CN107574262A - A kind of fluorescence RT RAA primers, probe and detection method for being used to detect A (H 1 N 1) virus - Google Patents
A kind of fluorescence RT RAA primers, probe and detection method for being used to detect A (H 1 N 1) virus Download PDFInfo
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- CN107574262A CN107574262A CN201711005640.1A CN201711005640A CN107574262A CN 107574262 A CN107574262 A CN 107574262A CN 201711005640 A CN201711005640 A CN 201711005640A CN 107574262 A CN107574262 A CN 107574262A
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Abstract
The invention provides a kind of application for the fluorescence RT RAA primers and fluorescence probe and fluorescence RT RAA primers and fluorescence probe of reverse transcription recombinase-mediated nucleic acid amplification technologies detection H1N1virus in H1N1virus is detected, fluorescence RT RAA primers and fluorescence probe can quickly, in qualitative detection throat swab sample H1N1virus, it can be detected under 39 DEG C of normal temperature, diagnostic result can be gone out in 20 minutes, greatly shorten detection time；Fluorescence RT RAA primers and fluorescence probe specificity is stronger, sensitivity is higher, fully meet epidemic situation quick diagnosis and complete monitoring, for the early diagnosis of epidemic situation, early treatment, reduce case fatality rate and control epidemic situation to race against time.
Description
Technical field
The invention belongs to beyond body nucleic acid detection field, and in particular to one kind is used to detect H1N1 stream in Pharyngeal swab samples
Fluorescence RT-RAA primers, probe and the detection method of Influenza Virus.
Background technology
Influenza A H1N1 is Acute respiratory infectious disease, and its pathogen is a kind of new H1N1virus,
Propagated in crowd.It is different from conventional or current seasonal current Influenza Virus, the virus stain include swine flu, bird flu and
The genetic fragment of three kinds of influenza viruses of human influenza.Crowd is generally susceptible to H1N1virus, and can infect people, people with people
Infect first stream after early symptom it is similar to common influenza, including generate heat, cough, having sore throat, physical distress, have a headache, feel cold with it is tired
Labor etc., some there is also diarrhoea or vomiting, courbature or it is tired, eyes are rubescent etc..Start within 2009, Influenza A H1N1 exists
It is extensive popular in global range.In the incubation period of Influenza A H1N1, grown compared with influenza, bird flu incubation period, incubation period duration 1~
7 days, mostly 1-3 days.The some patientss state of an illness can develop rapidly, break with tremendous force, unexpected high fever, body temperature more than 38 DEG C, it is or even secondary
Serious pneumonia.Therefore, be timely and effective discovery H1N1virus, by the virus prevention outside the gateway of a country, establish it is quick,
Sensitive, special H1N1virus detection method is very necessary.
With development of globalization, easily and efficiently transport facility makes tourism and country variant and regional animal, animal
The cross-border movement of product is increasingly frequent, and also the propagation to infectious disease and prevalence create advantage.In addition, Influenza A H1N1
Non-typical symptoms are presented in viral early infection, similar with other respiratory virus infection symptoms, it is difficult to distinguish.Therefore, establish fast
The laboratory method of speed diagnosis H 1 N 1 influenza A virus infection is particularly important.
Traditional detection method detection cycle length, and separation positive rate is low, is often delayed the control of the course of disease and epidemic situation.Point
Sub- biometric diagnostic method can detect the infection of virus, and rapid sensitive in viremia virusemia period, extensive at present
The detection applied to H1N1virus.At present, the H1N1virus detection method established both at home and abroad has
Real-time RT-PCR detection method, heminested PCR detection method, LAMP detection method etc., but PCR method needs to use the instrument of complexity
Device and well-equipped laboratory, and LAMP method primer is more complicated, it is necessary to special Software for Design, and obtained with LAMP method
To amplified production be fragment that some differ in size, can not Direct Cloning and sequencing, be only used for judging depositing for target gene
.
The content of the invention
It is an object of the present invention to provide a kind of primer pair for being used to detect H1N1virus, the primer pair
At least one of including following 1) -3)：
1) SEQ ID № in sequence table：SEQ ID № in nucleotide sequence shown in 1 and sequence table：Nucleotides sequence shown in 2
Row；
2) can be with SEQ ID № in sequence table under high high stringency conditions：The nucleotides sequence of 1 nucleotide sequence hybridization limited
Row；With can be with SEQ ID № in sequence table under high high stringency conditions：The nucleotide sequence of 2 nucleotide sequence hybridizations limited；
1) or 2) 3) nucleotide sequence with limiting has more than 90% homology, and has the nucleotides of identical function
Sequence.
Specifically, the homology is more than 95%；Specific again is more than 96%；Specific again is more than 97%；Again
Specific is more than 98%；Specific again is more than 99%.
Above-mentioned high high stringency conditions can be with 6 × SSC, 0.5%SDS solution, hybridize at 65 DEG C, then with 2 × SSC,
0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
It is described that there is identical function to specifically include the nucleotide sequence that can be used for detecting H1N1virus.
It is also another object of the present invention to provide a kind of probe for being used to detect H1N1virus, the probe bag
At least one of include following 1) -3)：
1) nucleotides sequence of the probe is classified as：
5'-CATTTGGGTAAATGTAACATTGCTGGCTGGANMCNGGGAAATCCAGAGTG-3',
Wherein, the N represents the nucleotides after any nucleotides or any modification, and the M represents the change of tool cyclic structure
Compound；
2) under high high stringency conditions can with 1) described in probe nucleotide sequence hybridization nucleotide sequence；
3) with 1) or 2) described in the nucleotide sequence of probe there is more than 90% homology, and there is identical function
Nucleotide sequence.
Specifically, the N represents the thymidylic acid FAM-dT for carrying fluorescein base group FAM or carried
Fluorescent quenching group BHQ thymidylic acid BHQ-dT, the M represents tetrahydrofuran.
Specifically, the homology is more than 95%；Specific again is more than 96%；Specific again is more than 97%；Again
Specific is more than 98%；Specific again is more than 99%.
Above-mentioned high high stringency conditions can be with 6 × SSC, 0.5%SDS solution, hybridize at 65 DEG C, then with 2 × SSC,
0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
It is described that there is identical function to specifically include the nucleotide sequence that can be used for detecting H1N1virus.
Specifically, the nucleotides sequence of the probe is classified as SEQ ID № in sequence table：Nucleotide sequence shown in 3.
A further object of the present invention is to provide a kind of composition for being used to detect H1N1virus, the combination
Thing include it is following 1) and 2)：
1) primer pair of the present invention；
2) any described probe of the present invention.
A further object is for the present invention provides a kind of kit for being used to detect H1N1virus, the reagent
At least one of box includes following 1) -3) described：
1) primer pair of the present invention；
2) any described probe of the present invention；
3) composition of the present invention.
Specifically, when the kit simultaneously include described primer pair and described probe when, described primer pair and
The mol ratio of described probe is 4：4：1.
It is also another object of the present invention to provide a kind of detection method of H1N1virus, the detection method is not
The method of diagnosis or treatment including disease；The detection method is including the use of following 1) -3) at least one of detected：
1) primer pair of the present invention；
2) any described probe of the present invention；
3) composition of the present invention.
At least one of specifically, the detection method also includes following 1) -2)：
1) when the detection method has used described primer pair and described probe simultaneously, described primer pair and institute
The mol ratio for the probe stated is 4：4：1；
2) RNA for extracting detected sample carries out RT-RAA reactions, and the reaction temperature of the RT-RAA reactions includes 39 DEG C；
Reaction time includes more than 20 minutes.
A further object of the present invention is to provide any primer pair of the present invention, any probe of the present invention, this hair
Bright any composition, any kit of the present invention, any detection method of the present invention are in detection H1N1 stream
Application in Influenza Virus, the application do not include the application for being related to the diagnosis or treatment method of disease.
The present invention's a further object is any primer pair of the offer present invention, any probe of the present invention, this hair
Bright any composition, any kit of the present invention, any detection method of the present invention are preparing detection A type
Application in H1N1 influenza virus Related products.
Beneficial effects of the present invention include：
It is an object of the present invention to provide one kind to be used for reverse transcription recombinase-mediated nucleic acid amplification technologies detection Influenza A H1N1
The fluorescence RT-RAA primers and fluorescence probe and fluorescence RT-RAA primers and fluorescence probe of virus are in detection H1N1 stream
Application in Influenza Virus, fluorescence RT-RAA primers and fluorescence probe can quickly, in qualitative detection throat swab sample A type
H1N1 influenza viruses.
Fluorescence RT-RAA primers and fluorescence probe provided by the invention, examined using fluorescence RT-RAA primers and fluorescence probe
When surveying H1N1virus, independent of the PCR instrument device of costliness, and detection time significantly contracts compared with PCR method or fluorescent PCR
It is short, and sensitivity even more high suitable with fluorescent PCR.
In RT-RAA courses of reaction, H1N1virus RNA synthesizes cDNA by reverse transcriptase first, recycles
Recombinase replaces PCR high-temperature denatured, completes to unwind to double-strand, the double-strand untied is combined by single strand binding protein, prevents DNA
Renaturation, then the extension by polymerase completion chain, using cDNA as templated synthesis purpose product.Reaction carries out 20 minutes at 39 DEG C
In addition, RAA technologies can also complete multi-primerses amplification, coordinate luminoscope, a set of RAA multi-fluorescences can be formed and detected in real time be
System, i.e., using the fluorescence labeling of different colours, detect different target gene, this is other constant temperature cores in same reaction
What sour amplification technique or Nested PCR Technique can not be compared.
The present invention can realize single tube scene, quick detection H1N1virus, and other detection techniques, which are compared, to be had
Advantages below：
1st, totally-enclosed reaction, monitor fluorescence in real time data, without subsequent treatment, avoid polluting, ensure testing result can
By property；2nd, it independent of expensive instruments such as PCR, or even can be detected under 37-39 DEG C of normal temperature, diagnosis knot can be gone out in 20 minutes
Fruit, greatly shorten detection time；3rd, fluorescence RT-RAA primers provided by the invention and fluorescence probe specificity is stronger, sensitivity more
Height, epidemic situation quick diagnosis and complete monitoring are fully met, be the early diagnosis of epidemic situation, early treatment, reduce case fatality rate and control epidemic situation
Race against time.
Brief description of the drawings
Fig. 1 is sensitivity experiment result figure.
Fig. 2 is specificity experiments result figure.
Fig. 3 is positive clinical sample detection result figure.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Do not make the experimental methods of molecular biology illustrated, equal reference in following embodiments《Molecular Cloning:A Laboratory guide》
Listed specific method is carried out in the book of (third edition) J. Pehanorm Brookers one, or is carried out according to kit and product description.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Embodiment 1, the design of fluorescence RT-RAA primer, probe for detecting H1N1virus
The special conservative region for being directed to H1N1virus is targeting regions, designs specific primer and fluorescence
RAA probes, its sequence are respectively：
Forward primer, such as SEQ ID № in sequence table：Nucleotide sequence shown in 1：
5'-GCGAACAATTCAACAGACACTGTAGACACAGTAC-3'
Reverse primer, such as SEQ ID № in sequence table：Nucleotide sequence shown in 2：
5'-CTAGGTGTTTCCACAATGTAGGACCATGAGC-3'
Oligonucleotide probe, such as SEQ ID № in sequence table：Nucleotide sequence shown in 3：
5'-CATTTGGGTAAATGTAACATTGCTGGCTGGA(FAM-dT)(THF)C(BHQ-dT)
GGGAAATCCAGAGTG-3'；
Wherein：
FAM-dT represents to carry fluorescein base group FAM (6-Carboxyfluorescein) thymidine
Acid；
THF represents tetrahydrofuran (tetrahydrofuran) connexon；
BHQ-dT represents to carry fluorescent quenching group BHQ (black hole quencher) thymidine deoxidation core
Thuja acid；
Embodiment 2, a kind of foundation for being used to detect the fluorescence RT-RAA methods of H1N1virus
(1) fluorescence RT-RAA reacts
1) extraction of sample rna：The sample rna can use the viral of German Kai Jie bioengineering Co., Ltd
RNA nucleic acid extraction kits, are extracted according to kit specification.
2) forward primer, reverse primer and the probe designed using embodiment 1, and RAA reaction kit (the present embodiment
The specific RAA basic agents box for employing Jiangsu Qi Tian genes bio tech ltd), prepared with the present embodiment step 1)
Sample rna to be detected is template, carries out amplified reaction, and wherein reaction system is as follows：
25 μ l RAA reaction buffers (offer of Jiangsu Qi Tian genes bio tech ltd RAA basic agents box)；
Forward primer, each 2 μ L of reverse primer (10 μM) of the design of embodiment 1；
The probe (10 μM) of 0.5 μ L embodiments 1 design；
Sample rna template to be detected prepared by 1 μ L steps 1)；
0.5 μ L M-MLV reverse transcriptases (ThermoFisher Products, 200U/ μ L)
Add 16.5 μ L distilled waters to 47.5 μ L, be well mixed, be then added to dry powder (the strange day gene biotechnology in Jiangsu
Co., Ltd RAA basic agents box provide) reaction tube in, mix again.Each pipe adds 2.5 μ L 280mM magnesium acetate solutions simultaneously
Mix.
(2) fluoroscopic examination
Above-mentioned reaction tube is positioned over RAAF-6100 fluorogenes detector (the strange limited public affairs of day gene biotechnology in Jiangsu
Department), 20min is reacted at 39 DEG C, carries out fluoroscopic examination.
Negative control：FAM passages are without amplification curve, and without Tt values；
Positive control：FAM passages have an amplification curve, Tt values≤8min
The above-mentioned requirement to positive control and negative control need to meet that otherwise, this is tested simultaneously in once experiment
It is invalid, it is necessary to re-start experiment.
4) sample detection result interpretation：
When the sample F AM passages detected have amplification curve, when quality control is normal, H1N1 influenza viruses sun can determine that
Property；
When the sample F AM passages detected are without amplification curve, and Tt values are shown as Undet or No Tt, and quality control is normal
When, it can determine that H1N1 influenza viruses feminine gender.
(3) sensitivity experiment
The minimum detection limit of the experimental verification detection method, uses concentration as 1.0 × 107copies/mL、1.0×
106copies/mL、1.0×105copies/mL、1.0×104copies/mL、1.0×103copies/mL、1.0×
102Copies/mL, 10copies/mL etc. positive plasmid show the detection method as test limit reference product by the experiment
Test limit reach 10copies/mL, specific experiment result is as shown in Figure 1.
10 in Fig. 17、106、105、104、103、102, 10 respectively represent concentration be 1.0 × 107copies/mL、1.0×
106copies/mL、1.0×105copies/mL、1.0×104copies/mL、1.0×103copies/mL、1.0×
102Copies/mL, 10copies/mL positive quality control product amplification curve, the moon is negative quality-control product, when abscissa represents reaction
Between, ordinate mv represents fluorescent value.Result shows shown in Fig. 1, and the test limit of the detection method reaches 10copies/mL.
(4) specificity experiments
The experimental selection is joined with the common causative that H1N1virus has identical infection site as specificity
Examine product.Specific reference material is respectively H3N2 influenza viruses, influenza B virus (influ B), H5N6 avian influenza virus, breathing
Road syncytial virus (RSV), rhinovirus (RV).Sample above is extracted using the Viral viral RNAs extracts kit of Qigen companies
In RNA tested as template.Specificity experiments result is as shown in Figure 2.
Abscissa represents the reaction time in Fig. 2, and ordinate mv represents fluorescent value.Result shows shown in Fig. 2, and this 5 kinds special
Property reference material do not have amplification curve, illustrate that the specificity of the detection method is good.
(5) positive clinical sample detection is tested
5 Influenza A H1N1 diseases that laboratory selection Ningbo international travel health care center complex laboratory preserves
Malicious positive sample (H1N1-1019, H1N1-1020, H1N1-10217, H1N1-776, H1N1-782) detected to this method,
Checking.Specific experiment result is as shown in Figure 3.
Abscissa represents the reaction time in Fig. 3, and ordinate mv represents fluorescent value.Result shows shown in Fig. 3, the detection method
Expanding effect is excellent, has good performance.
Embodiment described above only expresses embodiments of the present invention, and its description is more specific and detailed, but can not
Therefore the limitation to the scope of the claims of the present invention is interpreted as, as long as the skill obtained using the form of equivalent substitution or equivalent transformation
Art scheme, it all should fall within the scope and spirit of the invention.
Sequence table
<110>Ningbo international travel health care center
<120>A kind of fluorescence RT-RAA primers, probe and detection method for being used to detect A (H 1 N 1) virus
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 34
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gcgaacaatt caacagacac tgtagacaca gtac 34
<210> 2
<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ctaggtgttt ccacaatgta ggaccatgag c 31
<210> 3
<211> 50
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (32)..(32)
<223> n=FAM-dT
<220>
<221> misc_feature
<222> (33)..(33)
<223> m=THF
<220>
<221> misc_feature
<222> (35)..(35)
<223> n=BHQ-dT
<400> 3
catttgggta aatgtaacat tgctggctgg anmcngggaa atccagagtg 50
Claims (10)
1. a kind of be used to detect the primer pair of H1N1virus, it is characterised in that the primer pair includes following 1) -3)
At least one of：
1) SEQ ID № in sequence table：SEQ ID № in nucleotide sequence shown in 1 and sequence table：Nucleotide sequence shown in 2；
2) can be with SEQ ID № in sequence table under high high stringency conditions：The nucleotide sequence of 1 nucleotide sequence hybridization limited；With
Can be with SEQ ID № in sequence table under high high stringency conditions：The nucleotide sequence of 2 nucleotide sequence hybridizations limited；
1) or 2) 3) nucleotide sequence with limiting has more than 90% homology, and has the nucleotide sequence of identical function.
2. a kind of be used to detect the probe of H1N1virus, it is characterised in that the probe includes following 1) -3) in
It is at least one：
1) nucleotides sequence of the probe is classified as：
5'-CATTTGGGTAAATGTAACATTGCTGGCTGGANMCNGGGAAATCCAGAGTG-3',
Wherein, the N represents the nucleotides after any nucleotides or any modification, and the M represents the compound of tool cyclic structure；
2) under high high stringency conditions can with 1) described in probe nucleotide sequence hybridization nucleotide sequence；
3) with 1) or 2) described in the nucleotide sequence of probe there is more than 90% homology, and there is the nucleosides of identical function
Acid sequence.
3. probe according to claim 2, it is characterised in that the nucleotides sequence of the probe is classified as SEQ ID in sequence table
№：Nucleotide sequence shown in 3.
4. a kind of be used to detect the composition of H1N1virus, it is characterised in that the composition include it is following 1) and
2)：
1) primer pair described in claim 1；
2) any described probe of Claims 2 or 3.
5. a kind of be used to detect the kit of H1N1virus, it is characterised in that the kit includes following 1) -3)
It is at least one of described：
1) primer pair described in claim 1；
2) any described probe of Claims 2 or 3；
3) composition described in claim 4.
6. kit according to claim 5, it is characterised in that when the kit simultaneously including described primer pair and
During described probe, the mol ratio of described primer pair and described probe is 4：4：1.
A kind of 7. detection method of H1N1virus, it is characterised in that：The detection method does not include the diagnosis of disease
Or the method for the treatment of；The detection method is including the use of following 1) -3) at least one of detected：
1) primer pair described in claim 1；
2) any described probe of Claims 2 or 3；
3) composition described in claim 4.
8. detection method according to claim 7, it is characterised in that：The detection method also includes following 1) -2) in
It is at least one：
1) when the detection method simultaneously used described primer pair and described probe when, described primer pair with it is described
The mol ratio of probe is 4：4：1；
2) RNA for extracting detected sample carries out RT-RAA reactions, and the reaction temperature of the RT-RAA reactions includes 39 DEG C；Reaction
Time includes more than 20 minutes.
9. composition, right will described in any probe of primer pair, Claims 2 or 3 described in claim 1, claim 4
Ask any kits of 5-6, any detection methods of claim 7-8 answering in H1N1virus is detected
With the application does not include the application for being related to the diagnosis or treatment method of disease.
10. composition, right described in any probe of primer pair, Claims 2 or 3 described in claim 1, claim 4
It is required that any kits of 5-6, any detection methods of claim 7-8 are preparing detection H1N1virus phase
Close the application in product.
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| CN201711005640.1A CN107574262A (en) | 2017-10-25 | 2017-10-25 | A kind of fluorescence RT RAA primers, probe and detection method for being used to detect A (H 1 N 1) virus |
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|---|---|---|---|
| CN201711005640.1A CN107574262A (en) | 2017-10-25 | 2017-10-25 | A kind of fluorescence RT RAA primers, probe and detection method for being used to detect A (H 1 N 1) virus |
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| CN108315478A (en) * | 2018-02-13 | 2018-07-24 | 吴斌 | The probe and kit of RAA Fluorometric assay hydrophobins |
| CN111534636A (en) * | 2020-04-09 | 2020-08-14 | 中国人民解放军东部战区疾病预防控制中心 | Primer pair, probe, kit and detection method for detecting 2019-nCoV and application thereof |
| CN112063726A (en) * | 2020-09-21 | 2020-12-11 | 杭州市疾病预防控制中心 | Primer, probe and detection method for detecting oseltamivir drug resistance mutation |
| CN112831605A (en) * | 2021-02-19 | 2021-05-25 | 杭州宝临生物科技有限公司 | Multienzyme isothermal amplification detection kit and application thereof |
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