CN110358815A - Method and its kit a kind of while that detect multiple target nucleic acids - Google Patents
Method and its kit a kind of while that detect multiple target nucleic acids Download PDFInfo
- Publication number
- CN110358815A CN110358815A CN201910652239.XA CN201910652239A CN110358815A CN 110358815 A CN110358815 A CN 110358815A CN 201910652239 A CN201910652239 A CN 201910652239A CN 110358815 A CN110358815 A CN 110358815A
- Authority
- CN
- China
- Prior art keywords
- primer
- nucleic acids
- target nucleic
- fluorescent
- melting curve
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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 present invention provides method that is a kind of while detecting multiple target nucleic acids, the method is by expanding target nucleic acids different in same fluorescence channel with the primer of different fluorescent markers, so that the melting curve of the amplified production of target nucleic acids different in same fluorescence channel has different fusing point Tm values, to realize that the amplified production melting curve based on fluorescent primer detects multiple target nucleic acids simultaneously.The present invention detects more target nucleic acids using the melting curve based on product, and detection while more targets may be implemented, and greatly improves detection flux and efficiency, reduces testing cost；The mark fluorescent directly on primer reduces the use of probe, avoids non-specific binding between primed probe and reduces amplification efficiency, improves detection sensitivity.
Description
Technical field
The present invention relates to technical field of molecular biology, in particular to a kind of method for detecting multiple target nucleic acids simultaneously and
Its kit.
Background technique
Clinically common cause pathogeny imcrobe infection symptom has respiratory tract infection, alimentary infection, urogenital tract at present
Infection etc., and cause the pathogenic microorganism of these symptoms complex, including bacterium, virus, mycoplasma, Chlamydia, fungi etc.,
Great difficulty is caused to medical diagnosis on disease and treatment.If infection associated diseases cannot get quick diagnosis, can only be cured via clinic
Raw experience and existing knowledge, which is given, treats, and these treatment methods cause multiple resistance to often along with the abuse of broad-spectrum antibiotic
The appearance of medicine bacterium and the generation of hospital acquired infections.Multiple pathogens simultaneously detect strategy and pathogen it is quick, quasi-
Really identification is most important in disease control.
Traditional the pathogenic microorganism examination method and technology is complicated, positive rate is low, and takes time and effort, fraction of pathogens body culture
Condition is harsh cannot even to cultivate or need the restriction conditions such as technical professional's operation that it is made to be dfficult to apply to clinic early
Phase diagnosis and guiding treatment；Immunofluorescence technique, serological test isosensitivity and specificity are also rarely fulfilled that clinical diagnosis
Needs.With the development of biology new technology, it is indispensable that molecular diagnostic techniques are increasingly becoming Clinical microorganism laboratory
Inspection technology.Due to unknown pathogen body-sensing dye and more pathogen mixed infections in clinical sample, conventional list pathogen nucleic acid inspection
Survey method generally requires repeatedly to be screened, relatively time consuming laborious.Multi-target detection can be realized the quick of multiple pathogen
Identify and diagnose, reduces inspection cost.Multi-target detection has many advantages, such as high efficiency, systematicness and economical and convenient, extensively
The general detection applied to pathogenic microorganism.Common multi-target detection technique has following several.
Real-time fluorescent PCR technology is accumulated by the way that different fluorophors is added in PCR reaction system using fluorescence signal
The entire PCR process of real-time monitoring.Have many advantages, such as High sensitivity, high specific, effectively solve PCR pollution problem, is quick, is mesh
The method of preceding detection of nucleic acids extensive utilization.But due to being limited by fluorescent PCR instrument channel currently on the market, single tube can only at most be examined
4-5 target is surveyed, is not able to satisfy the demand of the clinically relevant multiple pathogens of a certain syndrome or gene screening simultaneously.
Biochip technology is also known as DNA microarray, be by micro-processing technology, by a large amount of DNA probes be fixed to silicon wafer,
On the solid supports such as slide, the base of sample then is obtained by the detection and analysis to hybridization signal with the sample hybridization of label
Because of sequence information.Technique can carry out detection and analysis simultaneously to a genes up to up to ten thousand, have the spies such as micromation, high throughput
Point.But there are still some problems for biochip technology, and if detection sensitivity is low, specificity is poor, and chip manufacturing is at high cost, and needs
Expensive detecting instrument is wanted, these problems make genetic chip fail to be widely applied to laboratory research is mainly limited at present
In the detection and identification of clinical pathogenic microorganism.
High throughput sequencing technologies are to develop swift and violent biotechnology in recent years, by DNA (or cDNA) random fragmentation,
Adjunction head, prepares sequencing library, by carrying out extension to clone ten hundreds of in library, detects corresponding signal, most
Sequence information is obtained eventually.The technology flux is big, high sensitivity, detection, unknown pathogen especially in some new hair disease pathogens
Body identifies to play a significant role in the prevention and control with burst disease.But the current technology still has some problems to be solved: surveying
The mass data that sequence generates needs the personnel of profession to analyze；The time of sequencing and cost still restrict it clinically
It is widely applied.
Isothermal duplication is that one kind can realize the quick detection of nucleic acid, detection timeliness is fast, and only at a constant temperature
Need to realize at a constant temperature more and more attention has been paid to.Mode that there are many kinds of nucleic acid isothermal amplification detections at present, than
Such as LAMP, HDA, RPA, the constant-temperature amplifications mode such as NASBA.Different isothermal duplication systems is different according to application scenarios, etc.
Warm amplification system, usually using some chemical reagent, such as the neutral red dye of LAMP inspection, the nucleic acid dye that electrophoresis is used,
Some other nucleic acid isothermal use molecular beacon system and Taqman self-quenching probe, these signal detection systems due to by
To the limitation of instrument channel quantity, Multiple detection cannot achieve.
In view of clinically to easy quick, high sensitivity, specificity is got well and the demand of multiplex detection technology, and shows at present
There is technology to have its limitation, clinical demand cannot be fully met, therefore we need to develop one kind quickly, it is accurate and low
The Multiple detection technology of cost.
Summary of the invention
To solve the above-mentioned problems, the present invention provides method that is a kind of while detecting multiple target nucleic acids, and the method is logical
It crosses and target nucleic acids different in same fluorescence channel is expanded with the primer of different fluorescent markers, so that same fluorescence channel
The melting curve of the amplified production of interior different target nucleic acids has different fusing point Tm values, and the phase in same fluorescence channel
The Tm value of the amplified production of adjacent difference target nucleic acids differs 2-20 DEG C, preferably differs 2-8 DEG C, is based on fluorescent primer to realize
Amplified production melting curve detect multiple target nucleic acids simultaneously.
In one embodiment, the amplification includes fluorescent PCR amplification, unwindase relies on amplification or recombinase relies on and expands
Increase.
In one embodiment, the target nucleic acids include DNA and/or RNA, and when including RNA, the method is also wrapped
Include reverse transcription step.
In one embodiment, fluorescent marker preferably marks at 5 ' ends of the primer to the sequence between 3 ' ends
In the middle position of the primer 5 ' to 3 '.
In one embodiment, spacerarm is introduced in the primer of the fluorescent marker, reduces fluorescence-causing substance fusing point Tm value；
And/or lock nucleic acid is introduced in the primer of the fluorescent marker, improve fluorescence-causing substance fusing point Tm value；And/or the fluorescent marker
It include hypoxanthine base in primer.
In one embodiment, when the primer includes upstream primer and downstream primer, the upstream primer and institute
It states downstream primer and an identical fluorophor is marked respectively.
In one embodiment, the method realizes the detection of multiple targets by more fluorescence channels.
In one embodiment, when the quantity of the target is not less than 4, in two kinds of targets of same fluorescence channel
Fluorescence-causing substance by fusing point Tm value cannot distinguish when, by simultaneously judge the fluorescence-causing substance fusing point Tm value of another corresponding channel come
Distinguish different targets.
In one embodiment, the G+C content of the primer of the fluorescent marker is 30%-65%, and Tm value is 45 DEG C -65
℃。
In one embodiment, the present invention provide it is a kind of use in the above-mentioned methods be used for while detecting multiple targets
The kit of nucleic acid.
Main advantages of the present invention are as follows:
1. detecting more target nucleic acids using the melting curve based on product, detection while more targets may be implemented, significantly
Detection flux and efficiency are improved, testing cost is reduced；
2. the mark fluorescent directly on primer reduces the use of probe, avoids non-specific binding between primed probe and drop
Low amplification efficiency improves detection sensitivity；
3. avoiding the use of quenching group using itself being quenched for fluorophor, detection fluorescence signal enhancing improves detection
Sensitivity；
4. primer synthesis cost substantially reduces in detection architecture, patient's expense can be reduced, in benefits subjects.
Detailed description of the invention
It in order to more clearly explain the technical solutions in the embodiments of the present application, below will be to needed in the embodiment
Attached drawing is briefly described, it should be apparent that, the accompanying drawings in the following description is only some embodiments as described in this application, right
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings
Its attached drawing.
Fig. 1 is the principle of the present invention figure；
Fig. 2 is the ratio for indicating primer mark reporter group (A1-A4) and fluorescent quantitative PCR (B1-B4) amplification curve
Compared with figure, wherein A1/B1:108,A2/B2:107,A3/B3:106,A4/B4:105Copy/ml；
Fig. 3 is to indicate that the melting curve (A) of CT primer mark reporter group and TaqMan probe melting curve (B) compare
Figure；
Fig. 4 is the channel the FAM melting curve figure for indicating CT different modes mark fluorescent reporter group；
Fig. 5 is the product melting curve figure indicated with FAM Air conduct measurement CT (A is indicated) and UU (B is indicated)；
Fig. 6 be indicate with FAM Air conduct measurement CT (A is indicated), UU (B is indicated), IC internal standard (C is indicated) product melting curve
Figure；
Fig. 7 is the product melting curve figure indicated with HEX Air conduct measurement NG (A is indicated) and HSV2 (B is indicated)；
Fig. 8 is the amplification curve diagram for indicating the HDA isothermal detection of venereal disease UU；
Fig. 9 is the melting curve figure for indicating the HDA isothermal detection of venereal disease UU；
Figure 10 is the amplification curve diagram for indicating the RPA isothermal detection of venereal disease NG；
Figure 11 is the melting curve figure for indicating the RPA isothermal detection of venereal disease NG；
Figure 12 is to indicate CT modification fluorescent primer amplification curve and melting curve figure；
Figure 13 is to indicate NG modification fluorescent primer amplification curve and melting curve figure, and wherein the left side is amplification curve diagram, right
Side is melting curve figure；
Figure 14 is to indicate that CT modifies two kinds of fluorophor primer amplification curves and melting curve figure, and wherein the left side is that amplification is bent
Line chart, the right are melting curve figure；
Figure 15 is to indicate that UU modifies 3 fluorophor primer amplification curves and melting curve figure, and wherein the left side is to melt song
Line chart, the right are amplification curve diagram；
Figure 16 is to indicate that swin flu H1N1 detects amplification curve diagram；
Figure 17 is to indicate that swin flu H1N1 detects melting curve figure.
Specific embodiment
In order to make those skilled in the art more fully understand the technical solution in the application, below in conjunction with embodiment to this
Invention is described further, it is clear that described embodiments are only a part of embodiments of the present application, rather than whole implementation
Example.Based on the embodiment in the application, obtained by those of ordinary skill in the art without making creative efforts
All other embodiment, shall fall within the protection scope of the present application.
It is conventional method in that art unless otherwise specified in following embodiments.In following embodiment, material used,
It unless otherwise specified, is that this field conventional biochemical reagent company is commercially available.
The principle of 1 the method for the present invention of embodiment
As shown in Figure 1, curve A is the change in fluorescence figure of the method for the present invention in Fig. 1, curve B is the probe in detecting side Taqman
The change in fluorescence figure of method.The curve B change in fluorescence of the fluorescence curve figure A of the method for the present invention and the probe in detecting side Taqman in Fig. 1
Process has certain similitude, but change in fluorescence of the invention becomes apparent from, and signal strength is higher.
The method of the present invention includes two processes of amplification procedure such as PCR process and melting curve analysis:
Amplification procedure: primer mark reporter group carries out amplification reaction.Mark fluorescent group primed DNA is single-stranded, fluorescence
Intensity can increase after label chain is complementary sequence hybridization, its background signal value is lower when single-stranded, as shown in figure 1 shown in A1；
After the combination complementary with template of primer with reporter group, DNA double is extended to form as primer and under the action of archaeal dna polymerase
Chain, fluorescent value gradually rise, and the plateau of amplification curve are finally reached, as shown in figure 1 shown in A2；The two processes constitute amplification
Process.
Melting curve process: shown in melting curve primitive curve two processes of A3-A4 as shown in figure 1, when temperature is lower than production
When object Tm corresponding temperature, i.e., shown in the A3 of Fig. 1, double-strand as the temperature rises, fluorescent value variation less, i.e. report base
Group is also present in double-stranded DNA, so being in high fluorescence state of value.When temperature reaches A3~A4's in product melting-point diagram 1
When near boundary, that is, when arrival product fusing point Tm value, DNA double chain is quickly opened, and fluorescence signal value sharply declines.Figure
1 A4 show the signal value change procedure that temperature is higher than double-stranded products Tm value, to form only band report due to being substantially at this stage
The DNA for accusing group is single-stranded, so fluorescent value is below the fluorescent value of other states.To in A3-A4 whole process, fluorescent value is carried out
The negative derivation of signal, so that it may obtain melting curve as shown in A in fig. 3.For different target nucleic acids, design amplification is different
The specificity amplification primer of length DNA product marks different fluorescence channels, realizes multiple melting curve detection.
2 the method for the present invention of example is compared with the PCR melting curve method based on Taqman probe
Single fluorescent dye primer melting curve method and the PCR melting curve method detection sand based on Taqman probe is respectively adopted
Chlamydia oculogenitale (Chlamydia trachomatis, abbreviation CT).
1. primed probe designs
It selects CT genome the preceding paragraph conserved sequence (GenBank:CP015307.1), in building and PMD19 plasmid vector,
As standard sequence, respectively to the conservative design upstream and downstream primer and TaqMan probe, wherein single fluorescent dye primer melts song
Upstream primer F mark fluorescent group in collimation method, the Taqman probe 5 ' in the PCR melting curve method based on TaqMan probe are held
With 3 ' end difference mark fluorescent groups and quenching group, see Table 1 for details.The chlamydia trachomatis plasmid 10 of building is detected respectively7~
103copies/ml。
1 chlamydia trachomatis primer and probe of table
2.PCR reaction and melting curve analysis
Two kinds of PCR reaction systems are prepared respectively, and reaction system and reaction condition are provided that
A, fluorescent primer reaction system: 1 × PCR buffer, 0.1mM MgSO4,200 μM of dNTPs, 0.1 μM of CT-F-
FAM, 0.1 μM of downstream primer, 0.75U polymerase.
B, Taqman probe in detecting system: 1 × PCR buffer, 200 μM of dNTPs, 0.20 μM of primer CT-F and CT-R,
0.12 μM of probe CT-P, 2U polymerase.
Reaction condition setting are as follows: 50 DEG C 2 minutes；95 DEG C 10 minutes；95 DEG C 15 seconds, 55 DEG C of annealing extend, and are collected simultaneously glimmering
Light repeats 40 circulations；40-95 DEG C of melting curve analysis, every 0.03 DEG C of detection first order fluorescence signal, 60 DEG C cool down 1 minute.
3. interpretation of result
Two kinds of system detections, as shown in Figure 2, two kinds of detection mode sensitivity are consistent for testing result.But melting curve result
It has been shown that, the corresponding melting curve signal of Taqman probe is weaker, and the melting curve baseline that fluorescent primer amplification generates is smooth, letter
Number strong, curve is preferable, and Tm value is consistent with theory T m.
The optimization of 3 fluorescent primer melting curve method of example
A variety of mark modes are had rated in this example, evaluate fluorophor, the upstream and downstream that different number is marked on primer
Primer distinguishes mark fluorescent group, downstream primer marks influence of the different fluorophors to detection effect.
1. the design of primer mark mode
Sequence and mark mode such as the following table 2:
The a variety of mark modes of 2. primer of table
The combination of 3. different primers of table label
2. detection architecture and reaction condition
Reaction system: 1 × PCR buffer, 0.1mM MgSO4,200 μM of dNTPs, 0.1 μM of upstream primer, under 0.1 μM
Swim primer, 0.75U polymerase.Its upstream and downstream primer combination is as shown in table 3.
3. testing result
The fluorophor of single primer mark different number was attempted to compare in CT upstream primer list mark fluorescent group, on
Downstream primer all marks the amplification curve sensitivity of reporter group best, and when reaching plateau, it is bent that fluorescence signal value increases melting
Line melting peakss signal value is also higher.
As shown in figure 4, the amplification curve and melting curve for combining 3 are all in 6 kinds of combinations of CT primer mark reporter group
It is best；Although combination 2 amplification curves and melting curve signal value it is stronger, its sensitivity be 6 kinds combination in most
Difference.
It is compared with upstream primer list mark fluorescent group, upstream and downstream primer all marks the amplification curve sensitivity of reporter group
Higher, melting curve melting peakss signal value is also higher.
Upstream primer flag F AM, downstream primer mark different reporter groups, the upstream and downstream primer of different fluorescence channels,
There is same products Tm melting curve with single upstream primer.
The single fluorescence channel multiplex detection of example 4
In this example, the method for the present invention can realize single channel multiplex detection, detect flux provider to improve single channel
Method.By taking the pathogen CT and UU that detect sexually transmitted disease as an example.
1. design of primers
Specific primer is designed for the conservative region of CT and UU respectively, and in the upstream primer of each detection target gene
One fluorescent reporter gene of label such as uses italic T in the sequence, and two amplification gene product lengths of setting are inconsistent, sequence
It is as shown in table 4 respectively.
The chlamydia trachomatis (CT) and Ureaplasma urealyticum (UU) primer that table 4. designs
2. reaction system and reaction condition
Reaction system: 1 × PCR buffer, 0.1mM MgSO4,200 μM of dNTPs, 0.1 μM of upstream primer, under 0.1 μM
Swim primer, 0.75U polymerase.
Reaction condition setting are as follows: 50 DEG C 2 minutes；95 DEG C 10 minutes；95 DEG C 15 seconds, 60 DEG C of annealing extend, and are collected simultaneously glimmering
Light repeats 40 circulations；40-95 DEG C of melting curve analysis, every 0.03 DEG C of detection first order fluorescence signal, 60 DEG C cool down 1 minute
3. interpretation of result
The double plasmid template evaluation result of this example detection CT and UU is as shown in Figure 5.It is detection CT template pair shown in Fig. 5 A
The product melting curve peak answered, Tm value are 70 DEG C；Be shown in B detection UU template corresponding product melting curve peak be built into it is double
Melting curve, Tm value are 78 DEG C.Double melting curve peak figure is clearly separated, and melting curve baseline is smooth, and only template produces
The corresponding melting peakss of object illustrate that designed single channel multiplex detection mode can be completed.
More than the 5 fluorescence channel multiplex detections of example
Single channel marks in reporter group, and the detection of double melting curve may be implemented.Then we are carrying out multichannel
Multiple detection expands evaluation.
1. design of primers
The channel FAM: CT (70 DEG C of Tm value ≈), UU (78 DEG C of Tm value ≈), IC internal reference (83 DEG C of Tm value ≈)
The channel VIC: NG (75 DEG C of Tm value ≈), HSV2 (87 DEG C of Tm value ≈).
5. Multiple detection primer sequence of table
2. reaction system and reaction condition
Reaction system: 1 × PCR buffer, 0.1mM MgSO4,200 μM of dNTPs, 0.1 μM of primer mark reporter group
Upstream primer, 0.1 μM of downstream primer, 0.75U polymerase, water.
Reaction condition setting are as follows: 50 DEG C 2 minutes；95 DEG C 10 minutes；95 DEG C 15 seconds, 55 DEG C of annealing extend, and are collected simultaneously glimmering
Light repeats 40 circulations；40-95 DEG C of melting curve analysis, every 0.03 DEG C of detection first order fluorescence signal, 60 DEG C cool down 2 minutes
3. interpretation of result
5 kinds of common venereal diseases pathogen detection melting curve results are as shown in fig. 6-7.Wherein: the CT in Fig. 6 expression channel FAM
The product melting curve figure of (A expression), UU (B expression), internal standard IC (C)；The NG (A expression) and HSV2 (B in Fig. 7 expression channel HEX
Indicate) product melting curve figure.
It can be seen that from the above testing result, venereal disease Multiple detection system detects chlamydia trachomatis, ureaplasma urealyticum, Neisser leaching
It is bent that coccus, herpes simplex virus type 2 and internal standard human genome sequence (IC), each pathogen and internal reference have corresponding feature to melt
Line peak illustrates that the respiratory tract Multiple detection system established can detect various common sexual reverses with accurate and effective.
Example 6HDA mode detects UU target
This example has rated label reporter group primer in HDA class constant temperature using ureaplasma urealyticum (UU) as detection target
The function and effect of detection system.
Specificity amplification primer pair is designed for UU, and marks fluorescent reporter group on a primer.Detection architecture packet
Include for primer, template, ddH2O, buffer and single strand binding protein SSB, DNA helicase, triphosphoric acid dezyribonucleoside,
Archaeal dna polymerase, MutL and buffer, Taq polymerase.Reaction condition includes 65 DEG C, 1 minute, 60 circulating collection fluorescence.95℃
5 minutes, 40 DEG C 2 minutes, 40 DEG C → 95 DEG C 0.03 DEG C/s gradually rise temperature, and in entire temperature-rise period collect fluorescence letter
Number, reach 95 DEG C after, 60 DEG C 1 minute, terminate operation.
The HDA design of primers sequence of table 6.UU
| Primer | Sequence | Mark mode | SEQ ID number |
| UU-F-zFAM | CCACTTAAATCCTAAGGTTCCAGA | TKilobase marker FAM fluorophor | SEQ ID NO:27 |
| UU-R | CAGCTGCAATTGTTTGGCTA | / | SEQ ID NO:28 |
Amplification curve and solubility curve based on HDA isothermal duplication detection technique and fluorescent dye primer detection UU are shown in respectively
Fig. 8-9.Fluorescent dye primer can be expanded effectively in HDA system and form preferable melting curve.
Example 7:RPA mode detects NG amplification target detection
By taking common venereal disease Neisseria gonorrhoeae (NG) as an example, specific primer, one report base of upstream primer label are designed
Group, corresponding special downstream primer carry out RPA isothermal duplication together.Its amplification system includes that the pair of specificity is drawn
Object, recombinase, binding protein (SSB), strand displacement archaeal dna polymerase expand buffer system, template, water.Augmentation detection response procedures
Including two steps of isothermal duplication and liquation, wherein amplification program includes: 37 DEG C, 1 minute 60 circulation, and 95 DEG C 5 points
Clock, 40 DEG C 2 minutes, 40 DEG C → 95 DEG C 0.03 DEG C/s gradually rise temperature, and collect fluorescence signal in entire temperature-rise period, arrive
Up to after 95 DEG C, 60 DEG C 1 minute, terminate operation.
The RPA design of primers sequence of table 7.NG
Amplification curve and melting curve method based on RPA isothermal duplication detection technique and fluorescent dye primer detection NG nucleic acid
Figure 10 and Figure 11, concentration 10 are seen respectively5Copy/ml concentration have target gene NG plasmid, can expand within 20 minutes to
Up to threshold value.
Example 8: special modification fluorescent primer improves the specificity of detection
The reason of multiplex PCR difficulty is done is because non-specific primer mistakenly starts nucleic acid synthesis.The present invention draws fluorescence
Object carries out special modification, and introducing hypoxanthine base, can reduce primer non-specific amplification such as in primer, to detect CT and NG is
Example.
1. design of primers
Table 8.CT and NG modify fluorescent primer sequence and mark mode
2. reaction system and reaction condition
Amplification reaction solution includes PCR buffer, dNTP, dUTP, upstream fluorescent primer, downstream primer, heat-resistant dna polymerization
Enzyme, ddH2O.Detecting program includes amplification step program: 50 DEG C of 2min, 95 DEG C of 10min；95 DEG C of 15s of 40 circulations, 55 DEG C
30s, and in 55 DEG C of progress phosphor collections.Melting curve analysis program: 40 DEG C of 2min, 40 DEG C → 95 DEG C 0.03 DEG C/s gradually rise
Temperature, and fluorescence signal is collected in entire temperature-rise period, after reaching 95 DEG C, 60 DEG C 1 minute, terminate operation.
3. interpretation of result
Testing result is shown in Figure 12-13.The primer of hypoxanthine modification can normally expand target gene template as the result is shown, put down
Row is repeated 8 times detection, and preferably, CV < 5%, wherein CT is 0.95%, NG 0.24% to repeatability.Melting curve also presents single
One melting curve peak, corresponding product Tm CV < 5%, it is 0.03% that wherein CT, which is 0.03%, NG,.
Example 9: the detection method of the permutation and combination of the same a variety of fluorescent markers of primer
In above-mentioned a variety of examples, the primer of each label only marks a kind of fluorescent reporter gene, a fluorescence channel inspection
The gene target of corresponding product Tm is surveyed, for example 2 or 3 Tm points of the target opened, four channels are arranged in each fluorescence channel
It is able to detect that 8 or 12 gene targets.However for more Multiple detection, because adjacent in same fluorescent marker channel
Target to be measured fluorescence-causing substance fusing point Tm value difference it is smaller, be also not enough to be applicable in the detection of part, it is logical in fluorescence signal value
On road and product Tm two-dimensional space, the requirement of Multiple detection can't be met.So on this basis, we have proposed another
A kind of mode of outer label, on a fluorescent primer, two kinds or three kinds fluorescent reporter genes of label, by carrying out arrangement group
Conjunction achievees the purpose that Multiple detection.
Tabulated for marking two ways it is as follows, when result judges, for same target determinand, channel 1
It is consistent with the fluorescence-causing substance fusing point Tm value in channel 2, by judging that the fluorescence-causing substance fusing point Tm value in corresponding two channels is true simultaneously
Fixed final target determinand.Such method is suitable for more multiple detection, two kinds of targets to be measured of especially same fluorescence channel
Substance fluorescence-causing substance fusing point Tm value is adjacent relatively close, it is more difficult to it distinguishes, it can be molten by judging the fluorescence-causing substance of another corresponding channel simultaneously
Point Tm value accurate judgement target substance to be measured.
| Channel 1 | Channel 2 | TM value | As a result judge |
| FAM | VIC | 70℃ | Target 1 |
| FAM | ROX | 71℃ | Target 2 |
| FAM | CY5 | 72℃ | Target 3 |
| VIC | ROX | 70℃ | Target 4 |
| VIC | CY5 | 71℃ | Target 5 |
| ROX | CY5 | 72℃ | Target 6 |
For the feasibility of verification method, We conducted a variety of fluorophors of single primer mark to be evaluated.
Reaction system: 1 × PCR buffer, 0.1mM MgSO4,200 μM of dNTPs, 0.1 μM of primer mark reporter group
Upstream primer, 0.1 μM of downstream primer, 0.75U polymerase, water.
Reaction condition setting are as follows: 50 DEG C 2 minutes；95 DEG C 10 minutes；95 DEG C 15 seconds, 55 DEG C of annealing extend, and are collected simultaneously glimmering
Light repeats 45 circulations；40-95 DEG C of melting curve analysis, every 0.03 DEG C of detection first order fluorescence signal, 60 DEG C cool down 2 minutes
Flag F AM and the VIC fluorescent reporter group simultaneously in the upstream primer of detection solution urea Chlamydia (CT), and downstream
Primer amplification detects sequence of the band containing CT gene target, and carries out melting curve analysis.A variety of fluorescence signal labels, have base
This consistent amplification curve, and the melting curve peak figure with same products Tm.It is due to different fluorescence signal values channel, institute
Launch wavelength is inconsistent, causes the height of signal value inconsistent.Product Tm is consistent, and evaluation result is as shown in figure 14, it was demonstrated that its is glimmering
Too many interference is not present in optical channel between each other, is the desired result that we want.So can be marked on a primer
A variety of fluorescence signal values channel.
The a variety of fluorophor mark modes of 9 primer of table and sequence
Example 10: a kind of drop produces or increases the detection method of product Tm
As the multiple detection method of melting curve detection pathogen, the product Tm of same channels is debugged, it is made to detect two
The product Tm of a gene target pathogen can be clearly separated has effect very much.Being often used adjusting melting curve Tm method includes
By controlling the sequence length of product, G/C content very, and adjusts the ion concentration of PCR reaction detection system, such as magnesium
Ion.We provide the method for new debugging product Tm a kind of herein, by introducing spacerarm (Spacer) on primer,
It has the function of that product Tm can be reduced, and in addition in primer, introduces the effect that lock nucleic acid (LNA) specifically improves product Tm.
A blocking agent of the spacerarm as primer is equivalent to the base number for reducing product, to have the function that reduce product Tm；
Lock nucleic acid is a kind of class oligonucleotide derivative, and the 2' and 4' carbon on a part of ribose links together, and 4 '-C pass through shrink
Effect forms the structure of rigidity, reduces the flexibility of ribose structure, so as to arrive to the effect for improving Tm.With CT and UU into
Row evaluation display, is added Sapcer, can reduce product Tm1~2 DEG C, CT and UU reduce by 1.77 DEG C and 1.03 DEG C respectively.And add
Entering lock nucleic acid can be improved greater than 2 DEG C, CT and UU product Tm increases 2.21 DEG C and 2.14 respectively.
Table 10 plus spacerarm primer and the product Tm difference results being not added
The product Tm difference results that table 11 locks nucleic acid LNA primer and is not added
A kind of example 11: multiple labeling method improving melting curve signal value peak
The method that primer multiple labeling improves melting curve signal value, in primer mark method, above example is mostly glimmering with 1
Light reporter group carries out verifying evaluation.We carry out multiple labeling evaluation, that is, at it using the primer of UU (solution urea Chlamydia)
3 fluorescent reporter groups of label on primer.Their amplification curve and melting curve is as shown in figure 15, as the result is shown melting curve
Signal value Rm=1807.62, much higher than the control group melting curve signal value Rm=146.29 of single fluorescent marker.Primer mark
Mode and sequence are as shown in the table:
12 primer multiple labeling mode of table and sequence
| Primer | Sequence | Mark mode | SEQ ID number |
| UU-F-zFAM | CCACTTAAATCCTAAGGTTCCAGA | TKilobase marker FAM fluorophor | SEQ ID NO:37 |
| UU-F-3zFAM | CCACTTAAATCCTAAGGTTCCAGA | TKilobase marker FAM fluorophor | SEQ ID NO:38 |
| UU-R | CAGCTGCAATTGTTTGGCTA | / | SEQ ID NO:39 |
Example 12: reverse transcription system detects influenza A virus
In this example, the method for the present invention can realize the detection to RNA target mark.To detect the A type stream in respiratory pathogen
For Influenza Virus H1N1.
1. design of primers
Specific primer is designed for the conservative region of H1N1, and label one in the upstream primer of detection target gene
Fluorescent reporter gene such as uses italic in the sequenceT, sequence is as shown in table 13.
The H1N1 sequence that table 13 designs
2. reaction system and reaction condition
Reaction system: 1 × RT-PCR buffer, 0.1mM MgSO4,200 μM of dNTPs, 0.1 μM of upstream primer, 0.1 μM
Downstream primer, 0.75U polymerase, 60U reverse transcriptase.
Reaction condition setting are as follows: 50 DEG C 30 minutes；95 DEG C 10 minutes；95 DEG C 15 seconds, 60 DEG C of annealing extend, and are collected simultaneously glimmering
Light repeats 40 circulations；40-95 DEG C of melting curve analysis, every 0.03 DEG C of detection first order fluorescence signal, 60 DEG C cool down 1 minute
3. interpretation of result
The nucleic acid amplification curve and melting curve of this example detection swin flu H1N1 sample are shown in Figure 16 and Figure 17 respectively.Figure 16 expands
Increasing curve smoothing, Figure 17 corresponding Product characteristics melting curve peak Tm value is 72 DEG C,；Illustrate that the method for the present invention can be realized to RNA
The detection of target.
It should be understood that the present invention disclosed is not limited only to specific method, scheme and the substance of description, because these
It is alterable.It will also be understood that purpose of the terminology used here just for the sake of the specific embodiment scheme of description, rather than
It is intended to limit the scope of the invention, the scope of the present invention is limited solely by the attached claims.
Those skilled in the art, which will also be appreciated that or be able to confirm that, uses no more than routine experiment, institute herein
The many equivalents for the specific embodiment of the invention stated.These equivalents are also contained in the attached claims.
Sequence table
<110>Jiangsu Hong Weitesi Pharmaceutical Technology Co., Ltd
<120>a kind of method and its kit for detecting multiple target nucleic acids simultaneously
<160> 41
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
agcgctgcga atagaaaaag t 21
<210> 2
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cgtttctatt gcttgagcgt a 21
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
agcgctgcga atagaaaaag t 21
<210> 4
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cgtttctatt gcttgagcgt a 21
<210> 5
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tgctatagca ctatcaagcc ttccc 25
<210> 6
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
cgctgcgaat agaaaaagt 19
<210> 7
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
cgctgcgaat agaaaaagt 19
<210> 8
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
cgtttctatt gcttgagcgt a 21
<210> 9
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cgtttctatt gcttgagcgt a 21
<210> 10
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
cgtttctatt gcttgagcgt a 21
<210> 11
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
cgtttctatt gcttgagcgt a 21
<210> 12
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
cgtttctatt gcttgagcgt a 21
<210> 13
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
ctatagcact atcaagcctt c 21
<210> 14
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
cgtttctatt gcttgagcgt a 21
<210> 15
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ccacttaaat cctaaggttc caga 24
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
cagctgcaat tgtttggcta 20
<210> 17
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ctatagcact atcaagcctt c 21
<210> 18
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
cgtttctatt gcttgagcgt a 21
<210> 19
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
ccacttaaat cctaaggttc caga 24
<210> 20
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
cagctgcaat tgtttggcta 20
<210> 21
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
gcagtgactt tctcagcaac atgtcga 27
<210> 22
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
cccagtgctg tagagctgtc c 21
<210> 23
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
ctgctatgac tatcaaccct gc 22
<210> 24
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
tgagcaaggc agtattcaag c 21
<210> 25
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
accgtcgccc tatacagctt aa 22
<210> 26
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
ggaagacccc gaggactcg 19
<210> 27
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
ccacttaaat cctaaggttc caga 24
<210> 28
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
cagctgcaat tgtttggcta 20
<210> 29
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
ctgctatgac tatcaaccct gc 22
<210> 30
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
tgagcaaggc agtattcaag c 21
<210> 31
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
agcgctgcga atagaaaact atagcact 28
<210> 32
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
cgtttctatt gcttgagcgt a 21
<210> 33
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
ctgctatgac tatcaaccct gcctacctag c 31
<210> 34
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
tgagcaaggc agtattcaag c 21
<210> 35
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
ctatagcact atcaagcctt c 21
<210> 36
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
cgtttctatt gcttgagcgt a 21
<210> 37
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
ccacttaaat cctaaggttc caga 24
<210> 38
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
ccacttaaat cctaaggttc caga 24
<210> 39
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
cagctgcaat tgtttggcta 20
<210> 40
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
gatcttgagg ctctcatgga at 22
<210> 41
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
aaagcgtcta cgctgcagt 19
Claims (10)
1. a kind of method for detecting multiple target nucleic acids simultaneously, which is characterized in that the method passes through in same fluorescence channel
Different target nucleic acids are expanded with the primer of different fluorescent markers, so that target nucleic acids different in same fluorescence channel
The melting curve of amplified production has different fusing point Tm values, and the expansion of the adjacent different target nucleic acids in same fluorescence channel
The Tm value for increasing production object differs 2-20 DEG C, preferably differs 2-8 DEG C, to realize the amplified production melting curve based on fluorescent primer
Multiple target nucleic acids are detected simultaneously.
2. the method according to claim 1, wherein the amplification includes fluorescent PCR amplification, unwindase dependence expansion
Increase or recombinase relies on amplification.
3. the method according to claim 1, wherein the target nucleic acids include DNA and/or RNA, when including
When RNA, the method also includes reverse transcription steps.
4. the method according to claim 1, wherein fluorescent marker is at 5 ' ends of the primer between 3 ' ends
Sequence, preferably label is in the middle position of the primer 5 ' to 3 '.
5. being reduced the method according to claim 1, wherein introducing spacerarm in the primer of the fluorescent marker
Fluorescence-causing substance fusing point Tm value；And/or lock nucleic acid is introduced in the primer of the fluorescent marker, improve fluorescence-causing substance fusing point Tm value；With/
It or include hypoxanthine base in the primer of the fluorescent marker.
6. the method according to claim 1, wherein when the primer includes upstream primer and downstream primer,
An identical fluorophor is marked in the upstream primer and the downstream primer respectively.
7. the method according to claim 1, wherein the method realizes multiple targets by more fluorescence channels
Detection.
8. the method according to the description of claim 7 is characterized in that when the quantity of the target be not less than 4 when, in same fluorescence
When the fluorescence-causing substance of two kinds of targets in channel cannot be distinguished by fusing point Tm value, by judging the glimmering of another corresponding channel simultaneously
Photoproduct fusing point Tm value distinguishes different targets.
9. -8 any method according to claim 1, which is characterized in that the G+C content of the primer of the fluorescent marker is
30%-65%, Tm value are 45 DEG C -65 DEG C.
10. the reagent of multiple target nucleic acids is used for while detected used in a kind of the method any in claim 1-9
Box.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910652239.XA CN110358815B (en) | 2019-07-18 | 2019-07-18 | Method for simultaneously detecting multiple target nucleic acids and kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910652239.XA CN110358815B (en) | 2019-07-18 | 2019-07-18 | Method for simultaneously detecting multiple target nucleic acids and kit thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110358815A true CN110358815A (en) | 2019-10-22 |
| CN110358815B CN110358815B (en) | 2023-05-23 |
Family
ID=68220985
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910652239.XA Active CN110358815B (en) | 2019-07-18 | 2019-07-18 | Method for simultaneously detecting multiple target nucleic acids and kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110358815B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111534624A (en) * | 2020-06-17 | 2020-08-14 | 台州市中心医院（台州学院附属医院） | RPA-based rapid detection method for ureaplasma urealyticum |
| CN112280839A (en) * | 2020-10-16 | 2021-01-29 | 李凯 | Real-time PCR technology for detecting more target points than fluorescent channels and application thereof |
| CN114606298A (en) * | 2020-12-08 | 2022-06-10 | 厦门致善生物科技股份有限公司 | Method for detecting length of one or more nucleic acid molecule amplification products in sample |
| CN114875116A (en) * | 2022-04-27 | 2022-08-09 | 广州博懿瑞生物科技有限公司 | Self-quenching fluorescence primer and design method and application thereof |
| CN116875664A (en) * | 2023-06-21 | 2023-10-13 | 浙江大学 | Real-time fluorescence loop-mediated isothermal nucleic acid amplification detection method |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1683564A (en) * | 2005-03-01 | 2005-10-19 | 中华人民共和国青岛出入境检验检疫局 | Method for detecting bird flu and newcastle disease virus by composite quantitative polyase chain reaction |
| WO2011147309A1 (en) * | 2010-05-24 | 2011-12-01 | 上海天昊生物科技有限公司 | Kit and method for determining copy numbers of multiplex genes |
| CN104293937A (en) * | 2014-09-28 | 2015-01-21 | 广东省妇幼保健院 | Group of probes, detection kit and detection method for detecting thalassemia gene point mutation based on liquid chip of locked nucleic acid sensibilization |
| CN105154534A (en) * | 2015-07-30 | 2015-12-16 | 中国人民解放军第三军医大学第一附属医院 | Real-time constant-temperature index amplification method of tiny RNAs |
| CN107488715A (en) * | 2017-08-14 | 2017-12-19 | 福建医科大学孟超肝胆医院 | PNPLA3 kit for detecting susceptibility genes and method based on self-quenching probe melting curve |
| CN107630098A (en) * | 2017-11-13 | 2018-01-26 | 湖南圣湘生物科技有限公司 | Fluorescent PCR detection architecture, kit and detection method for joint-detection various respiratory road bacterium |
| CN108998555A (en) * | 2018-08-15 | 2018-12-14 | 青岛啤酒股份有限公司 | Method based on four color Multiplex fluorescent PCRs identification hop varieties |
| CN109486964A (en) * | 2018-12-12 | 2019-03-19 | 沈阳农业大学 | For the individual identification of donkey and the microsatellite rapid detection method of paternity test |
| CN109576352A (en) * | 2018-11-25 | 2019-04-05 | 江苏宏微特斯医药科技有限公司 | Single tube detects method, probe and its kit of multiple object to be measured nucleic acid sequences |
| CN109750091A (en) * | 2019-03-13 | 2019-05-14 | 江苏宏微特斯医药科技有限公司 | Single tube detects the method and its kit of one or more object to be measured nucleic acid sequences |
-
2019
- 2019-07-18 CN CN201910652239.XA patent/CN110358815B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1683564A (en) * | 2005-03-01 | 2005-10-19 | 中华人民共和国青岛出入境检验检疫局 | Method for detecting bird flu and newcastle disease virus by composite quantitative polyase chain reaction |
| WO2011147309A1 (en) * | 2010-05-24 | 2011-12-01 | 上海天昊生物科技有限公司 | Kit and method for determining copy numbers of multiplex genes |
| CN104293937A (en) * | 2014-09-28 | 2015-01-21 | 广东省妇幼保健院 | Group of probes, detection kit and detection method for detecting thalassemia gene point mutation based on liquid chip of locked nucleic acid sensibilization |
| CN105154534A (en) * | 2015-07-30 | 2015-12-16 | 中国人民解放军第三军医大学第一附属医院 | Real-time constant-temperature index amplification method of tiny RNAs |
| CN107488715A (en) * | 2017-08-14 | 2017-12-19 | 福建医科大学孟超肝胆医院 | PNPLA3 kit for detecting susceptibility genes and method based on self-quenching probe melting curve |
| CN107630098A (en) * | 2017-11-13 | 2018-01-26 | 湖南圣湘生物科技有限公司 | Fluorescent PCR detection architecture, kit and detection method for joint-detection various respiratory road bacterium |
| CN108998555A (en) * | 2018-08-15 | 2018-12-14 | 青岛啤酒股份有限公司 | Method based on four color Multiplex fluorescent PCRs identification hop varieties |
| CN109576352A (en) * | 2018-11-25 | 2019-04-05 | 江苏宏微特斯医药科技有限公司 | Single tube detects method, probe and its kit of multiple object to be measured nucleic acid sequences |
| CN109486964A (en) * | 2018-12-12 | 2019-03-19 | 沈阳农业大学 | For the individual identification of donkey and the microsatellite rapid detection method of paternity test |
| CN109750091A (en) * | 2019-03-13 | 2019-05-14 | 江苏宏微特斯医药科技有限公司 | Single tube detects the method and its kit of one or more object to be measured nucleic acid sequences |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111534624A (en) * | 2020-06-17 | 2020-08-14 | 台州市中心医院（台州学院附属医院） | RPA-based rapid detection method for ureaplasma urealyticum |
| CN112280839A (en) * | 2020-10-16 | 2021-01-29 | 李凯 | Real-time PCR technology for detecting more target points than fluorescent channels and application thereof |
| CN114606298A (en) * | 2020-12-08 | 2022-06-10 | 厦门致善生物科技股份有限公司 | Method for detecting length of one or more nucleic acid molecule amplification products in sample |
| WO2022120914A1 (en) * | 2020-12-08 | 2022-06-16 | 厦门致善生物科技股份有限公司 | Method for measuring length of amplification product of one or more nucleic acid molecules in sample |
| CN114875116A (en) * | 2022-04-27 | 2022-08-09 | 广州博懿瑞生物科技有限公司 | Self-quenching fluorescence primer and design method and application thereof |
| CN114875116B (en) * | 2022-04-27 | 2023-08-29 | 广州博懿瑞生物科技有限公司 | Self-quenching fluorescence primer and design method and application thereof |
| CN116875664A (en) * | 2023-06-21 | 2023-10-13 | 浙江大学 | Real-time fluorescence loop-mediated isothermal nucleic acid amplification detection method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110358815B (en) | 2023-05-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110358815A (en) | Method and its kit a kind of while that detect multiple target nucleic acids | |
| CN109576352A (en) | Single tube detects method, probe and its kit of multiple object to be measured nucleic acid sequences | |
| CN111500771B (en) | Primer group and kit for detecting novel coronavirus SARS-CoV-2 | |
| CN110317861A (en) | A kind of kit detecting pathogen | |
| US20080261198A1 (en) | Diagnostic Primers and Method for Detecting Avian Influenza Virus Subtype H5 and H5n1 | |
| CN110408725B (en) | Kit for multiple detection of respiratory pathogens | |
| CN105018646B (en) | A kind of primer, probe and the kit of detection bovine epizootic fever virus | |
| CN109652516A (en) | A kind of structure and purposes of double chain oligonucleotide nucleic acid probe | |
| CN110273026B (en) | Multiple detection kit and detection method for respiratory tract infection | |
| CN110331232B (en) | Multiple nucleic acid detection kit for respiratory pathogens | |
| CN106399577B (en) | A kind of real-time fluorescence PCR detection method for the double target nucleic acids of interest present detections of single channel | |
| CN114134219A (en) | Multiple nucleic acid detection system and preparation method and application thereof | |
| Wang et al. | Recombinase-aided amplification–lateral flow dipstick assay—a specific and sensitive method for visual detection of avian infectious laryngotracheitis virus | |
| CN106636454B (en) | Real-time fluorescent multiplex RT-PCR method for simultaneously detecting human coronavirus 229E, OC43, NL63 and HKU1 | |
| CN111518959A (en) | Digital PCR detection method and kit for novel coronavirus | |
| CN113186342B (en) | 18 ally oneself with respiratory virus nucleic acid and unite detection device | |
| CN111826464A (en) | Primer probe for detecting multiple gastrointestinal viruses in one tube, screening method and kit | |
| WO2019001187A1 (en) | Multi-liquid phase gene chip detection primer, kit and method for rapidly distinguishing five pathogens in mouse respiratory tracts | |
| CN110878381A (en) | Primer composition, kit and method for detecting mycoplasma bovis and infectious bovine rhinotracheitis virus | |
| CN105154584B (en) | A kind of the HRM non-marked detecting probe method and its primer and probe of quick differentiation PRRSV classical strains and variation strain | |
| CN114381550A (en) | Multi-target nucleic acid detection kit and detection method for HPV typing | |
| CN112575118A (en) | Method for simultaneously detecting various diarrhea viruses by using melting curve | |
| CN116162734B (en) | Specific primer, probe and kit for rapidly detecting African swine fever wild strain and gene deletion strain | |
| CN110724763A (en) | Fluorescent quantitative PCR detection method for human adenovirus and bocavirus and application thereof | |
| CN107406891A (en) | Pcr method |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |