CN108504775A - A kind of detection A type, the kit of influenza B virus and its application method based on micro-fluidic chip - Google Patents
A kind of detection A type, the kit of influenza B virus and its application method based on micro-fluidic chip Download PDFInfo
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- CN108504775A CN108504775A CN201810209134.2A CN201810209134A CN108504775A CN 108504775 A CN108504775 A CN 108504775A CN 201810209134 A CN201810209134 A CN 201810209134A CN 108504775 A CN108504775 A CN 108504775A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The present invention relates to molecular biology fields, more particularly to a kind of kit and its application method of detection A type/influenza B virus based on micro-fluidic chip, including the micro-fluidic nucleic acid detection chip of more flux of active control flow path, prepackage dry powder detection reagent, positive quality control product；It is added in the well of micro-fluidic nucleic acid detection chip in use, sample DNA/RNA is extracted template altogether, covers sample-adding port lid, be put into micro-fluidic chip detector and detect；It carries out PCR amplification and carries out result judgement.The present invention has the advantage that compared with common fluorescent PCR methods, immunization and bacterial cultivation etc.：High specificity：The primed probe of the present invention is designed for specific conservative's regional sequence of A type/influenza B virus, high specificity；Sensibility is high：The present invention can detect the objective gene sequence of 10copies/ μ L concentration；It is easy to preserve：The present invention's pre-installs powdered reagent in micro-fluidic chip, and it is convenient to preserve.
Description
Technical field
The present invention relates to molecular biology fields, and in particular to a kind of detection A type based on micro-fluidic chip, B-mode stream
The kit and its application method of Influenza Virus.
Background technology
Influenza virus (influenza virus, Flu), abbreviation influenza virus are divided into first (A), second (B), third
(C) three type, the bovine influenza virus just found in recent years will be classified as fourth (D) type.Influenza virus can cause people, fowl, pig, horse, bat
Equal many animals infection and morbidity, are the cause of diseases of human influenza, bird flu, swine flu, equine influenza et al. and animal epidemic.Human influenza
Mainly caused by influenza A virus and influenza B virus, mainly cause the infection of the upper respiratory tract, can also cause children with
The severe influenza of the lower respiratory tract infection of adult, predominantly pneumonia, infant is often accompanied by bronchitis, high fever, once in a while with brain
The complication such as inflammation, tympanitis, myocarditis.Influenza virus is mainly propagated through airborne droplet, and influenza A virus is often with popular form
Occur, pandemics can be caused；Influenza B virus often results in local eruption and prevalence.It can be quick using molecular diagnostic techniques
Detection influenza virus has clinical treatment certain directive significance to carry out auxiliary diagnosis.
In main the first and second the third influenza virus, A type is easiest to cause prevalence, B-mode to take second place, and the third type seldom causes prevalence.
Influenza A virus (Flu A) infection morbidity degree is immune related to individual, and classical symptom is with chilly, lasting high fever, headache
It is main, while with laryngalgia, cough, nasal obstruction, generally having the constitutional symptoms such as ache all over, is weak.Incidence height, case fatality rate bottom,
Death is usually caused by concurrent bacterial sexuality contaminates.1~7 day incubation period, Flu-A often cause to be very popular, per there are one high every year
Peak, Virus type show as alternately popular rule.Document report epizootic modeling positive rate is 20%~40%, and in non-streaming
The positive rate in row season is 2%~20%.
Influenza B virus (FluB) infection onset is rapid, and patient's chilly, fever, body temperature rise within a few hours to 24 hours
Up to peak, 39~40 DEG C even higher.With headache, Muscular stiffness is weak, anorexia.Respiratory symptom is lighter, dry throat larynx
Bitterly, dry cough can have diarrhea.Blushing, eye conjunctiva outer canthus is congested, and congestion of throat, soft palate has folliculus etc..3~7 days incubation periods,
B virus often causes limitation popular, and in flu victims, influenza B virus infection accounts for about 10%~40%.Document report stream
Positive rate is 10%~30% between the departure date, in non-epidemic season positive rate 2%~10%.
Micro-fluidic chip (microfluidics) or chip lab (Lab-on-a-Chip) refer to biology and changing
It sample preparation involved in fields, biology and the chemical reaction such as learns, the basic operation units such as separation, detection or is integrated into substantially
On the chip of one piece several square centimeters (or even smaller), to complete different biological or chemical reaction process, and to its product
A kind of technology analyzed.It is suitable in this engineering philosophy various until organic and inorganic small molecule from nucleic acid, protein
Reaction, separation and the detection of different type molecule.Micro-fluidic chip have liquid flowing controllable, consumption sample and reagent it is few,
The features such as improving to analyze speed tenfold hundreds of times, it can carry out a samples up to a hundred within a few minutes or even shorter time
While analyze, and can with the pretreatment of canbe used on line sample and analysis overall process.
Being directed to influenza A virus and the detection method of influenza B virus currently on the market mainly has fluorescent PCR method, exempts from
Epidemic disease method and bacterial cultivation.Fluorescent PCR method, by fluorescence signal, is supervised in real time to PCR processes during PCR amplification
It surveys, qualitative or quantitative testing goal gene；Immunization is that specific binding occurs come testing goal albumen by antigen-antibody；Training
Foster method is pathogen to be cultivated on defined medium, then carry out observation analysis to the result of generation.
Being directed to influenza A virus and the fluorescent quantificationally PCR detecting kit of influenza B virus currently on the market has very
It is more, it is detected for some most commonly seen hypotype design primer probes of influenza A virus and influenza B virus.Its group
At salt ion buffer solution, enzyme, primer, probe, quality-control product is generally comprised, it is in charge of is stored at -20 DEG C in liquid form, when use
It needs to melt multitube reagent, be mixed according to a certain percentage, be configured to detection reaction solution, sample nucleic acid is then added,
It is put into fluorescence quantitative PCR instrument and is detected, testing result is finally analyzed according to amplification curve.
Immunization detection reagent is directed to the specific antibody that pathogen in sample causes body to generate, the detection of antibody
Have window phase, be easy to fail to pinpoint a disease in diagnosis at the initial stage of a disease, and sensitivity, specificity it is relatively low, sample is easy to pollute and interfering substance is more, concentration
It all be easy to cause result misjudgement when excessively high or too low, needs repetition detection, check that can just make a definite diagnosis, the confidence level of detection is poor；
It is taken generally at one day or more using cultivation detection, culture effect is poor, and many pathogen can not cultivate, when being delayed optimal treatment
Machine.
Although there are common fluorescence PCR detection reagent preferable sensitivity and specificity, kit to be generally more
Pipe liquid reagent is constituted, and usually needs to be stored in -20 DEG C of environment, needing proportionally to mix each pipe reagent in operation makes
With, there is higher requirement for preserving traffic condition, operating method etc., it is easy to cause to examine because preserving improper and operation error
It is insincere to survey result.Simultaneously because there are many subtype category of influenza A virus and influenza B virus, detection kit is general
It is directed to influenza A virus and the most commonly seen hypotype of influenza B virus, it, may sometimes because of the variation of virus stain
The case where will produce missing inspection.Result after being detected due to fluorescence quantitative PCR instrument needs professional to analyze amplification curve, has
When have human error generation.Finally, because the lifting speed of fluorescence quantitative PCR instrument is slower, detection time is generally 1~2
A hour takes longer.
Invention content
In order to solve drawbacks described above in the prior art, the present invention provides a kind of to influenza A virus and influenza B disease
Poison is quick and precisely detected, and can in various environment the easy-to-use detection A type based on the micro-fluidic chip/B-mode stream of simplicity
The kit and its application method of Influenza Virus, it is ensured that timeliness, specificity and the sensitivity of detection.
The present invention draws for design specificity on influenza A virus and the respective conserved sequence M genes of influenza B virus
Physical prospecting needle can detect known each subtype influenza virus, and mark different fluorescence signals on probe, can simultaneously detect simultaneously
Distinguish influenza A virus and influenza B virus.In order to solve the problems, such as Cord blood and use cumbersome, the present invention
Kit in detection reagent to be dry powder can preserve in advance in the micro-fluidic chip under 4 DEG C of low temperature or room temperature, use
When only need the sample of nucleic acid that has extracted is added and can go up machine testing.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of kit of detection A type/influenza B virus based on micro-fluidic chip, including active control flow path
The micro-fluidic nucleic acid detection chip of more flux, prepackage dry powder detection reagent, positive quality control product；It is with micro-fluidic runner, including one
Item goes out sample sprue and several decimated streams roads, each decimated streams road split settings, and each decimated streams road corresponds to a reaction chamber,
The reagent of the energy each reaction chamber of decile.Due to being expanded using micro-fluidic chip, the specific surface of reaction intracavity liquid is increased
Product, and heat transfer is faster, reagent can be made rapidly to carry out heating and cooling, and can control the close of reaction solution by instrument and chip cooperation
Closing property avoids leaking chemical pollution.After the adding mouth of detection chip is added in sample, it is introduced into out sample sprue, then flow into each point of sample
Runner.
In the micro-fluidic nucleic acid detection chip in different reaction chambers respectively be preinstalled with detection influenza A virus and
The detection reagent dry powder of influenza B virus；
The ingredient of the detection reagent includes：PCR buffer solutions, trehalose, bovine serum albumin(BSA), dATP, dUTP, dCTP,
DGTP, MgCl2, HotStart Taq enzymes, reverse transcriptase, UNG enzymes, influenza A virus (Influenza A virus) and second
The primer and Taqman fluorescence probe of the specific and conserved sequence of type influenza virus (Influenza B virus)；
Dry powder is made by drying process after the detection reagent mixing；It is made again after all reagent components are mixed
Dry powder, and use special dry powder drying process：
By prepared detection reagent solution, 8h or more is frozen under the conditions of being put into -80 DEG C；
The detection reagent frozen is put into vacuum freeze drier, drying process is:
- 50 DEG C, 1h, 1 atmospheric pressure；
- 40 DEG C, 5h, ＜ 10Pa；
- 10 DEG C, 2h, ＜ 10Pa；
0 DEG C, 2h, ＜ 10Pa；
30 DEG C, 5h, ＜ 10Pa；
After the completion of drying, powdered reagent is obtained；Powdered reagent is divided in different in micro-fluidic nucleic acid detection chip
In reaction chamber.Dry powder drying process can keep the activity of dry powder formulation as far as possible, improve drying efficiency, while after reagent mixing again
Dry powder is made to facilitate subsequent detection to operate.The micro-fluidic runner PCR detection kit of this dry powder, in the prior art
Without corresponding reagent kit product.
The specific primer sequence is：Influenza A virus (Influenza A virus), NO.1~2 SEQ ID；
Influenza B virus (Influenza B virus), NO.4~5 SEQ ID；
The specificity T aqman probe sequences are：Influenza A virus (Influenza A virus), SEQ ID
NO.3；Influenza B virus (Influenza B virus), SEQ ID NO.6；Each primer sequence such as the following table 1.
1 primer sequence of table
Final concentration of 100~1000nM of the primer in amplification system；End of the probe in amplification system is dense
Degree is 50~500nM；
The trehalose in amplification system final concentration of 1%~10%；The bovine serum albumin(BSA) is in amplification system
In final concentration of 0.1%~5%；Trehalose and bovine serum albumin(BSA), which belong to, additionally to be added, and can improve amplification efficiency, is protected
Hold the stability of reagent.
Final concentration of 0.5U~the 5U of the HotStart Taq enzymes, reverse transcriptase, UNG enzymes in amplification system；
Final concentration of 0.1mM~the 2mM of described dATP, dUTP, dCTP, dGTP in amplification system；
The MgCl2Final concentration of 1.5mM~10mM in amplification system；
The plasmid of amplification gene sequence containing A type/influenza B virus in the positive quality control product；
Further, the present invention also provides the application method based on aforementioned fluorescent quantitative PCR detection kit, this method
Include the following steps：
(1) sample DNA/RNA 200 μ L of template (from extracting in the sputum, Nasopharyngeal swabs equal samples of people) are extracted altogether to be added
In the well of micro-fluidic nucleic acid detection chip, sample-adding port lid is covered, is put into micro-fluidic chip detector and detects.
(2) condition of pcr amplification reaction is：50 DEG C of 5~30min of reverse transcription；92~97 DEG C of 1~10min of pre-degeneration；92~
97 DEG C of 5~10s of denaturation；58~62 DEG C of 15~30s of annealing；30~45 cycles.
(3) availability deciding：
Negative control hole is the blank well for not adding primed probe, and testing result should be negative, and Positive control wells detect
For the positive, otherwise experiment is considered as invalid；
(4) result interpretation：
Instrument is provided according to the detection signal of corresponding reaction chamber as a result, directly judging influenza A virus and influenza B
The yin and yang attribute result of virus.
Detection pipe agent prescription is by optimizing and revising in the kit of the present invention, handles as dry powder, can be low at 4 DEG C
Is preserved 1 year or more under temperature or room temperature, does not influence detection result, when use, which only needs to be added sample, can go up machine testing, it is convenient easily
With.
This kit using microfluidic chip technology, after sample-adding all operations all completed by instrument, easy to operate, speed
Degree is fast, detection can be completed in 30~60min, and will not pollute.
The kit of the present invention uses Taqman fluorescence probe round pcrs, is examined for A type/influenza B virus
It surveys, the sequence of design primer and probe is all very conservative in the gene of influenza A virus and influenza B virus, Ke Yijian
Known various hypotypes, high specificity are measured, and testing result can be obtained in 2 hours, sensitivity is up to 100copies/ μ
L。
Present invention employs UNG enzymes/anti-pollution systems of Dutp, can reduce the pollution interference that previous PCR reaction product is brought.
Reverse transcriptase and HotStart Taq enzymes are used in mixed way by the present invention, are first carried out process of reverse-transcription and are synthesized cDNA,
PCR amplification is carried out again, does not add random primer, and one step of process is completed, and without uncapping, can reduce laboratory operating procedures, is saved
It saves time, while also avoiding the sample contamination that operation of uncapping may be brought.
Probe used in the present invention is the TaqMan probe of 5 ' end FAM labels, and mark fluorescent report is distinguished at probe both ends
Acid of the few core former times 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 is sent out is quenched group absorptions.In fluorescent PCR amplification procedure, when special PCR product with
5 ' end 5 prime excision enzyme activities of HotStart Taq enzymes when hybridization reaction occur for TaqMan probe simultaneously also probe cleavage, report base
The fluorimeter that the released fluorescence of group can be built in instrument detects.PCR often passes through a cycle,
Fluorescence signal is also as target fragment, and there are one the process that sync index increases, the power of fluorescence signal just represents template
The number of the copy number of RNA.Therefore the present invention cannot be only used for simple qualitative detection, also can be used as determining for sample concrete content
Amount detection.
In conclusion the present invention has the advantage that compared with common fluorescent PCR methods, immunization and bacterial cultivation etc.：
1. high specificity：The primed probe of the present invention is directed to specific conservative's regional sequence of A type/influenza B virus
Design, high specificity；Corresponding influenza A virus or influenza B virus can be detected by respectively devising a set of primed probe
All hypotypes.
2. sensibility is high：The present invention can detect the objective gene sequence of 10copies/ μ L concentration.
3. being easy to preserve：The present invention's pre-installs powdered reagent in micro-fluidic chip, and it is convenient to preserve.
4. parting detects：A type/influenza B virus is distinguished in a chip.
5. pollution-free：Detection process is " locked in " operation, greatly reduces the possibility of pollution and result error.
6. easy to operate quick：Without preparing detection reagent, it is only necessary to which sample-adding is primary, you can upper machine testing, from specimen transfer
It can be completed within 2 hours to obtaining a result.
7. result interpretation is clear, objective；If desired can also quantitative analysis be carried out to result.
8. safety：Do not include poisonous and harmful substance in whole system, the post-processing of PCR product is not necessarily to, to operator and ring
Border is all non-hazardous.
Specific implementation mode
The present invention is further described with reference to embodiment.
Embodiment 1
It is different that the detection reagent of detection influenza A virus and influenza B virus is preloaded onto micro-fluidic nucleic acid detection chip
Reaction chamber in；
The ingredient of the detection reagent includes：PCR buffer solutions, trehalose, bovine serum albumin(BSA), dATP, dUTP, dCTP,
DGTP, MgCl2, HotStart Taq enzymes, reverse transcriptase, UNG enzymes, influenza A virus (Influenza A virus) and second
The primer and Taqman fluorescence probe of the specific and conserved sequence of type influenza virus (Influenza B virus)；
Dry powder is made by drying process in the detection reagent, can be pre-installed in micro-fluidic chip；
Final concentration of each primer in amplification system is respectively preferably 100nM；Each probe is in amplification system
In final concentration be preferably 50nM；
Final concentration of 1% (mass percent) of the trehalose in amplification system；
Final concentration of the bovine serum albumin(BSA) in amplification system is preferably 0.2% (mass percent)；
The HotStart Taq enzymes, the final concentration of reverse transcriptase, UNG enzymes in amplification system are respectively 1U；
The final concentration of described dATP, dUTP, dCTP, dGTP in amplification system is respectively 0.5mM；
The MgCl2Final concentration of 5.5mM in amplification system；
The plasmid of amplification gene sequence containing A type/influenza B virus in the positive quality control product；
Two kinds of reaction systems are respectively：1) PCR buffer solutions, trehalose, bovine serum albumin(BSA), dATP, dUTP, dCTP,
DGTP, MgCl2, HotStart Taq enzymes, reverse transcriptase, UNG enzymes, influenza A virus (Influenza A virus) spy
The primer and influenza A virus Taqman fluorescence probe of anisotropic conserved sequence；
2) PCR buffer solutions, trehalose, bovine serum albumin(BSA), dATP, dUTP, dCTP, dGTP, MgCl2、HotStart
Taq enzyme, the specific and conserved sequence of reverse transcriptase, UNG enzymes, influenza B virus (Influenza A virus) primer and
Influenza B virus Taqman fluorescence probe.
1) each component for the reaction systems for detecting influenza A virus and influenza B virus respectively with 2) two kinds is mixed
The dry powder being fabricated to afterwards is separately positioned in two different reaction chambers (embodiment 1), can also be by influenza A virus and second
The Taqman fluorescence probe and PCR buffer solutions, trehalose, ox of type influenza virus specific conservative primer and two kinds of influenza viruses
Seralbumin, dATP, dUTP, dCTP, dGTP, MgCl2, after HotStart Taq enzymes, reverse transcriptase, UNG enzymes be mixed together
It is fabricated to dry powder to be arranged in the same reaction chamber, without any component in runner, PCR buffer solutions are the salt of the components such as KCl, Tris
Solution.
The operation of kit and result judgement：
(1) sample DNA/RNA 200 μ L of template (from extracting in the sputum, Nasopharyngeal swabs equal samples of people) are extracted altogether to be added
In the well of micro-fluidic nucleic acid detection chip, sample-adding port lid is covered, is put into micro-fluidic chip detector and detects.
(2) condition of pcr amplification reaction is：
50 DEG C of reverse transcription 30min；
95 DEG C of pre-degeneration 2min；
95 DEG C of denaturation 10s, 58 DEG C of annealing extend 30s, 40 cycles.
(3) availability deciding：
Negative control hole is the blank well for not adding primed probe, and testing result should be negative, and Positive control wells detect
For the positive, otherwise experiment is considered as invalid；
(4) result interpretation：
The data of No. 1 reaction chamber correspond to influenza A virus, and the data of No. 2 reaction chambers correspond to influenza B virus, instrument
The fluorescence intensity level of corresponding reaction chamber can be carried out data processing by software, directly judge influenza A virus and influenza B disease
The yin and yang attribute result of poison.
Embodiment 2
4 samples are detected with micro-fluidic chip, number 1~4 contains influenza nucleic acids.No. 1 sample contains Flu-A
Viral nucleic acid and influenza B virus nucleic acid, No. 2 samples contain influenza A nucleic acid, and No. 3 samples contain influenza B disease
Malicious nucleic acid, No. 4 samples only have the nucleic acid of normal person, the nucleic acid without A type or influenza B virus.It is identical according to embodiment 1
Method is detected operation, and testing result is shown in Table 2.
2 testing result of table
Testing result shows that this kit is used in micro-fluidic chip detection and can accurately detect and distinguish Flu-A
Virus and influenza B virus infection.
Embodiment 3
Five gradient samples of influenza A virus and influenza B virus mixing plasmid, five samples are detected with this kit
Concentration both in this is respectively 105、104、103、102With 10copies/ μ L.It is examined in the same manner as shown in Example 1
Survey (wherein, influenza A virus and influenza B virus specific conservative primer and the spy of the Taqman fluorescence of two kinds of influenza viruses
Needle and PCR buffer solutions, trehalose, bovine serum albumin(BSA), dATP, dUTP, dCTP, dGTP, MgCl2, HotStart Taq enzymes,
Reverse transcriptase, UNG enzymes are fabricated to dry powder and are arranged in the same reaction chamber after being mixed together).
Testing result show kit of the present invention can in a reaction chamber and meanwhile detect two kinds of pathogen, two fluorescence
Signal path is not interfere with each other；And it can detect that concentration is respectively two kinds of pathogen of 10copies/ μ L.Sample 1~5 is respectively
The two mixes plasmid gradient sample, and concentration is respectively 105、104、103、102With 10copies/ μ L.Mixing plasmid sample all detects
Two amplification curves, respectively two channels, and Ct values all ＜ 35, show that result is effective；Wherein 5 each plasmid concentration of sample is
10copies/ μ L show that two channels may detect that the pathogen of 10copies/ μ L.
Test result shows that this kit can detect influenza A virus, influenza B virus, and the two concentration simultaneously
Still to may detect that when 10copies/ μ L.
Embodiment 4
Detected respectively with this kit 5 kinds of hypotypes such as Influenza virus H1N1, H1N2, H3N2, H5N1 and H9N2 and
Influenza B virus sample.It is detected in the same manner as shown in Example 1.
Testing result shows that kit of the present invention can detect the above A type and each hypotype of influenza B virus.Test result
The primed probe high specificity for showing conservative region design of this kit to A type/influenza B virus, is capable of detecting when each
Popular hypotype.
The above is only a preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
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Claims (7)
1. a kind of kit of detection A type/influenza B virus based on micro-fluidic chip, which is characterized in that controlled including active
The micro-fluidic nucleic acid detection chip of more flux of flow path processed, prepackage dry powder detection reagent, positive quality control product；
Detection influenza A virus and B-mode stream are respectively provided in the micro-fluidic nucleic acid detection chip in different reaction chambers
The prepackage dry powder detection reagent of Influenza Virus；
Prepackage dry powder detection reagent ingredient include：PCR buffer solutions, trehalose, bovine serum albumin(BSA), dATP, dUTP, dCTP,
DGTP, MgCl2, HotStart Taq enzymes, reverse transcriptase, UNG enzymes, influenza A virus (Influenza A virus) and second
The primer and specificity T aqman fluorescence probes of the specific and conserved sequence of type influenza virus (Influenza B virus)；
The primer sequence of the specific and conserved sequence is：Influenza A virus (Influenza A virus), SEQ ID
NO.1~2；Influenza B virus (Influenza B virus), NO.4~5 SEQ ID；
The sequence of the specificity T aqman probes is：Influenza A virus (Influenza A virus), SEQ ID NO.3；
Influenza B virus (Influenza B virus), SEQ ID NO.6；
The plasmid of amplification gene sequence containing A type/influenza B virus in the positive quality control product.
2. a kind of kit of detection A type/influenza B virus based on micro-fluidic chip according to claim 1,
It is characterized in that, primer probe sequence is specially：
3. a kind of kit of detection A type/influenza B virus based on micro-fluidic chip according to claim 1,
It is characterized in that, final concentration of 100~1000nM of the primer in amplification system；End of the probe in amplification system is dense
Degree is 50~500nM；The trehalose in amplification system final concentration of 1%~10%；The bovine serum albumin(BSA) is expanding
Final concentration of 0.1%~5% in increasing system；The HotStart Taq enzymes, reverse transcriptase, UNG enzymes are in amplification system
Final concentration of 0.5U~5U；Final concentration of 0.1mM~the 2mM of described dATP, dUTP, dCTP, dGTP in amplification system；It is described
MgCl2Final concentration of 1.5mM~10mM in amplification system.
4. a kind of kit of detection A type/influenza B virus based on micro-fluidic chip according to claim 1,
It is characterized in that, dry powder is made by drying process in detection reagent, that is, pre-installs dry powder detection reagent；Dry powder preparation process is：
By prepared detection reagent solution, 8h or more is frozen under the conditions of being put into -80 DEG C；
The detection reagent frozen is put into vacuum freeze drier, drying process is:
- 50 DEG C, 1h, 1 atmospheric pressure；
- 40 DEG C, 5h, ＜ 10Pa；
- 10 DEG C, 2h, ＜ 10Pa；
0 DEG C, 2h, ＜ 10Pa；
30 DEG C, 5h, ＜ 10Pa；
After the completion of drying, powdered reagent is obtained；Different reactions powdered reagent being divided in micro-fluidic nucleic acid detection chip
Intracavitary.
5. a kind of kit of detection A type/influenza B virus based on micro-fluidic chip according to claim 1,
It is characterized in that, micro-fluidic nucleic acid detection chip has micro-fluidic runner, including one goes out sample sprue and several decimated streams
Road, each decimated streams road split settings, each decimated streams road correspond to a reaction chamber, the reagent decile of each reaction chamber.
6. a kind of kit of detection A type/influenza B virus based on micro-fluidic chip according to claim 1,
Be characterized in that, the probe be 5 ' end FAM label TaqMan probes, probe both ends distinguish mark fluorescent reporter group R and
The oligonucleotides of fluorescent quenching group Q, fluorescent reporter group R are located at 5 ' ends, and fluorescent quenching group Q is located at 3 ' ends；Two special
Property Taqman probes 5 ' hold using different fluorescent reporter group R label modifications.
7. a kind of use of the kit of detection A type/influenza B virus based on micro-fluidic chip described in claim 1
Method, which is characterized in that this approach includes the following steps：
(1) sample DNA/RNA is extracted 200 μ L of template altogether to be added in the well of micro-fluidic nucleic acid detection chip, covers sample-adding
Port lid is put into micro-fluidic chip detector and detects；
(2) condition of pcr amplification reaction is：50 DEG C of 5~30min of reverse transcription；
92~97 DEG C of 1~10min of pre-degeneration；
92~97 DEG C of 5~10s of denaturation；58~62 DEG C of 15~30s of annealing；30~45 cycles；
(3) availability deciding：
Negative control hole is the blank well for not adding primed probe, and testing result should be negative, and Positive control wells testing result is answered
For the positive, otherwise experimental result is considered as in vain；
(4) result interpretation：
Instrument directly judges the yin and yang attribute of influenza A virus and influenza B virus according to the detection signal of corresponding reaction chamber
As a result.
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