amplification amplifies millions of times.The advantage of the method is that the external rapid amplifying achieving DNA profiling amplifies, and identify for follow-up electrophoretic analysis, susceptibility is high.But the problem of the method is electrophoresis method qualification, often brings the pollution of PCR primer, cannot promote in clinical application.
Real-time fluorescence quantitative PCR (Real-time PCR) technology was released in 1996 by Applied Biosystems company of the U.S., because this technology not only achieves the leap of PCR from qualitative to quantitative, and compared with Standard PCR, it has, and specificity is stronger, effective solves PCR pollution problem, level of automation high, is used widely at present.So-called Real-Time Fluorescent Quantitative PCR Technique, refers to and add fluorophor in PCR reaction system, utilize fluorescent signal to accumulate the whole PCR process of Real-Time Monitoring, finally by typical curve, unknown template is carried out to the method for quantitative analysis.
The fluorescence chemical that quantitative fluorescent PCR uses can be divided into two kinds: fluorescence dye and fluorescent probe.Now its principle is summarized as follows:
1) SYBR fluorescence dye: in PCR reaction system, add excessive SYBR fluorescence dye, after SYBR fluorescence dye non-specifically mixes DNA double chain, emitting fluorescence signal, and the SYBR dye molecule do not mixed in chain can not launch any fluorescent signal, thus ensure the increase of fluorescent signal and the increase Complete Synchronization of PCR primer.The advantage of the method achieves stopped pipe reaction detection, detects product in real time and carry out amount, and without the need to electrophoresis detection, but fluorescent signal specificity is not strong, needs subsequent dissolution tracing analysis to carry out specificity analyses.Extend analysis time, technical requirements is high, and result judges that subjectivity is strong, and clinical expansion is limited.
2) TaqMan fluorescent probe: add a specific fluorescent probe during pcr amplification while adding pair of primers, this probe is an oligonucleotide, and two ends mark a reporter fluorescence group and a quenching fluorescence group respectively.When probe is complete, the fluorescent signal that reporter group is launched is quenched group absorptions; During pcr amplification, probe enzyme is cut degraded by the 5'-3' 5 prime excision enzyme activity of Taq enzyme, reporter fluorescence group is separated with quenching fluorescence group, thus fluorescence monitoring system can receive fluorescent signal, namely often increase a DNA chain, just have a fluorescence molecule to be formed, the accumulation and the PCR primer that achieve fluorescent signal form Complete Synchronization.As US Patent No. 5,723,591 reports, the advantage of the method achieves stopped pipe reaction detection, detects product in real time and carry out amount, and without the need to electrophoresis detection, speed is fast, and accuracy is high, high specificity.But the necessary veriform special probe of design and use of different detection targets, the synthesis expense of probe costly.
Multiplex PCR (multiplex PCR), it is in same PCR reaction system, add that two to above primed probe, the PCR reaction of the multiple nucleic acid fragment of augmentation detection simultaneously, its reaction principle, reaction reagent is identical with general PCR with operating process, detect while multiplex PCR is mainly used in multiple pathogenic microorganisms or qualification, detect while multiple pathogenic microorganisms or qualification, it is the Auele Specific Primer simultaneously adding multiple pathogenic microorganisms in same PCR reaction tubes, adopt fluorescence quantifying PCR method, can simultaneously high efficiency, economical, easy, quick detection multiple pathogens or which type pathogenic infection identifies be.But detect in application at single tube multiplex amplification, because fluorescence channel number is limited, five can only be detected at most.When single tube detects more than 6 targets, more than 2 targets are just needed to share a passage, these targets just need the identical fluorescence dye that mark is corresponding, every bar probe has certain background signal, many probe background signal accumulations, just there will be this passage background signal too high, have a strong impact on amplification curve line style, simultaneously many probe synthesis cost intensive.
Fluorescence dye solubility curve analytical procedure is adopted to identify PCR primer, target type in assay products, as described in number of patent application CN102559868A, adopt closed tube analysis technology, decrease the risk of product pollution, reduce testing cost, but extend analysis time, technical requirements is high, and result judges that subjectivity is strong.
In sum, although have multiple nucleic acids recognition sequence and quantitative detection technique both at home and abroad at present, but in substance detection and Multiple detection, industrial production cost control, simple process, technical compatibility still has certain limitation, so be necessary that developing a kind of different target can adopt same probe to realize the universal method of real-time fluorescence detection.
Summary of the invention
The object of the present invention is to provide a kind of employing primer correlating method, realize the real time fluorescent PCR method that general probe detects multiple gene or different target.
For achieving the above object, the present invention can take following technical proposals:
The real time fluorescent PCR method that employing primer association general probe of the present invention detects multiple gene or different target is: react for polymerase chain reaction, comprise special primer system, probe, polysaccharase, dNTPS, damping fluid;
Primer system comprises: target sequence special primer F, general amplification primer R0 and multiplexed sequence primer RC;
Probe is general probe P0, and different target can adopt same general probe to detect;
Multiplexed sequence primer comprises following structure from 5 ' end successively to 3 ' end: general amplification primer sequence R0 district, general probe sequence P0 district, target specific combination district Zone R;
The first round reacts, and the target specific combination district R of multiplexed sequence primer, is combined with target, extends, and produces nucleic acid chains L ', L ' containing general amplification primer sequence R0, general probe sequence P0, target sequence special primer F complementary sequence;
Second takes turns reaction, and target sequence special primer F and target sequence nucleic acid chains complementary strand L ' combines, extends, and produces nucleic acid chains L0, L0 containing general amplification primer sequence complementary sequence R0 ', general probe complement thereof P ';
Target sequence special primer F, general amplification primer R0 form upstream and downstream primer pair, carry out main reaction, repeat special amplification nucleic acid chains L0 and complementary strand L0 '.General probe complement thereof P ' district in general probe P0 bind nucleic acid chain L0, hybridization combines or is hydrolyzed generation nonspecific signal.
Described multiplexed sequence primer R camount be less than general amplification primer R 0amount, described multiplexed sequence primer R camount be 10nM to 100nM, general amplification primer R 0amount 50nM-400nM.
Know-why of the present invention: multiplexed sequence primer comprises following structure from 5 ' end successively to 3 ' end: general amplification primer sequence R 0district, general probe sequence P 0district, target specific combination district Zone R.When reactive system contains target to be detected, the target specific combination district R of multiplexed sequence primer is combined with target, extends, by general amplification primer sequence R 0district and general probe sequence P 0district connects into amplified production.Target sequence special primer F, in conjunction with this amplified production, extends and produces containing general amplification primer R 0with general probe P 0the product of land, enters the reaction of main amplification detection.Main iodine is conventional Fluorescence PCR, relies on general amplification primer sequence R 0, general probe sequence P 0, target sequence special primer F, amplify the new template strand produced, produce signal.
When reactive system is without target to be detected, multiplexed sequence primer cannot extend in conjunction with template, cannot by general amplification primer sequence R 0district and general probe sequence P 0district connects into amplified production, and then target sequence special primer F just cannot produce containing general amplification primer R 0with general probe P 0the product of land, cannot enter main iodine, thus cannot produce signal.
The invention has the advantages that production technique is simple, be convenient to control, decrease the absolute usage quantity of probe, reduce reagent cost.Its advantage is embodied in:
1) in substance detects, disparity items can share a probe, and production technique is simplified, and is convenient to control; 2) when Multiple detection, different target adopts same probe to realize special real-time fluorescence and detects, without the need to adding different probe, the absolute usage quantity of direct minimizing probe, avoid that multiprobe autofluorescent background is high causes the problem that detection curve is poor, susceptibility is low, use same probe to decrease reagent cost simultaneously.
Accompanying drawing explanation
Fig. 1 is reaction principle figure of the present invention.
Fig. 2 is the detected result of the embodiment of the present invention 1.
Fig. 3 is the detected result of the embodiment of the present invention 2.
Embodiment
As shown in Figure 1, primer association general probe of the present invention detects the real time fluorescent PCR method of multiple gene or different target, adopts polymerase chain reaction, comprises multiplexed sequence primer system, use same probe, also comprise polysaccharase, dNTPS, damping fluid;
Multiplexed sequence primer system comprises target sequence special primer F, general amplification primer R 0with multiplexed sequence primer R c; Probe is general probe P 0;
Multiplexed sequence primer R cgeneral amplification primer sequence R is comprised successively to 3 ' end from 5 ' end 0district, general probe sequence P 0district and target specific combination district Zone R;
The first round reacts: the target specific combination district R of multiplexed sequence primer is combined with target, extends, and produce target sequence nucleic acid chains complementary strand L ', L ' is containing general amplification primer sequence R 0, general probe sequence P 0, target sequence special primer F complementary sequence;
Second takes turns reaction: target sequence special primer F and target sequence nucleic acid chains complementary strand L ' combines, extends, and produces nucleic acid chains L 0, L 0containing general amplification primer sequence complementary sequence R 0', general probe complement thereof P ';
Third round is reacted: target sequence special primer F, general amplification primer R 0composition upstream and downstream primer pair, carries out main reaction, repeats to amplify nucleic acid chains L 0with complementary strand L 0'; General probe P 0bind nucleic acid chain L 0in general probe complement thereof P ' district, hybridization combines or is hydrolyzed and produces specific fluorescence signal.
What relate in this reaction is nucleic acid-templated for DNA or RNA; RNA template enzymatic system, except Taq polysaccharase, also needs mlv, AMV, Tth enzyme etc. to have the enzyme of reverse transcriptase activity.
Below by specific embodiment, the present invention is further detailed explanation.
Carry out design verification with HPV18 type, high-risk 12 types of HCV RNA, HPV respectively in following examples, HCV RNA adopts the universal sequence different from HPV18, to verify method of design versatility; Verify that multiple aspect is applied with high-risk 12 types of HPV.
embodiment 1
The application that primer association general probe system in the present invention detects in HPV18 type.
Experimental design: in the high, medium and low value sample of contrast HPV18 type, primer association general probe detection system of the present invention and conventional quantitative fluorescent PCR system susceptibility.
Design of primers: according to the HPV18 type L1 gene conserved sequence design primer on NCBI gene bank (http://www.ncbi.nlm.nih.gov).Primer sequence sees attached list 1.
Quantitative fluorescent PCR system to more solito: 1*PCR buffer, 2.5mM magnesium ion, 0.5U Taq archaeal dna polymerase, 200 μMs of dNTP, upstream and downstream primer (SEQ ID NO 1-2) amount is 200nM, and detection probes FAM BHQ1 marks (SEQ ID NO 3) amount for 100nM.
Primer association general probe detection system: 1*PCR buffer, 2.5mM magnesium ion, 0.5U Taq archaeal dna polymerase, 200 μMs of dNTP, upstream primer (SEQ ID NO 1) amount is 200nM, multiplexed sequence primer R cthe amount of (SEQ ID NO 6) is 20nM, general amplification primer (SEQ ID NO 4) R 0amount 200nM, it is 50nM that detection probes FAM BHQ1 marks the amount of (SEQ ID NO 5).
HPV trace routine: 95 DEG C of denaturations 5 minutes; 95 DEG C of sex change 15 seconds, 52 DEG C of annealing 15 seconds, 72 DEG C of annealing 15 seconds, and fluorescence is detected when 52 DEG C of annealing, repeat 45 circulations.
Result interpretation: HPV18 high, medium and low concentration clinical sample all can effectively increase in the quantitative fluorescent PCR system and universal primer probe system of the present invention of routine, and two kinds of reaction system sensitivity are close.
Concrete outcome is as shown in Figure 2: two kinds of high, medium and low value clinical samples of systems amplification HPV18, expanding effect is close.Wherein, system 1 represents conventional quantitative fluorescent PCR system, and system 2 represents the universal primer probe reaction system in the present invention.
embodiment 2
The application that primer association general probe system in the present invention detects at HCV RNA.
Experimental design: in the high, medium and low value serum sample of contrast HCV RNA, primer association general probe detection system of the present invention and conventional quantitative fluorescent PCR system susceptibility.
Design of primers: according to HCV 5 ' the UTR sequence design primer on NCBI gene bank (http://www.ncbi.nlm.nih.gov).Primer sequence sees attached list 2.
Quantitative fluorescent PCR system to more solito: 1*RT-PCR single stage method buffer, 2.5mM magnesium ion, 10U M-MLV ThermoScript II, 0.5U Taq archaeal dna polymerase, 200 μMs of dNTP, upstream and downstream primer (SEQ ID NO 7-8) amount is 200nM, and detection probes FAM BHQ1 marks (SEQ ID NO 9) amount for 100nM.
Primer association general probe detection system: 1*PCR buffer, 2.5mM magnesium ion, 0.5U Taq archaeal dna polymerase, 200 μMs of dNTP, upstream primer (SEQ ID NO 7) amount is 200nM, multiplexed sequence primer R cthe amount of (SEQ ID NO 12) is 20nM, general amplification primer (SEQ ID NO 10) R 0amount 200nM, it is 50nM that detection probes FAM BHQ1 marks the amount of (SEQ ID NO 11).
HCV trace routine: 42 DEG C of 30min; 95 DEG C of denaturations 5 minutes; 95 DEG C of sex change 15 seconds, 52 DEG C of annealing 15 seconds, 72 DEG C of annealing 15 seconds, and fluorescence is detected when 52 DEG C of annealing, repeat 45 circulations.
Result interpretation: HCV high, medium and low concentration clinical sample all can effectively increase in the quantitative fluorescent PCR system and universal primer probe system of the present invention of routine, and two kinds of reaction system sensitivity are close.
Concrete outcome is as shown in Figure 3: two kinds of high, medium and low value clinical samples of systems amplification HCV, expanding effect is close.Wherein, system 1 represents conventional quantitative fluorescent PCR system, and system 2 represents the universal primer probe reaction system in the present invention.Universal primer probe in the present invention all can normally work in DNA and RNA project, and the design of universal primer probe has very large handiness.
embodiment 3
Primer association general probe system in the present invention carries out the application of multiple fluorescence quantitative detection in high-risk 14 types of HPV.
Experimental design: in contrast HPV high-risk 14 types (HPV16,18,31,33,35,39,45,51,52,56,58,59,66,68) sample, primer association general probe detection system of the present invention and conventional quantitative fluorescent PCR system susceptibility; By HPV low danger common type (HPV6, HPV11) positive sample checking native system detection specificity.
Design of primers: design primer respectively according to the high-risk 14 type L1 gene conserved sequences of the HPV on NCBI gene bank (http://www.ncbi.nlm.nih.gov), primer sequence sees attached list 3.
Quantitative fluorescent PCR system to more solito: 1*PCR buffer, 2.5mM magnesium ion, 0.5U Taq archaeal dna polymerase, 200 μMs of dNTP, upstream and downstream primer (SEQ ID NO 13-40) amount is 200nM, and detection probes FAM BHQ1 marks (SEQ ID NO 41-54) amount for 100nM.
Primer association general probe detection system: 1*PCR buffer, 2.5mM magnesium ion, 0.5U Taq archaeal dna polymerase, 200 μMs of dNTP, upstream primer (SEQ ID NO 13-26) amount is 200nM, multiplexed sequence primer R cthe amount of (SEQ ID NO 55-68) is 20nM, general amplification primer (SEQ ID NO 69) R 0amount 200nM, it is 50nM that detection probes FAM BHQ1 marks the amount of (SEQ ID NO 70).
HPV trace routine: 95 DEG C of denaturations 5 minutes; 95 DEG C of sex change 15 seconds, 52 DEG C of annealing 15 seconds, 72 DEG C of annealing 15 seconds, and fluorescence is detected when 52 DEG C of annealing, repeat 45 circulations.
The high-risk 14 type clinical samples of result interpretation: HPV all can effectively increase in the quantitative fluorescent PCR system and universal primer probe system of the present invention of routine.
Concrete outcome is as shown in subordinate list 4: though the high-risk 14 type Multiple detections of HPV use the universal primer probe system expanding effect in the present invention and there is faint difference to the sensitivity impinged upon in single amplification, but, only need a probe just can detect multiple row in the whole reaction system of the present invention and survey target, effectively solve the too high problem of many probe backgrounds in multiple fluorescence quantitative PCR reaction, can extensively should in multiple fluorescence quantitative PCR; And by carrying out cross specificity checking to HPV low risk positive sample, show that this primer association general probe detection system specificity is also verified.
Subordinate list 1:
Subordinate list 2:
Subordinate list 3:
Subordinate list 4:
。

Claims (8)

1. adopt primer to associate a real time fluorescent PCR method for general probe detection multiple gene or different target, it is characterized in that:
The first, react for polymerase chain reaction, comprise special primer system, probe, polysaccharase, dNTPS, damping fluid;
The second, primer system comprises: target sequence special primer F, general amplification primer R0 and multiplexed sequence primer RC;
3rd, probe is general probe P0, and different target can adopt same general probe to detect;
4th, multiplexed sequence primer comprises following structure from 5 ' end successively to 3 ' end: general amplification primer sequence R0 district, general probe sequence P0 district, target specific combination district Zone R; The target specific combination district R of multiplexed sequence primer is combined with target, extends, and general amplification primer sequence R0 district and general probe sequence P0 district are connected into amplified production; Target sequence special primer F, in conjunction with this amplified production, extends the product produced containing general amplification primer R0 and general probe P0 land, enters the reaction of main amplification detection;
5th, main iodine is the Fluorescence PCR of routine, relies on general amplification primer sequence R0, general probe sequence P0, target sequence special primer F, amplifies the new template strand produced, produces signal.
2. employing primer association general probe according to claim 1 detects the real time fluorescent PCR method of multiple gene or different target, it is characterized in that: described target sequence special primer F is positive strand primer or negative strand primer, described general amplification primer sequence R 0marriage chain in contrast.
3. employing primer according to claim 1 association general probe detects the real time fluorescent PCR method of multiple gene or different target, it is characterized in that: described general probe is hydrolysis probes or hybridization probe or molecular beacon probe.
4. employing primer according to claim 1 association general probe detects the real time fluorescent PCR method of multiple gene or different target, it is characterized in that: relate in described reaction nucleic acid-templated be DNA or RNA; Described RNA template enzymatic system comprises the enzyme that Taq polysaccharase and mlv/AMV/Tth have reverse transcriptase activity.
5. the real time fluorescent PCR method not adopting primer to associate general probe detection multiple gene or different target according to claim 1, is characterized in that: described general probe P 0length be 18bp to 50bp.
6. employing primer association general probe according to claim 1 detects the real time fluorescent PCR method of multiple gene or different target, it is characterized in that: described general amplification primer R 0length be 20bp to 100bp.
7. employing primer association general probe according to claim 1 detects the real time fluorescent PCR method of multiple gene or different target, it is characterized in that: described multiplexed sequence primer R camount be less than general amplification primer R 0amount.
8. employing primer association general probe according to claim 1 detects the real time fluorescent PCR method of multiple gene or different target, it is characterized in that: described multiplexed sequence primer R camount be less than general amplification primer R 0amount, described multiplexed sequence primer R camount be 10nM to 100nM, general amplification primer R 0amount 50nM-400nM.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220399A (en) * 2016-12-14 2018-06-29 李保伟 A kind of fluorescence quantifying PCR method based on general probe technology
CN112969707A (en) * 2018-09-07 2021-06-15 克罗玛科德公司 Universal tail primers with multiple binding motifs for multiplex detection of single nucleotide polymorphisms
EP3697931A4 (en) * 2017-10-16 2021-10-13 Chromacode, Inc. Methods and compositions for nucleic acid detection
CN114457196A (en) * 2022-02-28 2022-05-10 上海中医药大学附属龙华医院 Method for detecting multiple high-risk HPV (human papillomavirus) through high-throughput two-dimensional PCR (polymerase chain reaction) single closed tube

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CN102154505A (en) * 2011-04-20 2011-08-17 苟德明 Method and primers for detecting mi ribonucleic acid (miRNA) and application of method

Patent Citations (1)

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CN102154505A (en) * 2011-04-20 2011-08-17 苟德明 Method and primers for detecting mi ribonucleic acid (miRNA) and application of method

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220399A (en) * 2016-12-14 2018-06-29 李保伟 A kind of fluorescence quantifying PCR method based on general probe technology
CN108220399B (en) * 2016-12-14 2023-04-14 李保伟 Fluorescent quantitative PCR method based on universal probe technology
EP3697931A4 (en) * 2017-10-16 2021-10-13 Chromacode, Inc. Methods and compositions for nucleic acid detection
CN112969707A (en) * 2018-09-07 2021-06-15 克罗玛科德公司 Universal tail primers with multiple binding motifs for multiplex detection of single nucleotide polymorphisms
EP3847181A4 (en) * 2018-09-07 2022-06-15 Chromacode, Inc. Universal tail primers with multiple binding motifs for multiplexed detection of single nucleotide polymorphisms
CN114457196A (en) * 2022-02-28 2022-05-10 上海中医药大学附属龙华医院 Method for detecting multiple high-risk HPV (human papillomavirus) through high-throughput two-dimensional PCR (polymerase chain reaction) single closed tube

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