CN106086236A - Detect test kit and the method for influenza virus H1, H3, H5, H7 simultaneously - Google Patents
Detect test kit and the method for influenza virus H1, H3, H5, H7 simultaneously Download PDFInfo
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Abstract
The invention discloses test kit and the method using quantitative fluorescent PCR simultaneously to detect tetra-kinds of hypotypes of influenza virus H1, H3, H5 and H7, test kit includes specific primer and the probe of corresponding each influenza virus sub-strain.The test kit that the present invention provides is easy to use, and reagent used is few, the high specificity of detection, highly sensitive；Detection method achieves same sample, can carry out the detection of 4 kinds of influenza virus typings, enormously simplify operating process, decreases the process of repetitive operation, time-consuming, alleviates the labour force that repetitive operation consumes, and effectively save cost, it is achieved rapid screening.
Description
Technical field
The invention belongs to field of biological technology detection, be specifically related to detect the reagent of influenza virus H1, H3, H5, H7 simultaneously
Box and method.
Background technology
Influenza virus (influenza virus), is the representative of orthomyxoviridae family (Orthomyxoviridae)
Kind, it is called for short influenza virus, including human influenza virus and animal influenza virus, human influenza virus is divided into first (A), second (B), third (C)
Three types, are the pathogen of influenza (influenza).Influenza is called for short influenza, is primarily referred to as in crowd, by common influenza
The viral Acute respiratory infectious disease of one that virus causes.Influenza infection is strong, infects rapidly, can cause annual popular,
And flu outbreak can be caused every decades in history, make a lot of people ill even dead at short notice.To influenza epidemic disease
The approach that the prevention and control of feelings are the most basic is exactly early discovery, early diagnosis, early treatment and the cut-out source of infection.As can be seen here, rapid sensitive
Influenza virus detection technique becomes the first barrier that protection people's life is healthy, prevention and control epidemic situation is the most popular.
Existing Fast Detection Technique mainly has two classes: the first kind is immunological method, including enzyme linked immunosorbent assay, exempts from
Epidemic disease fluorescence method, isotope marked antibodies method, latex agglutination, immunosensor method, immunodiffusion method and immunochromatography etc.；The
Two classes are the molecular biology methods based on nucleic acid, mainly have nucleic probe method and polymerase chain reaction method
(Polymerase Chain Reaction, PCR).Immunological method is to combine for detecting with antigen-antibody, and detection needs
Preparing specific antigen or antibody, preparation process is complicated, and needs the antigen keeping using or antibody to have immunogenic activity,
The sensitivity of this detection is relatively low, false negative easily occurs.
PCR method is the Typical Representative of nucleic acid amplification technologies, and this technology is to be ground by PE-Cetus company of U.S. human inheritance
Mullin studying carefully room et al. was in invention in 1985, and over the last couple of decades, round pcr obtains as the core of molecular biology
Having arrived swift and violent development and application, particularly in the Clinical detection virus of disease, round pcr is quick with it, sensitive and special
Advantage, the most day by day instead of traditional detection method, and make the detection that in the past cannot complete become possibility.
Multiplex PCR, refers to that addition two, to above primer, amplifies multiple core simultaneously in same PCR reaction system
The PCR reaction of acid fragment, has had the advantage that high efficiency, systematicness, economical and convenient.But successfully it is critical only that because of PCR and set
Counting out rational primer, suitable primer is to determine PCR specific amplification and the key of sensitivity.And multiplex PCR is to primer
Requiring more strict, each pair of primer such as multiplex PCR must have close annealing temperature so that all of target site can be used
Identical PCR program is effectively expanded in single reaction；And stable dimer and hair clip knot between it, can not be formed
Structure.Ensure that simultaneously amplified production be sized to separated by electrophoresis.
Existing general detection 2 weight, 3 weight Pathogen test are more, such as existing patent publication No. CN101895531B, entitled inspection
Surveying influenza virus A hominis H1 and/or the primer of H3 hypotype, describing the sequence between its primer chosen in theory can be very
Distinguish well H1, H2, H3, H5, H7 and H9 (300 sequences chosen, in the most correct hypotype assigning to this point), but real
In the middle of border, owing to relating to the condition of multi-primers design, cross reaction between primer easily occurs, is easily caused false-negative result,
Or cannot be distinguished by out this several typings, currently also it is only capable of detecting two kinds of typings of H1, H3.
There is presently no the detection method carrying out multiplex PCR for tetra-kinds of typings of influenza virus H1, H3, H5, H7.Because of multiple
PCR is harsh to the requirement of design of primers, and the tuple of detection is the most, and the condition of restriction is the most, will reach highly sensitive, high specificity
Multiple detection is also accomplished by paying the most, not only needs to design in early stage design of primers or probe to need manual analysis, experience to sentence
Disconnected, in addition it is also necessary to thousands of times and the experiment screening of up to ten thousand times, checking testing result.
Summary of the invention
For solving above-mentioned technical problem, the invention provides one group for detect simultaneously influenza virus influenza H1, H3, H5,
The nucleic acid of tetra-kinds of hypotypes of H7.The present invention also provide for one group detect for quantitative fluorescent PCR simultaneously influenza virus influenza H1, H3, H5,
The test kit of tetra-kinds of hypotypes of H7 and detection method thereof.
For achieving the above object, the present invention is by the following technical solutions:
One group of nucleic acid simultaneously detecting influenza virus H1, H3, H5, H7 hypotype for multiplex PCR, including influenza virus H1's
Upstream and downstream primer, the upstream and downstream primer of influenza virus H3, the upstream and downstream primer of influenza virus H5 and influenza virus H7 upper,
Downstream primer；
Wherein, the forward primer sequence of described influenza virus H1 as shown in SEQ ID No.1, downstream primer sequence such as SEQ
Shown in ID No.2；
The forward primer sequence of described influenza virus H3 as shown in SEQ ID No.4, downstream primer sequence such as SEQ ID
Shown in No.5；
The forward primer sequence of described influenza virus H5 as shown in SEQ ID No.7, downstream primer sequence such as SEQ ID
Shown in No.8；
The forward primer sequence of described influenza virus H7 as shown in SEQ ID No.10, downstream primer sequence such as SEQ ID
Shown in No.11.
One group of nucleic acid simultaneously detecting influenza virus H1, H3, H5, H7 hypotype for quantitative fluorescent PCR, including influenza virus
The upstream and downstream primer of H1 and probe, the upstream and downstream primer of influenza virus H3 and probe, the upstream and downstream primer of influenza virus H5 and
The upstream and downstream primer of probe and influenza virus H7 and probe；
The forward primer sequence of described influenza virus H1 as shown in SEQ ID No.1, downstream primer sequence such as SEQ ID
Shown in No.2, probe sequence is as shown in SEQ ID No.3；
The forward primer sequence of described influenza virus H3 as shown in SEQ ID No.4, downstream primer sequence such as SEQ ID
Shown in No.5, probe sequence is as shown in SEQ ID No.6；
The forward primer sequence of described influenza virus H5 as shown in SEQ ID No.7, downstream primer sequence such as SEQ ID
Shown in No.8, probe sequence is as shown in SEQ ID No.9；
The forward primer sequence of described influenza virus H7 as shown in SEQ ID No.10, downstream primer sequence such as SEQ ID
Shown in No.11, probe sequence is as shown in SEQ ID No.12.
A kind of test kit for detecting influenza virus H1, H3, H5, H7 hypotype simultaneously, this test kit includes: above-mentioned influenza
The upstream and downstream primer of virus H1, H3, H5, H7 and corresponding probe, 5 ' ends of each bar probe and 3 ' ends correspondence markings FAM-respectively
BHQ1, Texred-BHQ2, JOE-TAMRA and CY5-BHQ3.
Test kit as above, it is preferable that also include 2 × RT-PCR buffer (Buffer), 25 × reverse transcription and
Taq polymerase mixed enzyme (25 × RT/Taq mix).
A kind of method simultaneously detecting influenza virus H1, H3, H5, H7 hypotype for quantitative fluorescent PCR, the method is non-examining
The detection method of disconnected purpose, comprises the following steps:
(1) from sample, viral RNA is extracted；
(2) viral RNA extracting described step (1) carries out fluorescent quantitative PCR；Wherein, fluorescent quantitative PCR
Time, in reaction system, use the nucleotide sequence such as SEQ ID No.1 and SEQ ID of the upstream and downstream primer of influenza virus H1
Shown in No.2, the nucleotide sequence of probe is as shown in SEQ ID No.3, and its 5 ' end and 3 ' holds correspondence markings FAM and BHQ1 respectively；
The nucleotide sequence of the upstream and downstream primer of influenza virus H3 as shown in SEQ ID No.4 and SEQ ID No.5, the nucleoside of probe
Acid sequence is as shown in SEQ ID No.6, and its 5 ' end and 3 ' holds correspondence markings Texred and BHQ2 respectively；Influenza virus H5 is upper and lower
Trip primer nucleotide sequence as shown in SEQ ID No.7 and SEQ ID No.8, the nucleotide sequence of probe such as SEQ ID
Shown in No.9, its 5 ' end and 3 ' ends correspondence markings JOE and TAMRA respectively；The nucleotide sequence of influenza virus H7 upstream and downstream primer
As shown in SEQ ID No.10 and SEQ ID No.11, the nucleotide sequence of probe as shown in SEQ ID No.12, its 5 ' end and
3 ' ends correspondence markings CY5 and BHQ3 respectively；
(3) collecting fluorescence signal, select the fluoroscopic examination pattern of above-mentioned fluorophor, baseline adjustment takes 3～15 circulations
Fluorescence signal, with threshold line just above normal negative control peak set threshold line；
(4) result judges: testing sample fluorescence growth curve exceedes threshold line, and presents good logarithm growth, then sentence
Break as the positive, as without typical amplification curve, it is judged that for feminine gender.
Method as above, it is preferable that in described step (2), the reaction system of fluorescent quantitative PCR is specific as follows:
1 × RT-PCR Buffer,
1 × RT/Taq mix,
The final concentration of 80nmol/L of the forward primer of described influenza virus H1, the final concentration of 80nmol/ of downstream primer
L, the final concentration of 40nmol/L of probe；The final concentration of 400nmol/L of the forward primer of described influenza virus H3, downstream primer
Final concentration of 400nmol/L, the final concentration of 200nmol/L of probe；The forward primer of described influenza virus H5 is final concentration of
200nmol/L, the final concentration of 200nmol/L of downstream primer, the final concentration of 100nmol/L of probe；On described influenza virus H7
The trip final concentration of 200nmol/L of primer, the final concentration of 200nmol/L of downstream primer, the final concentration of 100nmol/L of probe；
Described viral RNA is 2 μ L；
Arrange nuclease free water is negative control simultaneously.
Method as above, it is preferable that in described step (2), the response procedures of fluorescent quantitative PCR is: 50 DEG C
30min, 95 DEG C of 5min, then 95 DEG C of 10s, 55 DEG C of 40s, carry out fluoroscopic examination at 55 DEG C, carry out 45 circulations altogether.
Method as above, it is preferable that during described step (4) result judges, described threshold value is 35, when Ct value≤35
Time, having obvious amplification curve is positive findings；During Ct value ＞ 40, it is negative findings without obvious amplification curve.
The invention provides the nucleic acid group of detection influenza virus H1, H3, H5 and H7 hypotype, this group nucleic acid can be used for single disease
The detection of poison hypotype is it can also be used to the multiplex amplification of several virus subtype synchronous detecting uses.For synchronous detecting these four stream
During Influenza Virus hypotype, its detection sensitivity is high, high specificity, through checking, cross reaction does not the most occur.
Present invention also offers, a kind of quantitative fluorescent PCR that is used for detects tetra-kinds of Asias of influenza virus H1, H3, H5 and H7 simultaneously
The test kit of type, establishes a kind of ratio more efficient, sensitive simultaneously, can check the influenza virus of four kinds of hypotypes simultaneously, be specially
The method for quick of influenza virus H1, H3, H5 and H7 hypotype.The method can be effectively improved the sensitivity of detection, it is to avoid false cloudy
Property.The detection kit provided is easy to use, easy and simple to handle, and automaticity is high, effectively substitutes tradition virus purification and obtains
Testing result, and the reagent used by test kit is few, enormously simplify operating process, decreases the process of repetitive operation, the most just subtracts
Lacked the pollution in operating process, it is to avoid repetitive operation and consume too much labour force, time-consuming, effectively save cost, real
Having showed rapid screening, the Detection results of used kit is good, and high specificity is highly sensitive.
The detection method provided uses the operation of complete stopped pipe, simple, convenient quick, by direct detection PCR process
The change of middle fluorescence signal obtains quantitative result it is not necessary to PCR post processing or electrophoresis detection, overcomes Standard PCR technology
Easily pollute, false positive occur, a non-specific amplification difficult problem can be prevented effectively from, and be suitable for the detection of high-volume sample.
By influenza infection, general being difficult to finds and controls, and the test kit that the present invention provides has higher detection
Sensitivity, and the detection of 1 sample can be realized the investigation to four kinds of subtype influenza virus, and mainly in detection sample to be tested
Whether containing the nucleic acid of influenza virus H1, H3, H5 and H7 hypotype, be not the diagnosis for disease, this detection belongs to non-diagnostic
The detection of property.But can aid in and find that influenza virus infection carrier the incubation period without condition symptoms occurs and do not falls ill in advance
Inapparent infection person, for timely and effective the propagation of influenza virus H1, H3, H5 and H7 hypotype and diffusion can be controlled, it is provided that effective
Technical support.
Accompanying drawing explanation
Fig. 1 is the amplification curve of the method detection influenza virus H1 hypotype sensitivity of the present invention.
Fig. 2 is the amplification curve of the method detection influenza virus H3 hypotype sensitivity of the present invention.
Fig. 3 is the amplification curve of the method detection influenze virus H 5 subtype sensitivity of the present invention.
Fig. 4 is the amplification curve of the method detection influenza virus H7 hypotype sensitivity of the present invention.
Fig. 5 is the standard curve of the method detection influenza virus H1 hypotype of the present invention.
Fig. 6 is the standard curve of the method detection influenza virus H3 hypotype of the present invention.
Fig. 7 is the standard curve of the method detection influenze virus H 5 subtype of the present invention.
Fig. 8 is the standard curve of the method detection influenza virus H7 hypotype of the present invention.
Detailed description of the invention
Below in conjunction with instantiation, the present invention is described in further details, not limitation of the invention, the present invention's
Embodiment is not limited to this, and the complementary series of the nucleotide sequence that the present invention provides also can realize the present invention, as without special theory
Bright, agents useful for same is conventional reagent, and the most all equivalents according to this area done by present disclosure belong to this
Bright protection domain.
Embodiment 1 primer, the design of probe
Screen the specific target gene of four kinds of influenza virus sub-strains the most respectively, according to testing goal, to every kind of virus from
GenBank downloads a plurality of virus gene sequence, compares, and chooses conserved region, uses Array Designer
4.0software software Aided Design amplimer and the hybridization probe of applicable quantitative fluorescent PCR reaction system.
Owing to the present invention is multiple fluorescence quantitative, the first selection of probe labelling is more crucial, and secondly software design is out
Probe will through screening, the probe signals of four genes can not interfere, this be accomplished by design probe time consider that four is right
All can not interfere between primer and four probes.
Fluorescence quantitative PCR detection is on the basis of regular-PCR detects, further by specific fluorescent probe, and should
Probe is an oligonucleotide, two ends one reporter fluorescence group of labelling and a quenching fluorescence group respectively.When probe is complete, report
The fluorescence signal accusing group transmitting is quenched group absorptions；During PCR amplification, the 5'-3' 5 prime excision enzyme activity of Taq enzyme is by probe enzyme
Cut degraded, make reporter fluorescence group separate with quenching fluorescence group, thus fluorescence monitoring system can receive fluorescence signal, the most often
Expand a DNA, just have a fluorescence molecule to be formed, it is achieved that the accumulation of fluorescence signal and PCR primer are formed the most same
Step.So the premise of fluorescence quantitative PCR detection is intended to carry out pcr amplification reaction, primer and product between amplified reaction need to be use up
Amount avoids cross reaction, further ensures that specific probe and each amplified production, primer the most should not have cross reaction.Again by
It is multiple fluorescence quantitative in the present invention, so the selection of probe labelling is the most crucial, not only will be to software design spy out
Pin will be through screening, and the probe signals of four genes can't interfere.
Separately design out a plurality of primer sequence of HA gene and the hybridization probe of influenza virus H1, H3, H5 and H7 subtype sepcific
Sequence, uses NCBI Blast on-line analysis software (http://www.ncbi.nlm.nih.gov/blast) to intending selection
Probe and primer combination carry out sequence homology and suitability analysis and evaluation, and by final this influenza virus selected of detection test
4 kinds of subtype sepcifics and the primer of applicable multiple fluorescence quantitative reaction system and probe combinations.
Amplimer for every kind of virus subtype design first has to carry out the augmentation detection of single virus, confirms single virus
After augmentation detection does not has non-specific amplification, then carrying out the amplification of multiplexed viral, design primer is accomplished by avoiding handing over as far as possible
Fork reaction, it is also contemplated that amplification condition to be tried one's best unanimously, gets rid of cross reaction eventually through hybridization；According to above-mentioned design,
Bioinformatic analysis and the Experience Design of inventor and great many of experiments detection experiment sieving result determine following sequence:
The amplimer of influenza virus H1 subtype sepcific to and hybridization probe: such as SEQ ID No.1, SEQ ID No.2 institute
The specific amplification primer pair shown, and the hybridization probe sequence as shown in SEQ ID No.3, specific as follows:
H1-F (SEQ IDNo.1): 5 '-AGCTCATGGTCYTACATTGTGG-3 ' wherein Y represents pyrimidine, be herein T or
C。
H1-R (SEQ IDNo.2): 5 '-CCTTTCRAATGATGACACTGAGC-3 ' wherein R for representing purine, is A herein
Or G.
H1-P (SEQ IDNo.3): 5 '-TGTTACCCAGGAGATTTCA-3 '
Wherein, in order to improve the recall rate to H1 subtype variation strain, at SEQ the 12nd alkali of ID No.1 forward primer sequence
Base Y represents pyrimidine, represents purine for T or C, SEQ the 7th base R of ID No.2 downstream primer sequence herein, is A or G herein；
The amplimer of influenza virus H3 subtype sepcific to and hybridization probe: such as SEQ ID No.4, SEQ ID No.5 institute
The specific amplification primer pair shown, and the specific probe sequence as shown in SEQ ID No.6, specific as follows:
H3-F (SEQ IDNo.4): 5 '-TGATGGAAAAAACTGCACACTG-3 '
H3-R (SEQ IDNo.5): 5 '-GCGTTCAACAAAAAGGTCCC-3 '
H3-P (SEQ IDNo.6): 5 '-AGACCCTCATTGTGATGGCT-3 '；
The special amplimer of influenze virus H 5 subtype to and hybridization probe: such as SEQ ID No.7, SEQ ID No.8 institute
The specific amplified upstream and downstream primer shown, and the hybridization probe sequence as shown in SEQ ID No.9, specific as follows:
H5-F (SEQ IDNo.7): 5 '-TGGTAYGGRTACCAYCATAGCAA-3 ', wherein Y is T or C, and R is A or G.
H5-R (SEQ IDNo.8): 5 '-GAGTTKACYTTATTRGTGATTCCAT-3 ', wherein K is G or T, and Y is T or C,
R A or G.
H5-P (SEQ IDNo.9): 5 '-AGTGGATAYGCTGCAGACA-3 ' wherein Y is T or C.
The amplimer of influenza virus H7 subtype sepcific to and hybridization probe: such as SEQ ID No.10, SEQ ID No.11
Shown specific amplification primer pair, and the specific probe sequence as shown in SEQ ID No.12, specific as follows:
H7-F (SEQ IDNo.10): 5 '-AGAATAGAATACAGATWGACCCAGT-3 ', wherein W is A or T.
H7-R (SEQ IDNo.11): 5 '-GTGCACYGCATGTTTCCATT-3 ', wherein Y is T or C.
H7-P (SEQ IDNo.12): 5 '-TTTAGCTTCGGGGCATCATGTTT-3 '.
Using above-mentioned degenerate primer and probe, the specificity being effectively improved detection avoids missing inspection simultaneously.
Through lot of experiment validation, when using above-mentioned primer, probe, the influenza virus that can be individually used for detecting each correspondence is sub-
The detection of type, 5 ' ends and the 3 ' ends of its probe can be selected for flag F AM-BHQ1, Texred-BHQ2, JOE-TAMRA and CY5-respectively
BHQ3.But when detecting the hypotype of four kinds of influenza virus, each probe labelling then answers correspondence markings these four syntagmatic simultaneously
That is the probe of FAM-BHQ1, Texred-BHQ2, JOE-TAMRA and CY5-BHQ3 labelling H1 respectively, the spy of H3 are used
Pin, the probe of H5, the probe of H7, each probe 5 ' end and 3 ' end labellings are misaligned, but can arbitrarily be combined, detection knot
The most unaffected, when the most independent virus subtype detects, probe also can be selected for other fluorophor such as VIC, NED etc.
As luminophore, use such as BHQ and MGB etc. as non-fluorescence quenching group.
Embodiment 24 weight influenza virus sub-strain fluorescent quantificationally PCR detecting kit
Influenza virus H1, H3, H5 and H7 hypotype fluorescent quantificationally PCR detecting kit includes following component:
2×RT-PCR Buffer；25×RT/Taq mix；
The forward primer sequence of influenza virus H1 as shown in SEQ ID No.1, downstream primer sequence such as SEQ ID No.2 institute
Show, probe sequence as shown in SEQ ID No.3,5 ' flag F AM of probe SEQ IDNo.3,3 ' labelling BHQ1；
The forward primer sequence of described influenza virus H3 as shown in SEQ ID No.4, downstream primer sequence such as SEQ ID
Shown in No.5, probe sequence as shown in SEQ ID No.6 wherein, the 5 ' labelling Texred of probe SEQIDNo.6,3 ' labellings
BHQ2；
The forward primer sequence of described influenza virus H5 as shown in SEQ ID No.7, downstream primer sequence such as SEQ ID
Shown in No.8, probe sequence is as shown in SEQ ID No.9, wherein, and the 5 ' labelling JOE of probe SEQIDNo.9,3 ' labelling TAMRA；
The forward primer sequence of described influenza virus H7 as shown in SEQ ID No.10, downstream primer sequence such as SEQ ID
Shown in No.11, probe sequence is as shown in SEQ ID No.12, wherein, the 5 ' labelling CY5 of probe SEQ IDNo.12,3 ' labellings
BHQ3。
Wherein, described 2 × RT-PCR Buffer and Hui Rui bio tech ltd, 25 × RT/Taq mix Shanghai
HR One-step qPCR Master Mix, Code:SJ-2106B；Primer, probe entrust victory base (Shanghai) trade in the English Weihe River limited
Company synthesizes.
The method of 34 kinds of subtype influenza virus fluorescence quantitative PCR detection of embodiment
Using quantitative fluorescent PCR to detect influenza virus H1, the method for H3, H5, H7 hypotype comprises the following steps simultaneously:
(1) viral RNA extracts
Viral RNA extracts and can be selected for QIAmp RNA Mini Kit extraction test kit (being such as purchased from QIAGEN company of Germany)
Extract.
(2) fluorescent quantitative PCR
Using the test kit configuration reaction system of embodiment 3 preparation, its component and volume thereof preferably employ component in table 1 below
Configuration.
Table 1 reaction system
When arranging positive control, the RNA using influenza virus H1, H3, H5, H7 hypotype to extract replaces sample rna, arranges the moon
Property comparison time, use nuclease free water replace sample rna.
Amplification condition: preferred following amplification condition: 50 DEG C of 30min, 95 DEG C of 5min, then 95 DEG C of 10s, 55 DEG C of 40s, 55
DEG C carry out fluoroscopic examination, carry out 45 circulations altogether.
(3) collect fluorescence signal signal, select the fluoroscopic examination pattern of FAM, Texred, JOE, CY5, baseline adjustment respectively
Take the fluorescence signal of 3～15 circulations, set threshold line with threshold line just above the peak of normal negative control；
(4) result judges: testing sample fluorescence growth curve exceedes threshold line, and presents good logarithm growth, to be measured
Fluorescent growth curve exceedes threshold line, and presents good logarithm growth, then be judged as the positive, as without typically expanding song
Line, it is judged that for feminine gender.
Further, in the present embodiment, threshold value is 35, and when Ct value≤35, having obvious amplification curve is positive findings, Ct value
＞ 40, hence it is evident that amplification curve is negative findings.
Above-mentioned quantitative fluorescent PCR react spendable instrument include ABI real-time PCR system (such as 7000,7300,7500,
7900 etc.)；BioRad Real Time PCR Detection System, Stratagene quantitative polumerase chain reaction instrument (such as MX4000, MX3000,
MX3005)。
The most in the present invention, as a example by ABI7500 quantitative real time PCR Instrument, the first to Article 4 inspection detection is by respectively
With FAM, Texred, JOE and Cy5 labelling.Article 1, sense channel detection influenza virus H1 hypotype, the inspection of its fluorescent dye FAM
Survey wavelength and be about 520nm；Article 2 sense channel detection influenza virus H3 hypotype, the detection wavelength of its fluorescent dye Texred is about
For 580nm；Article 3 sense channel detection influenze virus H 5 subtype, the detection wavelength of its fluorescent dye JOE is about 550nm；The
Article four, sense channel influenza virus H7 hypotype, the detection wavelength of its fluorescent dye CY5 is about 650nm.Following example use
Preferred embodiment illustrates the present invention.
The detection of embodiment 4 test kit of the present invention sensitivity
First, preparing of positive template is carried out as follows:
The bacterial strain of infected by influenza H1, H3, H5, H7 hypotype carries out the extraction of RNA, uses in embodiment 3 and is added without probe
System, other reaction reagent and amplification condition are constant, expand.Power taking swimming is accredited as the fragment of positive amplification, corresponding
Fragment after purification carry out in vitro transcription, SP6 5 × Buffer 20 μ L, rNTP s 30 μ L, enzyme action purified product 30 μ L,
SP6Enzyme Mix10 μ L, Nuclease-Free Water 10 μ L, mix homogeneously, of short duration centrifugal, put in PCR amplification instrument,
37 DEG C, react 4h.Add 1 μ L RQ1 DNase to 100 μ L system, put in PCR amplification instrument 37 DEG C, 15min.Again take out anti-
Answer thing, by phenol chloroform method extracting RNA, the positive template being precipitated as influenza virus obtained, molten with Nuclease-Free Water
Solve ,-80 DEG C of preservations.
In said process, corresponding steps the most all uses conventional method, wherein, the reagent such as test kit of employing
And source is as follows, the little extraction reagent kit of high-purity plasmid is TIANGEN company, and XhoI enzyme is that New England BioLabs is public
Department, it is QIAGEN company that plasmid reclaims purification kit, and SP6 in vitro transcription test kit is the test kit of Promega company.
By influenza virus H1, H3, H5, H7 hypotype positive template of above-mentioned acquisition, after measured, its concentration is respectively 283ng/
μ l, 182ng/ μ l, 262ng/ μ l, 238ng/ μ l, according to copy number copies/ μ l=(ng/ μ l × 10-9)×(6.02×
1023)/(660 × DNA length) formula calculating, the copy number of influenza virus H1, H3, H5, H7 hypotype positive template is respectively 6.19
×1010copies/μl、3.95×1010copies/μl、5.74×1010copies/μl、5.21×1010Copies/ μ l, is carried out
10 doubling dilutions, can obtain concentration from 6.19copies/ μ L to 6.19 × 109The H1 hypotype positive template sample of copies/ μ l
Product, concentration is from 3.95copies/ μ L to 3.95 × 109The H3 hypotype positive template sample of copies/ μ l, concentration from
5.74copies/ μ L to 5.74 × 109The H5 hypotype positive template sample of copies/ μ l, concentration from 5.21copies/ μ L to
5.21×109The H7 hypotype positive template sample of copies/ μ l.
Utilize embodiment 3 detection method that the positive template of above-mentioned influenza virus H1, H3, H5 and H7 hypotype is detected,
Wherein,
1) NTC: nuclease free water；
2) reaction system is prepared according to table 1 in embodiment 3, and the positive template after sample rna uses above-mentioned dilution enters
Row detection, each gradient does three Duplicate Samples.
Result judges: Ct value≤35, and having obvious amplification curve is positive findings, Ct value ＞ 40, hence it is evident that amplification curve is cloudy
Property result.Detection influenza virus H1 hypotype, H3 hypotype, H5 hypotype, the fluorescent value figure of positive gradient sample of H7 hypotype, as Fig. 1,
2, shown in 3,4.Result shows, the primer of present invention design and the detection sensitivity of probe in detecting influenza virus H1 hypotype are
6copies/ μ L, the detection sensitivity of H3 hypotype is 30copies/ μ L, and the detection sensitivity of H5 hypotype is 5copies/ μ L, H7
The detection sensitivity of hypotype is 50copies/ μ L.
Detect the canonical plotting of the positive gradient sample of influenza virus H1 hypotype, H3 hypotype, H5 hypotype, H7 hypotype, point
Not as shown in Fig. 5,6,7,8.As seen from the figure, influenza virus H1 hypotype, H3 hypotype, H5 hypotype, the R of H7 hypotype2Value is respectively
0.999,0.999,0.999 and 0.998, there is good linear relationship.
The specific detection of embodiment 5 test kit of the present invention
1) select the RNA that influenza virus H1, H3, H5, H7 subtype virus strain is extracted as positive control；
2) negative control: nuclease free water；
3) template: select influenza virus H1, H3, H5, H7 hypotype, H9 hypotype, influenza B, bocavirus, coronavirus,
Metapneumovirus, the RNA of respiratory syncytial virus, above-mentioned bacterial strains is China Inst. of Quarantine Inspection Sciences's health quarantine institute
Preserve.
4) reaction system is prepared according to table 2 in embodiment 3, carries out fluorescent quantitative PCR afterwards.
Result shows, the fluorescence quantifying PCR method of the four kinds of influenza subtype viruses set up and test kit, influenza virus
H1, H3, H5, H7 hypotype has obvious amplification curve, for the positive, sick for other in addition to influenza virus H1, H3, H5, H7 hypotype
Poison is negative, does not has specific amplification, shows that the primer of design and probe specificity are strong.
Embodiment 6: utilize test kit of the present invention that sample is detected
Viral RNA extracting solution is detected by the test kit of the present invention.The RNA of the specimen such as people's Nasopharyngeal swabs, tissue extracts
Flow process refers to WHO promulgated standard and carries out, it is recommended to use the High Pure Viral RNA Kit of Roche company (catalog number (Cat.No.):
11858882001) or OIAampViral RNA Mini Kit (catalog number (Cat.No.): 52904) of Qiagen company carries out Virus Sample
RNA extract operation.
For at present, heating crowd carries influenza virus H1, H3, H5 and H7 hypotype probability big, to heating crowd
The examination detection of middle influenza virus H1, H3, H5 and H7 hypotype is particularly important, and mainly whether carries heating crowd below
Some influenza virus H1, H3, H5 and H7 hypotypes are simulated detection.
Utilize test kit of the present invention, the RNA of the extraction of Nasopharyngeal swabs is simulated the detection of positive, wherein, mould
Plan positive is the Strain in throat swab added with influenza virus H1, H3, H5 and H7 hypotype, and specific experiment step is as follows:
(1) viral RNA extracts
Viral RNA extracts selects the RNA extraction test kit of QIAGEN company to extract.
A. homogenized:
From-80 DEG C of refrigerators or liquid nitrogen, take out throat swab sample, be placed in the zirconia particles being previously added 3 diameter 3mm
2mL round bottom centrifuge tube in, add 1.5mL lapping liquid (containing the cell culture medium of 10% mycillin), with the tissue of TOMY
4000 times/min of dismembyator shakes 60sec, until becoming homogenate, in 4 DEG C, 10,000rpm are centrifuged 10min.
B. supernatant is transferred to 1.5mL centrifuge tube, 4 DEG C, 12,000rpm, centrifugal 2min.Take 140 μ L of supernatant liquid for RNA
Extracting, remaining homogenate is placed in-40 DEG C of preservations.
Total RNAs extraction
C. the RNA selecting QIAGEN company extracts test kit, according to the quantity subpackage AVL liquid of sample, often pipe 560 μ L；
D. take 140 μ L lapping liquid supernatants and join the AVL liquid that 560 μ L subpackages are good, vortex concussion 15s, fully mix, room temperature
Hatch 10min；
The most often pipe is separately added into the dehydrated alcohol (96%-100%) of 560 μ L, and concussion 15s fully mixes, and is quickly centrifuged；
F. from Kit, take out the collecting pipe of the 2mL of band filter post, carry out labelling, take mixed liquor 630 μ L and join collecting pipe
In, 8000rpm is centrifuged 1min；
G. being placed in new 2mL collecting pipe by filter post, remaining liquid adds filter post, 8000rpm is centrifuged 1min；
H. moving in new collecting pipe by filter post, add 500 μ L AW1 liquid, 8000rpm is centrifuged 1min；
I. filter post is placed in a new collecting pipe, adds 500 μ L AW2 liquid, and 14,000rpm are centrifuged 3min；
J. moving on in clean 1.5mL EP pipe by filter post, add 60 μ L AVE, it is centrifugal that room temperature stands 2min, 8000rpm
1min, i.e. obtains RNA, is positioned over-80 DEG C of Refrigerator stores standby.
(2) fluorescent quantitative PCR
Configure reaction system and amplification condition according to embodiment 3, detect.Testing result draws, adds INFLUENZAInfluenza
The sample of the Strain of virus H1, H3, H5 and H7 hypotype, testing result is positive, illustrates that test kit of the present invention can be applicable to inspection
Survey the detection of influenza virus H1, H3, H5 and H7 hypotype in throat swab, and testing result is accurately, reliably.
Claims (8)
1. one group is detected influenza virus H1, the nucleic acid of H3, H5, H7 hypotype for multiplex PCR simultaneously, it is characterised in that include stream
The upstream and downstream primer of Influenza Virus H1, the upstream and downstream primer of influenza virus H3, the upstream and downstream primer of influenza virus H5 and influenza are sick
The upstream and downstream primer of poison H7；
Wherein, the forward primer sequence of described influenza virus H1 as shown in SEQ ID No.1, downstream primer sequence such as SEQ ID
Shown in No.2；
The forward primer sequence of described influenza virus H3 as shown in SEQ ID No.4, downstream primer sequence such as SEQ ID No.5 institute
Show；
The forward primer sequence of described influenza virus H5 as shown in SEQ ID No.7, downstream primer sequence such as SEQ ID No.8 institute
Show；
The forward primer sequence of described influenza virus H7 as shown in SEQ ID No.10, downstream primer sequence such as SEQ ID No.11
Shown in.
2. one group of nucleic acid simultaneously detecting influenza virus H1, H3, H5, H7 hypotype for quantitative fluorescent PCR, it is characterised in that bag
Include the upstream and downstream primer of influenza virus H1 and probe, the upstream and downstream primer of influenza virus H3 and probe, influenza virus H5 upper,
The upstream and downstream primer of downstream primer and probe and influenza virus H7 and probe；
The forward primer sequence of described influenza virus H1 as shown in SEQ ID No.1, downstream primer sequence such as SEQ ID No.2 institute
Showing, probe sequence is as shown in SEQ ID No.3；
The forward primer sequence of described influenza virus H3 as shown in SEQ ID No.4, downstream primer sequence such as SEQ ID No.5 institute
Showing, probe sequence is as shown in SEQ ID No.6；
The forward primer sequence of described influenza virus H5 as shown in SEQ ID No.7, downstream primer sequence such as SEQ ID No.8 institute
Showing, probe sequence is as shown in SEQ ID No.9；
The forward primer sequence of described influenza virus H7 as shown in SEQ ID No.10, downstream primer sequence such as SEQ ID No.11
Shown in, probe sequence is as shown in SEQ ID No.12.
3. the test kit for detection influenza virus H1, H3, H5, H7 hypotype simultaneously, it is characterised in that this test kit bag
Include: the upstream and downstream primer of influenza virus H1, H3, H5, H7 as claimed in claim 2 and corresponding probe, the 5 ' of each bar probe
End and 3 ' ends correspondence markings FAM-BHQ1, Texred-BHQ2, JOE-TAMRA and CY5-BHQ3 respectively.
4. test kit as claimed in claim 3, it is characterised in that also include 2 × RT-PCR buffer and 25 × reverse transcription
With Taq polymerase mixed enzyme.
5. the method simultaneously detecting influenza virus H1, H3, H5, H7 hypotype for quantitative fluorescent PCR, it is characterised in that should
Method is the detection method of non-diagnostic purpose, and it comprises the following steps:
(1) from sample, viral RNA is extracted；
(2) viral RNA extracting described step (1) carries out fluorescent quantitative PCR；Wherein, during fluorescent quantitative PCR,
In reaction system, use the nucleotide sequence such as SEQ ID No.1 and SEQ ID of the upstream and downstream primer of influenza virus H1
Shown in No.2, the nucleotide sequence of probe is as shown in SEQ ID No.3, and its 5 ' end and 3 ' holds correspondence markings FAM and BHQ1 respectively；
The nucleotide sequence of the upstream and downstream primer of influenza virus H3 as shown in SEQ ID No.4 and SEQ ID No.5, the nucleoside of probe
Acid sequence is as shown in SEQ ID No.6, and its 5 ' end and 3 ' holds correspondence markings Texred and BHQ2 respectively；Influenza virus H5 is upper and lower
Trip primer nucleotide sequence as shown in SEQ ID No.7 and SEQ ID No.8, the nucleotide sequence of probe such as SEQ ID
Shown in No.9, its 5 ' end and 3 ' ends correspondence markings JOE and TAMRA respectively；The nucleotide sequence of influenza virus H7 upstream and downstream primer
As shown in SEQ ID No.10 and SEQ ID No.11, the nucleotide sequence of probe as shown in SEQ ID No.12, its 5 ' end and
3 ' ends correspondence markings CY5 and BHQ3 respectively；
(3) collecting fluorescence signal, select the fluoroscopic examination pattern of above-mentioned fluorophor, baseline adjustment takes the glimmering of 3～15 circulations
Optical signal, sets threshold line with threshold line just above the peak of normal negative control；
(4) result judges: testing sample fluorescence growth curve exceedes threshold line, and presents good logarithm growth, then be judged as
The positive, as without typical amplification curve, it is judged that for feminine gender.
6. method as claimed in claim 5, it is characterised in that the reaction system of fluorescent quantitative PCR in described step (2)
Specific as follows:
1 × RT-PCR buffer,
1 × reverse transcription and Taq polymerase mixed enzyme,
The final concentration of 80nmol/L of the forward primer of described influenza virus H1, the final concentration of 80nmol/L of downstream primer, visit
The final concentration of 40nmol/L of pin；The final concentration of 400nmol/L of the forward primer of described influenza virus H3, downstream primer is the denseest
Degree is 400nmol/L, the final concentration of 200nmol/L of probe；The forward primer of described influenza virus H5 is final concentration of
200nmol/L, the final concentration of 200nmol/L of downstream primer, the final concentration of 100nmol/L of probe；On described influenza virus H7
The trip final concentration of 200nmol/L of primer, the final concentration of 200nmol/L of downstream primer, the final concentration of 100nmol/L of probe；
Described viral RNA is 2 μ L；
Arrange nuclease free water is negative control simultaneously.
7. method as claimed in claim 5, it is characterised in that the response procedures of fluorescent quantitative PCR in described step (2)
For: 50 DEG C of 30min, 95 DEG C of 5min, then 95 DEG C of 10s, 55 DEG C of 40s, carry out fluoroscopic examination at 55 DEG C, carry out 45 circulations altogether.
8. method as claimed in claim 5, it is characterised in that during described step (4) result judges, described threshold value is 35, when
During Ct value≤35, having obvious amplification curve is positive findings；During Ct value ＞ 40, it is negative findings without obvious amplification curve.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106868213A (en) * | 2017-03-21 | 2017-06-20 | 重庆迪安医学检验中心有限公司 | A kind of primer and its application for detecting parainfluenza virus and coronavirus simultaneously based on melting curve method single tube |
| CN106868225A (en) * | 2017-04-26 | 2017-06-20 | 广州和实生物技术有限公司 | The method and kit of a kind of utilization ligase detection influenza A virus series |
| CN107142335A (en) * | 2017-05-23 | 2017-09-08 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Reagent, detection method and the application detected for H7 subtype avian influenza virus |
| CN108950081A (en) * | 2018-08-10 | 2018-12-07 | 中国动物卫生与流行病学中心 | A kind of real-time fluorescence quantitative RT-PCR detection method of H7 subtype avian influenza virus |
| CN109468411A (en) * | 2018-12-05 | 2019-03-15 | 中国动物卫生与流行病学中心 | A kind of real-time fluorescence quantitative RT-PCR detection method of H5 subtype avian influenza virus |
| CN110283940A (en) * | 2019-06-27 | 2019-09-27 | 深圳市刚竹医疗科技有限公司 | Nucleic acid compositions, the detection kit of influenza virus and micro-fluidic chip |
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| CN104498622A (en) * | 2014-11-24 | 2015-04-08 | 湖北新纵科病毒疾病工程技术有限公司 | Primers, probes, and method used for influenza virus typing |
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Patent Citations (1)
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| CN104498622A (en) * | 2014-11-24 | 2015-04-08 | 湖北新纵科病毒疾病工程技术有限公司 | Primers, probes, and method used for influenza virus typing |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106868213A (en) * | 2017-03-21 | 2017-06-20 | 重庆迪安医学检验中心有限公司 | A kind of primer and its application for detecting parainfluenza virus and coronavirus simultaneously based on melting curve method single tube |
| CN106868225A (en) * | 2017-04-26 | 2017-06-20 | 广州和实生物技术有限公司 | The method and kit of a kind of utilization ligase detection influenza A virus series |
| CN107142335A (en) * | 2017-05-23 | 2017-09-08 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Reagent, detection method and the application detected for H7 subtype avian influenza virus |
| CN108950081A (en) * | 2018-08-10 | 2018-12-07 | 中国动物卫生与流行病学中心 | A kind of real-time fluorescence quantitative RT-PCR detection method of H7 subtype avian influenza virus |
| CN109468411A (en) * | 2018-12-05 | 2019-03-15 | 中国动物卫生与流行病学中心 | A kind of real-time fluorescence quantitative RT-PCR detection method of H5 subtype avian influenza virus |
| CN110283940A (en) * | 2019-06-27 | 2019-09-27 | 深圳市刚竹医疗科技有限公司 | Nucleic acid compositions, the detection kit of influenza virus and micro-fluidic chip |
| CN110283940B (en) * | 2019-06-27 | 2023-11-24 | 深圳市呈晖医疗科技有限公司 | Nucleic acid composition, influenza virus detection kit and microfluidic chip |
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