CN109957624A - PCR kit that is a kind of while detecting first, influenza B, parainfluenza - Google Patents
PCR kit that is a kind of while detecting first, influenza B, parainfluenza Download PDFInfo
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Abstract
Name of product: influenza A virus, influenza B virus and parainfluenza virus kit for detecting nucleic acid (fluorescent PCR method) technical field: the present invention relates to molecular biology fields, and in particular to a kind of for detecting influenza A virus, influenza B virus and the kit of parainfluenza virus and its application method.Technical problem: three kinds of viruses belong to Respirovirus, and symptom is closely similar, only strong and weak slight difference.The demand that the product of commercially available detection individual event pathogen is unable to satisfy quickly detection, distinguishes three kinds of pathogen.Technical solution: it according to fluorescent PCR method principle, according to three kinds of viral nucleic acid sequence features, selects its respective specific and conserved sequence and designs corresponding primer and TaqMan fluorescence probe.Main application: in the tissues such as cloacal swab, tracheae, lung, spleen or the lymph of this kit for poultry and the throat swab equal samples of patient, while qualitative detection is carried out to above-mentioned three kinds of viral nucleic acids.
Description
Technical field
The present invention relates to molecular biology fields, and in particular to one kind is for detecting influenza A virus, influenza B disease
The kit and its application method of poison and parainfluenza virus.
Background technique
Influenza virus (influenza virus) belong to orthomyxoviridae family (Orthomyxoviridae), it is single stranded RNA film
Virus.Influenza structural can be divided into coating, stromatin and core three parts from outer to inner.Influenza virus particles outer membrane has
Two types surface glycoprotein, a type are hemagglutinin (H), are divided into 15 hypotypes, and a type is neuraminidase (N), point
For 9 hypotypes.It is divided into first (A), second (B), the third (C) three type, and human influenza is mainly influenza A virus and influenza B virus
It is caused.
Wherein, influenza A virus is different according to H with N antigen, and is divided into many hypotypes, H can be divided into 17 hypotypes (H1~
H17), N has 10 hypotypes (N1~N10).Wherein only H1N1, H2N2, H3N2 main infection mankind, wherein H1, H5, H7 hypotype be
It is highly pathogenic.Influenza A virus is easiest to morph, and to human pathogenic's height, Zeng Duoci causes worldwide be very popular.Stream
The infection sources of sense is mainly patient, followed by subclinical infection person, propagated with airborne droplet based on, followed by pass through virus pollution
Tea set, tableware, towel etc. are propagated indirectly, and close contact is also one of the approach for propagating influenza.After patient's idiopathy in 5d
Virus is discharged from secretion such as nasal mucus, mouth saliva, sputums, it is infective stage about 1 week, most strong with 2~3d infectiousness at the beginning of disease.It is metainfective
Symptom is mainly shown as high fever, cough, runny nose, myalgia etc., and majority is with serious pneumonia, a variety of internal organs such as serious person's heart, kidney
Failure leads to death, and case fatality rate is very high.And pneumonia is the most common complication.
Influenza B virus is only propagated between people, in breaking out or small prevalence, does not cause worldwide flu outbreak.It is B-mode
It is similar with the clinical symptoms of Flu-A, but admission rate caused by influenza A virus is four times in influenza B virus.It is B-mode
Influenza usually has myositis and gastrointestinal symptom.The antigenic variation of influenza B virus is very slow.If big change has occurred in H and N
Different, new hypotype (qualitative change) is just produced, such as 2 hypotype of nineteen fifty-seven H2N2(first), the H3N2(Type A3 after nineteen sixty-eight).
Parainfluenza virus category paramyxovirus genus is also single stranded RNA, there is 4 types, and wherein I~type III is relatively conventional, baby children
Youngster's easy infection, IV type are more rare.Spectrum of disease caused by I type is very wide, there is laryngotracheobronchitis, bronchiolitis, branch gas
Guan Yan and pneumonia；The infection of II type may occur in which typical lower respiratory tract infection symptom, but in nonimmune inhibition or without merging chronic disease
Children in, laryngotracheobronchitis is the most common disease；Type III mainly causes bronchiolitis and pneumonia, newborn
Respiratory Syncytial Virus(RSV) is only second to the infection disease of young infant.Incubation period general 1~7 day.The infection sources of parainfluenza virus is also led
It to be patient and recessive patient, route of transmission is mainly droplet transmission.
As it can be seen that three kinds of viruses belong to Respirovirus, all unusual phases such as symptom after the infection sources, route of transmission and infection
Seemingly, only strong and weak slight difference.And the product for detecting individual event pathogen on the market is unable to satisfy quickly detection, distinguishes three kinds of cause of diseases
The demand of body.Based on the above circumstances, the demand for quickly detecting differentiation to these three pathogen is higher and higher, therefore, in order to more
It accurately and quickly diagnoses, it is necessary to which establishing one kind being capable of sensitive simultaneously, special and efficient detection influenza A virus, influenza B
The detection means of virus and parainfluenza virus.
Molecular diagnostic techniques can satisfy to influenza A virus, influenza B virus and parainfluenza virus quick diagnosis
Demand, and since their inhereditary material is all ribonucleic acid, it requires to carry out process of reverse-transcription after sample extraction, then carry out
Molecular Detection can quickly and accurately carry out pathogen so this kit can detect these three pathogen simultaneously
Screening, so that cooperation reduces the abuse of antibiotic medicine correctly to treat.
Summary of the invention
The technical problem to be solved by the present invention is to provide one kind and can while quickly detect influenza A virus, influenza B
The kit of virus and three kinds of pathogen of parainfluenza virus.
In order to solve the above technical problems, the technical solution adopted by the present invention is that, this is used to detect influenza A virus, B-mode
The kit of influenza virus and parainfluenza virus by mixed reactant (2X) (including Taq thermal starting archaeal dna polymerase, UNG enzyme,
TaqMan fluorescence probe, PCR buffer, dATP, dUTP, dCTP, dGTP, MgCl2 etc.), enzymatic mixture (20X) (Luna Wen Qi
Dynamic reverse transcriptase and mouse RNase inhibitor), forward primer, reverse primer, positive quality control product and formed without RNase water；Its
In, it include influenza A virus, influenza B virus and the specific and conserved sequence of parainfluenza virus in mixed reaction solution
Primer and TaqMan fluorescence probe；
The primer sequence of the specific and conserved sequence are as follows:
Influenza A virus, SEQ ID NO.1 and SEQ ID NO.2；
Influenza B virus, SEQ ID NO.4 and SEQ ID NO.5；
Parainfluenza virus, SEQ ID NO.7 and SEQ ID NO.8；
The TaqMan probe sequence of the specific and conserved sequence are as follows:
Influenza A virus, SEQ ID NO.3；
Influenza B virus, SEQ ID NO.6；
Parainfluenza virus, SEQ ID NO.9.
Above-mentioned technical proposal can quick and precisely examine influenza A virus, influenza B virus and parainfluenza virus
It surveys, and can be easy to be easy-to-use in various environment, ensure that the timeliness, specificity and sensitivity of detection；For Flu-A disease
Specific primer probe is designed on poison, influenza B virus and the respective conserved sequence of parainfluenza virus, and is marked on probe
Different fluorescence signals can detect simultaneously and distinguish influenza A virus, influenza B virus and parainfluenza virus；It solves
The problem of instrument compatibility and product residual contamination.Kit uses TaqMan probe Fluorescence PCR assay, for Flu-A
Virus, influenza B virus and parainfluenza virus are detected, and the sequence of design primer and probe is in influenza A virus, B-mode
It is all very conservative in the gene of influenza virus and parainfluenza virus, high specificity, and testing result can be obtained in 2 hours,
Sensitivity is up to 10copies/ μ L.
Wherein, SEQ ID NO.1:GAC CRA TCC TGT CAC CTC TGA C；
SEQ ID NO.2:GGG CAT TYT GGA CAA AKC GTC TAC G；
SEQ ID NO.3:FAM-TGC AGT CCT CGC TCA CTG GGC ACG-BHQ；
SEQ ID NO.4:TCC TCA ACT CAC TCT TCG AGC G；
SEQ ID NO.5:CGG TGC TCT TGA CCA AAT TGG；
SEQ ID NO.6:VIC-CCA ATT CGA GCA GCT GAA ACT GCG GTG-BHQ；
SEQ ID NO.7:CGCAGCTATCCGCTGC；
SEQ ID NO.8:GAGTACCTGGCAGCTT；
SEQ ID NO.9:ROX-CTAGCGTGTCCTATACAGGACT-BHQ.
Preferably, the detection reagent include mixed reactant (2X) (including Taq thermal starting archaeal dna polymerase, UNG enzyme,
TaqMan fluorescence probe, PCR buffer, dATP, dUTP, dCTP, dGTP, MgCl2 etc.), enzymatic mixture (20X) (Luna Wen Qi
Dynamic reverse transcriptase and mouse RNase inhibitor), forward primer, reverse primer, positive quality control product and without RNase water.
Using UNG enzyme/anti-pollution system of dUTP, the pollution interference of previous PCR reaction product bring can be reduced；Kit
Mixed reactant contain dUTP, pre-processed before qPCR/RT-qPCR with uracil dna glycosylase (UDG), pass through excision
Uracil base eliminates the product containing uracil previously expanded to generate not amplifiable DNA product.So mixed in reaction
The South Pole temperature-sensitive UDG that 0.025 unit/μ l NEB is added in object is closed, may be implemented to prevent product residual contamination.By reverse transcriptase
It is blended in a PCR reaction tube and uses with hot start Taq polymerase, in single tube, RNA is transcribed by reverse transcriptase first
CDNA, the archaeal dna polymerase that then DNA is relied on expand cDNA, are completed by qPCR quantitative.QPCR/RT- based on probe
Fluorescence increases when the cutting of rear 5' → 3' exonuclease is quenched in qPCR monitoring, target-specific probe, for each circulation in PCR
Middle measurement DNA cloning.Reliably detect the point fluorescence of fluorescence signal in background at one, quantization loop or Ct value can be with
It determines.Ct value can be used for assessing between two or more samples or relative target abundance, calculates with reference to standard curve appropriate
Absolute object amount.Used probe is the TaqMan probe of 5 ' end fluorescent markers, and probe both ends distinguish mark fluorescent and report base
The oligonucleotides of group (R) and fluorescent quenching group (Q).When probe is complete, i.e. stochastic regime and without PCR product hybridized state
When, the fluorescence that reporter group issues is quenched group absorptions.In fluorescent PCR amplification procedure, when special PCR product with
5 ' end 5 prime excision enzyme activities of hot start Taq polymerase when hybridization reaction occur for TaqMan probe simultaneously also probe cleavage, reporter group
The fluorimeter that the fluorescence released can be built in instrument detects.PCR is every by a circulation, glimmering
Optical signal is also as target fragment, as soon as the process for having a sync index to increase, the power of fluorescence signal represents template DNA
Copy number number.Therefore the present invention cannot be only used for simple qualitative detection, also can be used as quantifying for sample concrete content
Detection.
Preferably, the Luna startup temperature reverse transcriptase is by design, thermostabilization more higher than many other RT
Property, optimal reaction temperature can be made to reach 55 DEG C.For difficult target/template, up to 60 °C of higher RT step can be used
Temperature.Luna startup temperature reverse transcriptase and Taq thermal starting archaeal dna polymerase are existed using a kind of temperature sensitive, reversible aptamers
45 DEG C or less inhibit its activity.Therefore, reaction can be established at room temperature and inhibits non-specific amplification.
Preferably, the mixed reactant of the kit is formulated by unique passive reference dye, can with it is various
QPCR instrument platform is compatible, and including the instrument for needing ROX to correct, additional dyestuff is no longer needed during experimental implementation.
Preferably, final concentration of 100~900nM of the primer in amplification system；The probe is in amplification system
In final concentration of 100~500nM.Taq thermal starting archaeal dna polymerase (1~2U/ μ L), Luna startup temperature reverse transcriptase, UNG enzyme
Final concentration of 0.5U~5U in amplification system.
Wherein, M=mol/L is concentration unit；W/v is mass volume ratio；In addition, the concentration of enzyme is in a reactant
1U in system.
Preferably, influenza A virus, influenza B virus and the expansion of parainfluenza virus are contained in the positive quality control product
Increase the plasmid of gene order.
Preferably, the detection reagent the preparation method comprises the following steps: being used for using standard desalination process purifying primer with meeting
qPCR /RT-qPCR.The sensitivity for analysis needed is potentially contributed to using HPLC or PAGE purifying to improve.
The invention solves another technical problem be to provide the application method of aforementioned agents box a kind of, this method includes
Following steps:
(1) before use:
1. extracting using required RNA and purification process preparing target RNA, concentration is determined by OD260 absorbance.
2. diluting RNA is used for standard curve, these should test preceding fresh preparation each, and can dilute in water or TE.
(2) specific steps:
1. defrosting kit reaction mixture and other reactive components at room temperature, are subsequently placed on ice.After thawing completely, pass through
It is inverted, liquid relief or mild be vortexed simply mix each component, for 96 orifice plates, it is proposed that end reaction volume is 20 μ l.
2. determining the total volume of right quantity reaction, 10% excess volume is added, and correspondingly prepare in addition to RNA template
The measurement mixture of all components.By liquid relief or vortex but softly it is thoroughly mixed.Liquid is collected by being simply centrifuged
Bottom of the tube.
3. aliquot is mixed into qPCR pipe or plate.To obtain optimum efficiency, it please ensure precisely consistent liquid relief amount
And reduce bubble to the greatest extent.
4. RNA template is added in qPCR pipe or plate.Seal pipe has optically transparent flat cover;Use optical clear
Diaphragm seal plate.It should be noted that correct sealing plate edge and corner, to prevent artifact caused by evaporation.
5. of short duration rotation pipe or plate are to remove bubble and collect liquid (2500 ~ 3000r/min).
(3) reaction setting: to obtain optimum, it is proposed that running each RNA standard items and three parts of sample.
(4) condition of pcr amplification reaction are as follows: 55 DEG C of reverse transcription 10min, 1 circulation；95 DEG C of initial denaturation 1min, 1 is followed
Ring；95 DEG C of denaturation 10s, it includes the read plate time that 60 DEG C of annealing, which extend 30s(), 40~45 circulations.
(5) availability deciding:
The Ct value that no RNase water detects is Undet or 40, and value≤35 Ct that positive quality control product detects, otherwise experiment view
It is invalid；
(6) result interpretation:
Pattern detection pipe Ct value is Undet or > 40, which is judged as negative, and sample rna extracts failure, sample to be tested
In without RNA or content lower than detection limit；
Pattern detection value≤35 pipe Ct and curve has apparent amplification region, the sample results are judged as that corresponding pathogen is positive,
Pattern detection success；
Pattern detection pipe Ct value is greater than 35 and to be less than or equal to 40 and curve when having apparent amplification region, need to recheck it is primary, if
Second of detection is then negative without Ct value, is positive if second of detection has Ct value；According to the above interpretation method, in conjunction with sample
Influenza A virus, influenza B virus and the parainfluenza virus of detection pipe detection correspond to the Ct value of fluorescence channel to judge three kinds
The testing result of pathogen.
SEQ ID NO.1:GACCRATCCTGTCACCTCTSR C；
SEQ ID NO.2:GRGCATTYT GGACAA AKCKTCTAC；
SEQ ID NO.3:FAM-TGCAGTCCTCGCTCACTGKGCACG-BHQ；
SEQ ID NO.4:CCTCA ACTCMCTCTTCGAGCG；
SEQ ID NO.5:CGGTGCTCTTGACCAMAT TGG；
SEQ ID NO.6:VIC-CCAATTCGAGCAGCTGAA ACTGCGK-BHQ；
SEQ ID NO.7:CGCAGCTAWCCGCTGC；
SEQ ID NO.8:AGTACCTGGCAGCWT；
SEQ ID NO.9:ROX-TAGCGTGTCCTATASAGGACT-BHQ.
Claims (9)
1. a kind of for detecting influenza A virus, influenza B virus and the kit of parainfluenza virus, which is characterized in that should
Kit by mixed reactant (2X) (including Taq thermal starting archaeal dna polymerase, UNG enzyme, TaqMan fluorescence probe, PCR buffer,
DATP, dUTP, dCTP, dGTP, MgCl2 etc.), (Luna startup temperature reverse transcriptase and mouse RNase inhibit enzymatic mixture (20X)
Agent), forward primer, reverse primer, positive quality control product and without RNase water form；It wherein, include A type stream in mixed reaction solution
Influenza Virus, the primer of influenza B virus and the specific and conserved sequence of parainfluenza virus and TaqMan fluorescence probe；
The primer sequence of the specific and conserved sequence are as follows:
Influenza A virus, SEQ ID NO.1 and SEQ ID NO.2；
Influenza B virus, SEQ ID NO.4 and SEQ ID NO.5；
Parainfluenza virus, SEQ ID NO.7 and SEQ ID NO.8；
The TaqMan probe sequence of the specific and conserved sequence are as follows:
Influenza A virus, SEQ ID NO.3；
Influenza B virus, SEQ ID NO.6；
Parainfluenza virus, SEQ ID NO.9.
2. according to claim 1 for detecting influenza A virus, influenza B virus and the reagent of parainfluenza virus
Box, which is characterized in that final concentration of 100~900nM of the primer described in the mixed reaction solution in amplification system；The probe
Final concentration of 100~500nM in amplification system, Taq thermal starting archaeal dna polymerase (1~2U/ μ L), Luna startup temperature reverse
Record the final concentration of 0.5U~5U of enzyme, UNG enzyme in amplification system.
3. according to claim 1 for detecting influenza A virus, influenza B virus and the reagent of parainfluenza virus
Box, which is characterized in that the Luna startup temperature reverse transcriptase is the thermal stability more higher than many other RT by designing,
Optimal reaction temperature can be made to reach 55 DEG C, for difficult target/template, up to 60 °C of higher RT step temperature can be used
Degree；The aptamers that Luna startup temperature reverse transcriptase and Taq thermal starting archaeal dna polymerase utilize one kind temperature sensitive, reversible are 45
DEG C or less inhibit its activity；Therefore, reaction can be established at room temperature and inhibits non-specific amplification.
4. according to claim 1 for detecting influenza A virus, influenza B virus and the reagent of parainfluenza virus
Box, which is characterized in that the mixed reactant is formulated by unique passive reference dye, can be flat with various qPCR instruments
Platform is compatible, and including the instrument for needing ROX to correct, additional dyestuff is no longer needed during experimental implementation.
5. according to claim 1 for detecting influenza A virus, influenza B virus and the reagent of parainfluenza virus
Box, which is characterized in that the mixed reactant contains dUTP, uses uracil dna glycosylase before qPCR/RT-qPCR
(UDG) pre-process, by excision uracil base with generate not amplifiable DNA product eliminate previously expanded containing uracil
Product；So the South Pole temperature-sensitive UDG of 0.025 unit/μ L NEB is added in the reactive mixture, may be implemented to prevent product
Residual contamination.
6. according to claim 1 for detecting influenza A virus, influenza B virus and the reagent of parainfluenza virus
Box, which is characterized in that contain influenza A virus, influenza B virus and the amplification of parainfluenza virus in the positive quality control product
The plasmid of gene order；It is according to claim 1 to be used to detect influenza A virus, influenza B virus and parainfluenza virus
The kit of poison, which is characterized in that using no RNase water as negative control.
7. according to claim 1-5 for detecting influenza A virus, influenza B virus and parainfluenza virus
The kit of poison, which is characterized in that the preparation method of the detection reagent: using standard desalination process purifying primer to meet use
In qPCR/RT-qPCR；The sensitivity for analysis needed is potentially contributed to using HPLC or PAGE purifying to improve.
8. a kind of application method using kit described in any one of claims 1-6, method includes the following steps:
(1) before use:
1. extracting using required RNA and purification process preparing target RNA, concentration is determined by OD260 absorbance；2. diluting
RNA is used for standard curve, these should test preceding fresh preparation each, and can dilute in water or TE；
(2) specific steps:
1. defrosting kit reaction mixture and other reactive components at room temperature, are subsequently placed on ice；After thawing completely, pass through
It is inverted, liquid relief or mild be vortexed simply mix each component, for 96 orifice plates, it is proposed that end reaction volume is 20 μ L；2. determining
The total volume of right quantity reaction, is added 10% excess volume, and correspondingly prepare the measurement of all components in addition to RNA template
Mixture by liquid relief or vortex but is softly thoroughly mixed, liquid is collected into bottom of the tube by being simply centrifuged；3. by equal point of examination
Sample is mixed into qPCR pipe or plate, to obtain optimum efficiency, please be ensured precisely consistent liquid relief amount and be reduced bubble to the greatest extent；4. will
RNA template is added in qPCR pipe or plate, and seal pipe has optically transparent flat cover;With optical transparent film sealing plate, answer
Correct sealing plate edge and corner are paid attention to, to prevent artifact caused by evaporation；5. of short duration rotation pipe or plate are to remove bubble and receive
Collect liquid (2500 ~ 3000r/min)；
(3) reaction setting: to obtain optimum, it is proposed that running each RNA standard items and three parts of sample；
(4) condition of pcr amplification reaction are as follows: 55 DEG C of reverse transcription 10min, 1 circulation；95 DEG C of initial denaturation 1min, 1 circulation；95
DEG C denaturation 10s, it includes the read plate time that 60 DEG C of annealing, which extend 30s(), 40~45 recycle；
(5) availability deciding: the Ct value that no RNase water detects is Undet or > 40, and the Ct value that positive quality control product detects
≤ 35, otherwise experiment is considered as invalid；
(6) result interpretation: pattern detection pipe Ct value is Undet or 40, which is judged as negative, and sample rna, which extracts, to be lost
It loses, be lower than in sample to be tested without RNA or content and detect limit；Pattern detection value≤35 pipe Ct and curve has apparent amplification region,
The sample results are judged as that corresponding pathogen is positive, pattern detection success；Pattern detection pipe Ct value be greater than 35 and be less than etc.
In 40 and curve have apparent amplification region when, need to recheck primary, be negative if second of detection is without Ct value, if second is examined
It is positive that survey, which has Ct value then,；According to the above interpretation method, the influenza A virus detected in conjunction with pattern detection pipe, influenza B disease
Poison corresponds to the Ct value of fluorescence channel with parainfluenza virus to judge the testing result of three kinds of pathogen.
9. the application method of kit according to claim 7 characterized by comprising sample to be detected is provided, it is described
Sample to be detected is the tissues and the throat swab of patient etc. such as cloacal swab, tracheae, lung, spleen or the lymph of chicken.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811402860.2A CN109957624A (en) | 2018-11-23 | 2018-11-23 | PCR kit that is a kind of while detecting first, influenza B, parainfluenza |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811402860.2A CN109957624A (en) | 2018-11-23 | 2018-11-23 | PCR kit that is a kind of while detecting first, influenza B, parainfluenza |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342503A (en) * | 2014-10-29 | 2015-02-11 | 福建国际旅行卫生保健中心 | Method for simultaneously detecting twelve kinds of common respiratory viruses |
| CN107365876A (en) * | 2017-08-07 | 2017-11-21 | 南京岚煜生物科技有限公司 | For detecting the kit and its application method of 10 respiratory tract infection pathogen |
| CN107937613A (en) * | 2017-12-21 | 2018-04-20 | 北京卓诚惠生生物科技股份有限公司 | Respiratory system common seven kinds of influenza virus pathogen real-time fluorescence multiple PCR primer probes and kit |
-
2018
- 2018-11-23 CN CN201811402860.2A patent/CN109957624A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342503A (en) * | 2014-10-29 | 2015-02-11 | 福建国际旅行卫生保健中心 | Method for simultaneously detecting twelve kinds of common respiratory viruses |
| CN107365876A (en) * | 2017-08-07 | 2017-11-21 | 南京岚煜生物科技有限公司 | For detecting the kit and its application method of 10 respiratory tract infection pathogen |
| CN107937613A (en) * | 2017-12-21 | 2018-04-20 | 北京卓诚惠生生物科技股份有限公司 | Respiratory system common seven kinds of influenza virus pathogen real-time fluorescence multiple PCR primer probes and kit |
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Application publication date: 20190702 |