CN107034310A - It is a kind of while detecting the primer sets of a variety of influenza viruses, probe groups and its kit - Google Patents
It is a kind of while detecting the primer sets of a variety of influenza viruses, probe groups and its kit Download PDFInfo
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- CN107034310A CN107034310A CN201710059374.4A CN201710059374A CN107034310A CN 107034310 A CN107034310 A CN 107034310A CN 201710059374 A CN201710059374 A CN 201710059374A CN 107034310 A CN107034310 A CN 107034310A
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Abstract
The invention provides a kind of while detect the primer sets of a variety of influenza viruses, probe groups, the sequence such as SEQ ID NO of the primer sets：Shown in 1~8, the sequence such as SEQ ID NO of probe groups：Shown in 9~12；The influenza virus is influenza A virus, influenza B virus, seasonal current Influenza Virus H1 hypotypes and H3 hypotypes；On this basis, there is provided a kind of while detecting the kit of a variety of influenza viruses, direct amplification and multiplex PCR are combined together by the present invention, the direct RT PCR to doubtful flu casess or epidemiology survey throat swab sample can be realized, nucleic acid extraction step is saved than conventional method, beneficial to automation integrated detection is realized, efficiency is improved.Had a clear superiority in quick discriminating detection influenza virus the infected's application aspect, easy to operate, detection time is short, and testing cost is relatively low.Available for reply epidemic situation field quick detection, with extensive and actual application value.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly, to a kind of while detecting a variety of influenza viruses
Primer sets, probe groups and its kit.
Background technology
Influenza is a kind of viral infection, mainly influences nose, larynx, bronchus, and influence lung once in a while.Infection is typically lasted for
About one week, be characterized in burst hyperpyrexia, DOMS, headache and serious discomfort, dry cough, laryngalgia and rhinitis.Coughed by contaminating patient
Or the spittle and particulate produced when sneezing, virus is easy to propagating between men.During seasonal epidemic, influenza is past
Toward rapid propagation.Majority dye patient rehabilitation within one to two week, without carrying out therapeutic treatment.But, to child, the elderly and trouble
There are other serious conditions persons, infection can cause serious complication, pneumonia and death according to inherent situation.Seasonal current Influenza Virus
With influenza a virus infection at most, secondly it is influenza B virus.It is again sub- with H1 hypotypes and H3 wherein in influenza A virus
Type Strain is epidemic strain or alternately popular.At present, in clinical and laboratory, immunology and molecular Biological Detection influenza virus
It is the most commonly used.It is more typical for ELISA detection reagents, fluorescence immunoassay detection reagent and real-time PCR detection reagent.Wherein
It is that the detection of nucleic acids product represented is usually, using throat swab as sample, first to carry out sample nucleic acid using real-time PCR detection reagent
Extraction process is operated, then carries out RT-PCR amplification operations, and two steps are indispensable.Direct TRAP is one kind in amplified reaction
System and core starting materials are optimized on the basis of transformation, realize virolysis, nucleic acid release, RNA reverse transcriptions, PCR expansion
Increase the technical method that step is carried out with pipe, prior art lacks the kit of a tube reaction for a variety of influenza viruses of detection.
The content of the invention
The technical problems to be solved by the invention are that the drawbacks described above for overcoming prior art to exist is detected simultaneously there is provided a kind of
Primer sets, the probe groups of a variety of influenza viruses.
Second object of the present invention is to provide the kit containing the primer sets.
The purpose of the present invention is achieved by the following technical programs：
It is a kind of while detect the primer sets of a variety of influenza viruses, probe groups, the sequence such as SEQ ID NO of the primer sets：1~8
It is shown, the sequence such as SEQ ID NO of probe groups：Shown in 9~12；The influenza virus is influenza A virus, influenza B disease
Poison, seasonal current Influenza Virus H1 hypotypes and H3 hypotypes.
The present invention also provides a kind of while detecting the kit of a variety of influenza viruses, and the kit contains described primer
Group and probe groups；The influenza virus is influenza A virus, influenza B virus, seasonal current Influenza Virus H1 hypotypes and H3 sub-
Type.
By preparing each reagent needed for RT-PCR, nucleic acid extraction and RT-PCR are combined and carry out single step reaction, this hair
Bright to devise a kind of kit, the kit is by direct RT-PCR reaction solutions A, direct RT-PCR reaction solutions B, Sample Dilution
Liquid, positive quality control product and negative quality-control product composition.
Wherein, primer sets and probe groups are in direct RT-PCR reaction solutions A, it is preferable that the direct RT-PCR reaction solutions A
Also contain Tris-HCl, MgCl2、NP-40、Tween20、KCl。
It is highly preferred that the composition of the direct RT-PCR reaction solutions A is：0.2~0.5 μM of SEQ ID NO：Shown in 1~8
Primer, 0.1~0.3 μM of SEQ ID NO：Probe shown in 9~12,10~60mM Tris-HCl, 2~6mM MgCl2, 5~
12mM NP-40,2~6mM Tween20,20~38mM KCl.
Preferably, the direct RT-PCR reaction solutions B contains reverse transcriptase, Taq archaeal dna polymerases, dNTPs and RNase suppression
Preparation.
It is highly preferred that directly RT-PCR reaction solutions B composition is：Reverse transcriptase, the Taq of 5U/ reactions of 2.5U/ reactions
Archaeal dna polymerase, the RNase inhibitor of dNTPs, 20U/ reaction of 0.6mm/ reactions.
Preferably, the Sample dilution contains Tris-HCl, EDTA；Tris-HCl concentration is 80~100mM, EDTA
Concentration be 0.2~6mM；The positive quality control product is influenza A virus, influenza B virus, seasonal current Influenza Virus H1 Asias
The amplified fragments of type and H3 hypotypes, content is 1.0 E5 copies/mL.
The negative quality-control product is physiological saline.
As a kind of specific embodiment, direct RT-PCR reaction solutions A of the present invention composition is：Above-mentioned primer sets
With probe groups, 10mM Tris-HCl（pH7.5）、2mM MgCl2、0.2%（V/V）NP-40,0.01%（V/V）Tween20,
30mM KCl, remaining is DEPC water.Appointing in its middle probe 5 ' end difference any flag F AM, HEX, Texas Red and Cy5
One kind, 3 ' end mark BHQ1.
The composition of the Sample dilution is：80mM Tris-HCl （pH8.5）, 0.2mM EDTA solution.
The negative quality-control product is into being grouped into：Physiological saline.
The present invention also provides and detects a variety of viral methods using the kit, comprises the following steps：
（1）Take 200 μ l Sample dilutions to add throat swab sample mesoscale eddies concussion 10s to obtain fully to infiltrate and elute virion
Dip lotion；
（2）Take the μ l of 28 μ l, RT-PCR reaction solutions B of RT-PCR reaction solutions A 2 to mix, PCR is added to as 1 RT-PCR reaction solution
In reaction tube；
（3）From step（1）In take 20 μ l dip lotions to add in above-mentioned PCR reaction tubes；
（4）PCR reaction tubes are placed on real-time fluorescence quantitative PCR instrument and carry out augmentation detection, reaction condition is 60 DEG C of 10min, 95
DEG C, 10min, 1 circulation；95 DEG C, 15s, 60 DEG C, 30s（Collect fluorescence signal）, 45 circulations.Fluorescence detector selection FAM,
VIC、Texas Red、Cy5；
（5）Result judgement：The corresponding amplification curve of tetra- passages of FAM, VIC, Texas Red, Cy5 is checked at amplification interface, if
There is amplification curve, then judge that correspondence fluorescence channel detects target as the positive accordingly, FAM passages correspondence influenza A virus, VIC
Passage correspondence influenza B virus, Texas Red passages correspondence seasonal current Influenza Virus H1 hypotypes, Cy5 correspondence seasonal influenzas
Viral H3 hypotypes.
Compared with prior art, the invention has the advantages that：
Present invention firstly provides a kind of while detect the primer sets of a variety of influenza viruses, probe groups, the sequence of the primer sets
Such as SEQ ID NO：Shown in 1~8, the sequence such as SEQ ID NO of probe groups：Shown in 9~12；The influenza virus is Flu-A
Virus, influenza B virus, seasonal current Influenza Virus H1 hypotypes and H3 hypotypes；There is provided a kind of detection simultaneously is many on this basis
The kit of influenza virus is planted, the present invention direct will be expanded and multiplex PCR be combined together, it is possible to achieve to doubtful influenza disease
The direct RT-PCR of example or epidemiology survey throat swab sample, nucleic acid extraction step is saved than conventional method, beneficial to realization certainly
Dynamicization integration detection, improves efficiency.On the basis of directly expanding, the discriminating purpose examined a pipe being realized again, quick more
Differentiate that detection influenza virus the infected's application aspect has a clear superiority, easy to operate, detection time is short, and testing cost is relatively
It is low.If collocation portable fluorescence detector can be applied to tackle epidemic situation field quick detection, with extensive and actual application
Value.
Brief description of the drawings
Fig. 1 is quality-control product amplification curve diagram.
Fig. 2 is the amplification curve diagram of influenza A virus H1 hypotype samples.
Fig. 3 is the amplification curve diagram of influenza A virus H3 hypotype samples.
Fig. 4 is the amplification curve diagram of influenza B virus sample.
Fig. 5 is the amplification curve diagram of influenza A virus H1 hypotype samples.
Fig. 6 is the amplification curve diagram of influenza A virus H3 hypotype samples.
Embodiment
The present invention is expanded on further with reference to Figure of description and specific embodiment.These embodiments are merely to illustrate this
Invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in lower example embodiment, generally according to this
Field normal condition or the condition advised according to manufacturer.Unless otherwise defined, all specialties and science used in text
Term is identical with meaning familiar to the person skilled in the art.
Embodiment 1
For influenza virus IVA, IVB, H1 and H3, specific primer is separately designed, its primer sequence is as follows：
IVA-F：5’-CTTCTAAGCGAGGTCGAACC-3’
IVA-R：5’-GTCTTAGCCATTCGATGAGT-3’
IVB-F：5’-CGACATCCAAAGCCAATTCG-3’
IVB-R：5’-CGGTGCTCTTGACCAAATTGC-3’
H1-F：5’-CCTCAGGGAGCTATAAACAGA-3’
H1-R：5’-TGACCTACTTTGGACACTCTCC-3’
H3-F：5’-CAACTGCAATTCTGAATGCATC-3’
H3-R：5’-GATCCTGTTTACATTTTGGAATG-3’
For every group of primer, its specific probe is designed, sequence is as follows：
IVAP：5’-CAGGCCCCCTCAAAGCCG-3’ （5’ FAM；3’ BHQ1）
IVBP：5’-AGACTCCCACCGCAGTTTCAGC-3’（ 5’ HEX；3’ BHQ1）
H1P：5’-CCTTTCCAGAATGTACACCCAGTC-3’ （5’ Texas Red；3’ BHQ1）
H3P：5’-CCAAATGGAAGCATTCCCAATGAC-3’ （5’ Cy5；3’ BHQ1）
RT-PCR reaction systems are：28 μ l, RT-PCR reaction solutions B of RT-PCR reaction solutions A 2 μ l, the μ l of sample elution liquid 20.
RT-PCR response procedures are：60 DEG C of 10min, 95 DEG C, 10min, 1 circulation；95 DEG C, 15s, 60 DEG C, 30s（Collect
Fluorescence signal）, 45 circulations.Fluorescence detector selection FAM, VIC, Texas Red, Cy5.
As a result show：Pattern detection is that VIC passages have amplification curve, is determined as that influenza B virus is positive.
Comparative example 1
Experimental method be the same as Example 1, it is unique unlike, primer sets and probe set sequences used are as follows：
IVA-F’： CGGAGGACTTGAATGGAATGATA
IVA-R’： TGTTTTGGAGTAAGTGGAGGTCC
IVB-F’： TGGAGCTGCCTATGAAGACC
IVB-R’： TTGCTGGAACATGGAAACC
H1-F’： GCTCCTGGGAAATCCAGAGT
H1-R’： TGGGTAACACGTCCCATTGT
H3-F’： GATTCCGATATCAAAACTCGG
H3-R’： AATTTTCCATTGATCTGGTCG
For every group of primer, its specific probe is designed, sequence is as follows：
IVAP’： TACTGCTTCTCCAAGCGAATCTCTG
IVBP’： CTGATCTAGGCTTGAATTCTGTGCC
H1P’： TCTTCACAGCAAGCTCATGGTCCTA
H3P’： CCTCTTTAGATCTGCAGCTTGTCC
As a result show：These four viruses effectively can not be all detected using the primer sets and probe groups of the comparative example.
Comparative example 2
Experimental method be the same as Example 1, it is unique unlike, primer sets and probe set sequences used are as follows：
IVA-F’： ATGAGAATGGGGGACCTCCA
IVA-R’： TCACTTCTTCAATCAGCCATCTTAT
IVB-F’： GATGGCTTCAGTGGACTAAATCAC
IVB-R’： ATTAATGAAGGGTCAAGTCCAACTC
H1-F’： CCCGTCTATTCAATCTAGAGGC
H1-R’： GTGATAACCGTACCATCCATCTA
H3-F’： TCTAAAGAGCACTCAAGCAGC
H3-R’： CCCTTCTACTTCTGAGAATTCC
For every group of primer, its specific probe is designed, sequence is as follows：
IVAP’： AGCTGTTCTCGCCATTTTCCGTTTC
IVBP’： ATCATTCATCTGTGAATGCCCAATC
H1P’： CCATCCCCCCTCAATAAAGCCG
H3P’： TTCTCATTGGTCCTTTCAATCACTC
As a result show：These four viruses effectively can not be all detected using the primer sets and probe groups of the comparative example.
The composition of the kit of embodiment 2 and preparation
It is a kind of while detect the kit of a variety of influenza viruses, contain：Direct RT-PCR reaction solutions A, direct RT-PCR reaction solutions
B, Sample dilution, positive quality control product, negative quality-control product.
Wherein RT-PCR reaction solutions A is constituted：The specific primer and probe sequence of four kinds of influenza viruses described in embodiment 1
（Primer is 15pmol, and probe is 10pmol）、10mM Tris-HCl （pH7.5）、2mM MgCl2、0.2%（V/V）NP-
40,0.01%（V/V）Tween20,30mM KCl, remaining is DEPC water.
Wherein RT-PCR reaction solutions B is constituted：Reverse transcriptase（2.5U/ reaction）, Taq archaeal dna polymerases（5U/ reacts）、
dNTPs（0.6mm/ reacts）, RNase inhibitor（20U/ reacts）.
Wherein Sample dilution, which is constituted, is：10mM Tris-HCl （pH8.5）, 0.2mM EDTA solution.
Wherein positive quality control product, which is constituted, is：By the plasmid containing four kinds of influenza viral amplification fragments（Such as SEQ ID NO：13）
Composition, content is 1.0E5 copies/mL.
Wherein negative quality-control product is into being grouped into：Physiological saline.
The method that influenza virus is detected using the kit, sample includes 1 H1 hypotype throat swabs positive sample, 1 H3
Hypotype throat swab positive sample, 1 influenza B virus throat swab positive sample, a Respiratory Syncytial Virus(RSV) throat swab are positive
Sample.Comprise the following steps：
（1）Take 200 μ l Sample dilutions to add throat swab sample mesoscale eddies concussion 10s fully to infiltrate and elute virion；
（2）Take the μ l of 28 μ l, RT-PCR reaction solutions B of RT-PCR reaction solutions A 2 to mix, PCR is added to as 1 RT-PCR reaction solution
In reaction tube；
（3）20 μ l dip lotions are taken to add in above-mentioned PCR reaction tubes；
（4）PCR reaction tubes are placed on real-time fluorescence quantitative PCR instrument and carry out augmentation detection, reaction condition is 60 DEG C of 10min, 95
DEG C, 10min, 1 circulation；95 DEG C, 15s, 60 DEG C, 30s（Collect fluorescence signal）, 45 circulations.Fluorescence detector selection FAM,
VIC、Texas Red、Cy5；
（5）Result judgement：The corresponding amplification curve of tetra- passages of FAM, VIC, Texas Red, Cy5 is checked at amplification interface, if
There is amplification curve, then judge that correspondence fluorescence channel detects target as the positive accordingly, FAM passages correspondence influenza A virus, VIC
Passage correspondence influenza B virus, Texas Red passages correspondence seasonal current Influenza Virus H1 hypotypes, Cy5 correspondence seasonal influenzas
Viral H3 hypotypes.
First, the influenza B virus nucleic acid combined test kit produced with Shanghai ZJ Bio-Tech Co., Ltd.
（Fluorescent PCR method）, influenza A virus H1 hypotypes and H3 hypotype nucleic acid combined test kits（Fluorescent PCR method）Two kinds of reagents are made
The contrast agents box detection expanded again after being extracted for sample nucleic acid.
As a result show：Influenza virus parting is commonly used on the market differentiates that detection reagent at least needs two kits jointly complete
Into；And two steps of nucleic acid extraction and amplification amount to needs the time for 180min or so, and all operations of the present invention amount to and needed
105min；And augmentation detection effect is suitable with the Detection results of contrast agents box.
SEQUENCE LISTING
<110>Nanfang Medical Univ
<120>It is a kind of while detecting the primer sets of a variety of influenza viruses, probe groups and its kit
<130> 2017
<160> 13
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> IVA-F
<400> 1
cttctaagcg aggtcgaacc 20
<210> 2
<211> 20
<212> DNA
<213> IVA-R
<400> 2
gtcttagcca ttcgatgagt 20
<210> 3
<211> 20
<212> DNA
<213> IVB-F
<400> 3
cgacatccaa agccaattcg 20
<210> 4
<211> 21
<212> DNA
<213> IVB-R
<400> 4
cggtgctctt gaccaaattg c 21
<210> 5
<211> 21
<212> DNA
<213> H1-F
<400> 5
cctcagggag ctataaacag a 21
<210> 6
<211> 22
<212> DNA
<213> H1-R
<400> 6
tgacctactt tggacactct cc 22
<210> 7
<211> 22
<212> DNA
<213> H3-F
<400> 7
caactgcaat tctgaatgca tc 22
<210> 8
<211> 23
<212> DNA
<213> H3-R
<400> 8
gatcctgttt acattttgga atg 23
<210> 9
<211> 18
<212> DNA
<213> IVAP
<400> 9
caggccccct caaagccg 18
<210> 10
<211> 22
<212> DNA
<213> IVBP
<400> 10
agactcccac cgcagtttca gc 22
<210> 11
<211> 24
<212> DNA
<213> H1P
<400> 11
cctttccaga atgtacaccc agtc 24
<210> 12
<211> 24
<212> DNA
<213> H3P
<400> 12
ccaaatggaa gcattcccaa tgac 24
<210> 13
<211> 358
<212> DNA
<213>4 kinds of influenza virus plasmids
<400> 13
agacaagacc aatcctgtca cctctgacta aagggatctt gggagttgta ttcacgctca 60
ccgtgcccag tgaaattcga gcagctgaaa ctgcggtggg agtcttatcc caatttggtc 120
aagagcaccg attatcacca gaatgtaata caaagtgtca aacacctcat ggagctataa 180
acagcagcct tcctttccag aatgtacacc cagtcacaat aggagagtgt ccaaaatatg 240
tcaagagtgc aaaatagatg cacccatcgg caaatgcaat tctgaatgca tcgctccaaa 300
tggaagcatt cccaatgaca aatcattcca aaatgtaaac aggatcacat acggggcc 358
Claims (9)
1. it is a kind of while detecting the primer sets of a variety of influenza viruses, probe groups, it is characterised in that the sequence of the primer sets is such as
SEQ ID NO：Shown in 1~8, the sequence such as SEQ ID NO of probe groups：Shown in 9~12；The influenza virus is Flu-A disease
Poison, influenza B virus, seasonal current Influenza Virus H1 hypotypes and H3 hypotypes.
2. it is a kind of while detecting the kit of a variety of influenza viruses, it is characterised in that the kit contains described in claim 1
Primer sets and probe groups；The influenza virus is influenza A virus, influenza B virus, seasonal current Influenza Virus H1 hypotypes
And H3 hypotypes.
3. kit according to claim 2, it is characterised in that the kit by direct RT-PCR reaction solutions A, directly
RT-PCR reaction solutions B, Sample dilution, positive quality control product and negative quality-control product composition.
4. kit according to claim 3, it is characterised in that the direct RT-PCR reaction solutions A also contains Tris-
HCl、MgCl2、NP-40、Tween20、KCl。
5. kit according to claim 3, it is characterised in that the direct RT-PCR reaction solutions B contains reverse transcription
Enzyme, Taq archaeal dna polymerases, dNTPs and RNase inhibitor.
6. kit according to claim 3, it is characterised in that the Sample dilution contains Tris-HCl, EDTA.
7. kit according to claim 3, it is characterised in that the positive quality control product is influenza A virus, B-mode
The amplified fragments of influenza virus, seasonal current Influenza Virus H1 hypotypes and H3 hypotypes；The negative quality-control product is physiological saline.
8. kit according to claim 4, it is characterised in that the composition of the direct RT-PCR reaction solutions A is：0.2
~0.5 μM of SEQ ID NO：Primer shown in 1~8,0.1~0.3 μM of SEQ ID NO：Probe shown in 9~12,10~60mM
Tris-HCl, 2~6mM MgCl2, 5~12mM NP-40,2~6mM Tween20,20~38mM KCl.
9. kit according to claim 4, it is characterised in that direct RT-PCR reaction solutions B composition is：2.5U/ it is anti-
Reverse transcriptase, the Taq archaeal dna polymerases of 5U/ reactions, the RNase inhibitor of dNTPs, 20U/ reaction of 0.6mm/ reactions answered.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107354237A (en) * | 2017-08-29 | 2017-11-17 | 无锡市疾病预防控制中心 | A kind of Rapid identification A type, B-mode, season H3 subtype influenza virus fluorescence PCR method |
| CN107447046A (en) * | 2017-08-29 | 2017-12-08 | 无锡市疾病预防控制中心 | A kind of Rapid identification A type, B-mode, H1N1 2009, season H3 subtype influenza virus quadruple fluorescence PCR method |
| CN109517927A (en) * | 2018-09-03 | 2019-03-26 | 连云港出入境检验检疫局检验检疫综合技术中心 | A kind of A type, influenza B virus rapid typing detection reagent box and its application |
| CN111808989A (en) * | 2020-06-18 | 2020-10-23 | 重庆浦洛通基因医学研究院有限公司 | Coronavirus/influenza virus/rhinovirus nucleic acid combined detection kit and use method thereof |
| EP3943612A4 (en) * | 2020-06-12 | 2022-03-30 | Da An Gene Co., Ltd. | Swab sample nucleic acid releaser and application thereof |
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| EP3943612A4 (en) * | 2020-06-12 | 2022-03-30 | Da An Gene Co., Ltd. | Swab sample nucleic acid releaser and application thereof |
| CN111808989A (en) * | 2020-06-18 | 2020-10-23 | 重庆浦洛通基因医学研究院有限公司 | Coronavirus/influenza virus/rhinovirus nucleic acid combined detection kit and use method thereof |
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