CN105755170A - Primer and probe as well as kit used for avian influenza virus universal type direct RT-PCR detection - Google Patents
Primer and probe as well as kit used for avian influenza virus universal type direct RT-PCR detection Download PDFInfo
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Abstract
The invention discloses a primer and a probe as well as a kit used for avian influenza virus universal type direct RT-PCR detection. The primer and the probe comprise a detecting primer and a detecting probe of the avian influenza virus as well as a probe of internal label RNA. With the adoption of the primer and the probe, a liquid sample can be directly added into a reaction system, so that release, reverse transcription and fluorescent quantitative PCR detection of the virus RNA are continuously and automatically realized in one PCR tube; the purposes of extracting the virus RNA and directly detecting the avian influenza virus in the extracted sample are realized, so that only 1.5 hours are needed for obtaining detected results from a to-be-detected sample; and the defect that RNA is needed to be extracted by conventional RT-PCR is effectively overcome, operation steps can be greatly reduced, the detection speed is greatly increased, the cost is reduced, and cross contamination is effectively avoided.
Description
Technical field
The present invention relates to virus detection techniques field, particularly relate to a kind of universal directly for avian influenza virus
The primer of RT-PCR detection and probe and kit.
Background technology
Europe checken pest or fowl plague are also in bird flu (Avian Influenza, AI), are sick by A type influenza
The one acute high degree in contact sexually transmitted disease that poison causes, is internationally recognized a kind of crushing disease, Ke Yijing
Pig or other zoochory, to people, therefore have highly important ecology and public hygienics meaning, especially
It is that highly pathogenic bird flu is classified as A class infectious disease by OIE.
Discovered in recent years bird flu not only serious harm bird, and endanger mammal, especially can feel
Dye people causing death, greatly threaten mankind's public health security.Avian influenza virus is sub-thread pair chain RNA
Virus, is divided into 8 sections.Wherein 7 sections i.e. M full length gene is 1027bp, encodes 2 kinds of albumen
M1 and M2.In the albumen of M gene code, M1 albumen has group specificity, be virus taxis and
The basis of diagnosis.
At present, the method for detection avian influenza virus includes viral separation and cultivation and Enzyme-linked Immunosorbent Assay
(ELISA) etc., but these methods still suffer from certain deficiency in sensitivity and specific aspect.Reverse transcription PCR
(RT-PCR) technology is owing to sensitivity is with the highest, has become as the common method of avian flu virus detection.
But conventional RT-PCR needs to carry out follow-up electroresis appraisal, cross pollution is easily occurred to produce false positive, and behaviour
When making trouble longer.By contrast, fluorescent quantitative PCR technique (real-time RT-PCR) has higher spirit
Quick property, and without PCR subsequent treatment, cross pollution can be prevented effectively from and improve detection efficiency, but carrying out
Viral RNA extracts and not only increases time and cost, it is difficult to realizes high flux detection, and extracts RNA
Process be still susceptible to RNA degraded or the event such as cross pollution, additionally for trace sample, extract RNA
Still suffer from bigger difficulty.Therefore, existing method based on RT-PCR detection avian influenza virus needs
Improve and development.
In order to improve avian flu virus detection efficiency, cost-effective, avoid cross pollution and carry out high flux inspection
Surveying, a kind of preferable solution is to manage to realize the RT-PCR detection that avian influenza virus directly expands, i.e.
Directly carry out RT-PCR reaction using the liquid sample gathered as detection object.But, be limited to primer and
Probe is easily affected by sample complicated factor, there is presently no and finds the primer and probe that can so use.
Summary of the invention
The present invention provides a kind of primer for the detection of avian influenza virus universal direct fluorescence RT-PCR and spy
Pin and kit.Use the primer of the present invention and probe or kit, and combine other conventional reagent component,
It is capable of in a PCR pipe continuously, automatically carries out the release of viral RNA, reverse transcription and fluorescence calmly
Amount PCR detection；1.5 hours are had only to obtaining testing result from obtaining detection sample；Sensitivity, standard
Really property can compare favourably with tradition RT-PCR；Overcome tradition RT-PCR to need to extract this loaded down with trivial details step of RNA
Rapid deficiency, reduces operating procedure, improves detection speed, cost-effective, is prevented effectively from cross pollution, energy
Carry out the high flux detection of avian influenza virus, to quickly finding bird flu epidemic situation, formulate prevention and control measure tool in time
There is important meaning.
According to the first aspect of the invention, the present invention provides a kind of for the universal direct amplification of avian influenza virus
RT-PCR detection primer and probe sequence, including:
The detection primer of a pair avian influenza virus, its sequence is as shown in SEQ ID NO:1 and SEQ ID NO:2；
Article one, the detection probe of avian influenza virus, its sequence is as shown in SEQ ID NO:3；3 ' ends of this sequence
Combined with fluorescent quenching group, 5 ' end combined with fluorescent reporter groups；
Article one, the probe of internal standard RNA, its sequence is as shown in SEQ ID NO:4；3 ' ends of this sequence combine glimmering
Optical quenching group, 5 ' end combined with fluorescent reporter groups.
As the preferred version of the present invention, the 3 ' ends of above-mentioned probe SEQ ID NO:3 combine BHQ1 (Black
Hole Quencher-1, black hole quencher dyes-1) fluorescent quenching group, 5 ' ends combine FAM
(6-carboxy-fluorescein, 6-Fluoresceincarboxylic acid) fluorescent reporter group；Above-mentioned probe SEQ ID NO:4
3 ' ends combine BHQ3 fluorescent quenching group, 5 ' ends combine CY5 fluorescent reporter group.
According to the second aspect of the invention, the present invention provides a kind of for the universal direct amplification of avian influenza virus
The kit of RT-PCR detection, including detection primer, detection probe and internal standard probe, above-mentioned detection primer
Sequence as shown in SEQ ID NO:1 and SEQ ID NO:2, the sequence such as SEQ ID of above-mentioned detection probe
Shown in NO:3,3 ' end combined with fluorescent quenching groups of this sequence, 5 ' end combined with fluorescent reporter groups；In above-mentioned
Mark probe is as shown in SEQ ID NO:4, and 3 ' end combined with fluorescent quenching groups of this sequence, 5 ' hold combined with fluorescent
Reporter group.
As the preferred version of the present invention, the 3 ' ends of above-mentioned probe SEQ ID NO:3 are combined with BHQ1 fluorescence
Quenching group, 5 ' ends are combined with FAM fluorescent reporter group；3 ' the ends of above-mentioned probe SEQ ID NO:4 combine
BHQ3 fluorescent quenching group, 5 ' ends combine CY5 fluorescent reporter group.
As the preferred version of the present invention, mentioned reagent box includes the universal inner mark solution of avian influenza virus；Excellent
Selection of land, above-mentioned inner mark solution is expressed by universal for avian influenza virus M gene is cloned into RNA pseudovirus
Prepare in carrier pAR and by the method that the plasmid pAR-AIV UN obtained is converted by Escherichia coli.
As the preferred version of the present invention, mentioned reagent box also includes reverse transcriptase and Taq archaeal dna polymerase.
As the preferred version of the present invention, mentioned reagent box also includes PCR reinforcing agent；Preferably, above-mentioned PCR
Reinforcing agent is selected from carnitine (L-carnitine), glycine betaine (Glycine betaine), trehalose
(D-(+)-trehalose), one or more in nonionic detergent NP-40.
As the preferred version of the present invention, the detection object of mentioned reagent box is that brush,throat liquid, cloaca are wiped
Sub-liquid, tissue exudates or allantoic fluid.
Compared with prior art, the method have the advantages that primer and the probe using the present invention,
Be capable of directly liquid sample being added reaction system, in a PCR pipe continuously, be automatically obtained virus
The release of RNA, reverse transcription and fluorescence quantitative PCR detection；Achieve without extracting viral RNA, directly
The purpose of avian influenza virus is detected, from obtaining sample to be detected to only obtaining testing result from the sample taked
Need 1.5 hours；Effectively overcome tradition RT-PCR and need to extract the deficiency of RNA, can greatly reduce
Operating procedure, increases substantially detection speed, cost-effective, is prevented effectively from cross pollution.
Additionally, the kit of the present invention can also contain multiple PCR reinforcing agent, include but not limited to carnitine,
Glycine betaine, trehalose, nonionic detergent NP-40, they can carry the amplification efficiency of high GC content gene,
Increase the reaction system tolerance to PCR inhibiting substances, effectively reduce the fluorescent quenching effect of inhibiting substances,
NP-40 additionally aids lytic virus shell to discharge RNA simultaneously.Use the primer of the present invention and probe
The sensitivity of RT-PCR detection method is not less than tradition RT-PCR, even in low concentration PBS or the feelings of body fluid
Under condition, sensitivity is higher than tradition RT-PCR.
Accompanying drawing explanation
Fig. 1 is the collection of illustrative plates of RNA pseudovirus expression plasmid carrier pAR-AIV UN in the embodiment of the present invention 1；
Fig. 2 is the RT-PCR detection method of the universal direct amplification of avian influenza virus in the embodiment of the present invention 2
Sensitivity comparison diagram with the traditional RT-PCR method detection throat swab liquid sample extracting RNA；
Fig. 3 is the RT-PCR detection method of the universal direct amplification of avian influenza virus in the embodiment of the present invention 2
Sensitivity comparison diagram with the traditional RT-PCR method detection allantoic fluid sample extracting RNA；
Fig. 4 is the RT-PCR detection method of the universal direct amplification of avian influenza virus in the embodiment of the present invention 3
Tolerance figure to variable concentrations throat swab liquid (10～40%)；
Fig. 5 is to use RT-PCR detection method of the present invention detection avian influenza virus in the embodiment of the present invention 4
Dynamic range；
Fig. 6 is the detection method detection avian influenza virus using RT-PCR of the present invention in the embodiment of the present invention 4
The linearity.
Detailed description of the invention
Combine accompanying drawing below by detailed description of the invention the present invention is described in further detail.
One of key of the present invention is: have found one group and can be used in the universal direct amplification of avian influenza virus
RT-PCR detection primer, probe sequence, specifically as shown in SEQ ID NO:1-4.Use the present invention's
Primer and probe or kit, and combine other conventional reagent component, it is possible to realize connecting in a PCR pipe
Continue, automatically carry out the release of viral RNA, reverse transcription and fluorescence quantitative PCR detection；From obtaining detection sample
This has only to 1.5 hours to obtaining testing result；Sensitivity, accuracy can compare favourably with tradition RT-PCR；
Overcome tradition RT-PCR and need to extract the deficiency of this tedious steps of RNA, reduce operating procedure, improve
Detection speed, cost-effective, it is prevented effectively from cross pollution, the high flux detection of avian influenza virus can be carried out,
To quickly finding bird flu epidemic situation, formulate prevention and control measure in time and have great importance.
3 ' the end combined with fluorescent quenching groups of the probe sequence SEQ ID NO:3 of the present invention, 5 ' end combined with fluorescent
Reporter group；3 ' the end combined with fluorescent quenching groups of probe sequence SEQ ID NO:4,5 ' end combined with fluorescent reports
Accuse group.
In order to avoid detection signal interferes with internal standard signal, the fluorescence report base of probe SEQ ID NO:3
Group is different from the fluorescent reporter group of probe SEQ ID NO:4.
In one embodiment of the invention, the 3 ' ends of probe SEQ ID NO:3 combine BHQ1 fluorescent quenching
Group, 5 ' ends combine FAM fluorescent reporter group；3 ' the ends of probe SEQ ID NO:4 combine BHQ3 fluorescence
Quenching group, 5 ' ends combine CY5 fluorescent reporter group.
In one embodiment of the invention, kit also includes the universal inner mark solution of avian influenza virus；Excellent
Selection of land, above-mentioned inner mark solution is expressed by universal for avian influenza virus M gene is cloned into RNA pseudovirus
Prepare in carrier pAR and by the method that the plasmid pAR-AIV UN obtained is converted by Escherichia coli.Specifically
Ground, preparation method includes:
(1) universal for avian influenza virus M gene is cloned in RNA pseudovirus expression vector pAR,
To plasmid pAR-AIV UN；
(2) plasmid pAR-AIV UN is converted e. coli bl21；
(3) recombinant bacterium step (2) obtained is in the LB fluid nutrient medium containing Amp, swings overnight incubation；
(4) taking after incubated overnight proceeds to bacteria log growth period, IPTG Fiber differentiation crosses liquid；
(5) collect thalline, slightly carry, essence carries pseudovirus, is prepared as pseudovirus positive reference substance mother liquor；
(6) pseudovirus positive reference substance mother liquor presses 10 times of gradient dilutions to 10-8, then led to by avian influenza virus
With type standard items, this dilution is carried out quantitatively, thus obtain the universal inner mark solution of avian influenza virus.
In one embodiment of the invention, kit also includes reverse transcriptase and Taq archaeal dna polymerase.Excellent
Selection of land, reverse transcriptase and Taq archaeal dna polymerase specifically directly RT-PCR enzyme mixture (Direct RT-PCR
Enzyme mix) form, i.e. include reverse transcriptase and the mixture of Taq archaeal dna polymerase, Ke Yizhi
Connect in RT-PCR reacts.Present invention preferably employs height endurability Taq archaeal dna polymerase, because original
In biological specimen, often contain in the material of a large amount of suppression PCR, such as whole blood (or serum, blood plasma) and contain
There are a large amount of immunoglobulin (Ig), hemoglobin, heme etc.；Containing more humic acid in soil, milk
In containing more lactoferrin etc..The kit of the present invention uses height endurability Taq archaeal dna polymerase, permissible
Tolerate the impact of these materials.
Below by specific embodiment, the present invention is described in further detail.Unless stated otherwise, below in fact
The technology used in example of executing is routine techniques known to those skilled in the art；The instrument used
Equipment and reagent etc., being those skilled in the art can be obtained as being purchased etc. by public approach.
The experiment material related in following example is as follows:
1. sample process:
Brush,throat liquid, cloacal swab liquid: in equipped with the centrifuge tube of swab, add 500 μ L PBS or lifes
Reason salt solution, acutely vibrate 30s, and extrusion liquid moves on to 6000rpm in centrifuge tube and is centrifuged 20s as far as possible, takes supernatant
Standby.
Tissue exudates, allantoic fluid: sample this 6000rpm and be centrifuged 1min, take supernatant standby.
2. reagent: Direct RT-PCR 2 × buffer mix (comprise 20% (V/V) glycerine, 50mM KCl,
10mM Tris-HCl (pH7.5), 1.5mM MgCl2, 0.8mM dNTPs, 0.4mM carnitine (L-carnitine),
0.5mM trehalose (D-(+)-trehalose), 0.1% (V/V) NP-40 (Nonidet P-40), 1wt%
BSA, 0.01% (V/V) Tween-20,1U/ μ L RNase inhibitor (RNase Inhibitor), its
Remaining is deionized water)；Direct RT-PCR enzyme mix (2.5U/ μ L Taq archaeal dna polymerase and 5U/ μ L
Reverse transcriptase is obtained by mixing by 2:1 volume ratio)；Viral RNA post extraction reagent kit.
3. instrument: quantitative real time PCR Instrument (ABI7500), refrigerated centrifuge (5804R, Eppendorf).
The RT-PCR detection method of the universal direct amplification of embodiment 1 avian influenza virus
1, avian influenza virus is universal RT-PCR detection primer and the design of probe
Bird flu nucleotide sequence according to GenBank, selects the conserved sequence district of M gene as amplification
Region, utilize ABI Primer Express software separately design the universal upstream and downstream primer of avian influenza virus and
The detection probe that avian influenza virus is universal, its sequence is as follows:
The sequence of the upstream primer (AIV UN-PF) that avian influenza virus is universal is:
5’-TGAACACGACCTAGACTGACCCAGAA-3’(SEQ ID NO:1)；
The sequence of the downstream primer (AIV UN-PR) that avian influenza virus is universal is:
5’-TGGCATGTTGGACACAATGTCAAG-3’(SEQ ID NO:2)；
The sequence of the detection Taqman probe (AIV UN-PB) that avian influenza virus is universal is:
5’-AGGCTTCACTCCTGCATTGTGACGGTG-3’(SEQ ID NO:3)。
3 ' ends of the detection probe that avian influenza virus is universal combine BHQ1 fluorescent quenching group, and 5 ' ends combine
FAM fluorescent reporter group.
Inventor is investigated the validity of other primer and probe sequence, finds to use other primer and probe
Under sequence peak optimization reaction system in the present embodiment and reaction condition, all can not successfully realize directly expanding
RT-PCR detection, and be respectively directed to these unsuccessful primers and other reaction system and anti-used instead by probe
Answer condition, the most all can not realize the purpose of the RT-PCR detection directly expanded.
2, the universal internal standard of avian influenza virus detects design and the preparation of inner mark solution of probe
Avian influenza virus universal internal standard detection probe (AIV UN is designed according to the method described in step 1
IC-PB), particular sequence is as follows:
5’-CCGGTGTCCGCATTGTGATCAC-3’(SEQ ID NO:4)。
3 ' ends of avian influenza virus universal internal standard detection probe combine BHQ3 fluorescent quenching group, 5 ' end knots
Close CY5 fluorescent reporter group.
According to " Molecular Cloning: A Laboratory guide (third edition) ", carry out avian influenza virus universal M gene gram
Grand and order-checking, M gene is cloned in RNA pseudovirus expression vector pAR, recombinant plasmid is named
pAR-AIV UN.RNA pseudovirus expression vector pAR is (purchased from EMD at pET32a carrier
Biosciences (Novagen)) MCS insert the maturase of bacteriophage MS2 and capsid protein gene
(RNA of MS2 is purchased from Shanghai company of Roche Diagnistics) is built into RNA pseudovirus plasmid vector, and collection of illustrative plates is such as
Shown in Fig. 1.
The plasmid pAR-AIV UN built is converted e. coli bl21 (DE3).Picking recombinant bacterium
Single bacterium colony, accesses in the 2mL LB fluid nutrient medium containing Amp (100 μ g/mL), 37 DEG C of shaken cultivation mistakes
Night；Take 500 μ L overnight culture and access in the 50mL LB fluid nutrient medium containing Amp (100 μ g/mL),
37 DEG C of shaken cultivation 2.0h are to bacteria log growth period；Derivant IPTG is added to final concentration in culture
For 1.0mmol/L, continue shaken cultivation 16h (overnight) and take out afterwards.
Bacterium solution after induction is moved to 10mL centrifuge tube, and 5000rpm collects thalline, every 10mL bacterium solution 3mL
PBS washes twice, finally with 1mL PBS suspension thalline；Add DNase and RNase A after ultrasonication to disappear
Change 2h.700 μ LPBS and 200 μ L 5M NaCl are added in 100 μ L pseudovirus crude extracts, mixing, ice
Upper placement 1h.Add 0.1g PEG6000, after fully dissolving, continue on ice to place 2h.12000rpm
4 DEG C of centrifugal 20min, move and abandon supernatant, add 500 μ L SM buffer solutions, at room temperature vortex oscillation 3min,
Being centrifuged 5min in 12000rpm after placing 5min, transfer supernatant is to new centrifuge tube.Add 300 μ L chlorine
Imitative, at room temperature vortex oscillation 1min, 12000rpm is centrifuged 2min, transfer supernatant to the most marked new from
In heart pipe, and add the sterile glycerol of 1/10 volume, fully mix, be prepared as pseudovirus positive reference substance female
Liquid.By 10 times of gradient dilutions of this reference substance to 10-8, by the universal standard items of avian influenza virus, this is diluted
Liquid is carried out quantitatively, thus obtains pseudovirus inner mark solution.
3, groping of RT-PCR reaction system is directly expanded
Direct RT-PCR 2 × buffer mix, Direct RT-PCR is comprised in 25 μ L reaction systems
Enzyme mix, upstream primer (SEQ ID NO:1), downstream primer (SEQ ID NO:2), avian influenza virus are logical
By the detection probe (SEQ ID NO:3) of type, avian influenza virus universal internal standard detection probe (SEQ ID NO:4)
And appropriate liquid sample.Series of parameters is groped, including primer concentration (0.2～0.4 μM), visits
Pin concentration (0.2～0.4 μM), the consumption (0.75～1.50 μ L/25 μ L) of Direct RT-PCR enzyme mix
Deng.Positive control and negative control are set；Wherein positive control uses 1 μ L AIV UN RNA to be template (100
μ L AIV sample viral RNA post extraction reagent kit extracts, and elutes with 100 μ L RNase-free water
RNA)；Negative control is template by 1 μ L AIV negative sample.Every kind of sample (includes that feminine gender, the positive are right
According to) each 3 repetitions.
Through groping, it is determined that directly expand the reaction system of RT-PCR.25 μ L reaction systems are wrapped
Containing 12.5 ± 0.25 μ L Direct RT-PCR 2 × buffer mix, 1.25 μ L Direct RT-PCR enzyme mix,
0.3 ± 0.1 μM of upstream primer (SEQ ID NO:1), 0.3 ± 0.1 μM of downstream primer (SEQ ID NO:2), SEQ
Taqman probe shown in ID NO:3 and SEQ ID NO:4 is all 0.2 ± 0.1 μM, 1.25～5 μ L detections
Sample, remaining is deionized water.Detection sample is the mixed liquor of sample to be checked and inner mark solution.Sample to be checked
It is 5～10:1 with the volume ratio of inner mark solution.The detection Ct value of inner mark solution is about 30.
Optimum directly amplification RT-PCR reaction system is: comprise 12.5 μ L in 25 μ L reaction systems
Direct RT-PCR 2 × buffer mix, 1.25 μ L Direct RT-PCR enzyme mix, 0.3 μM of upstream
Primer (SEQ ID NO:1), 0.3 μM of downstream primer (SEQ ID NO:2), SEQ ID NO:3 and SEQ ID
Taqman probe shown in NO:4 0.2 μM respectively, 1.25～5 μ L detect sample, and remaining is deionized water.
4, fluorescent quantitation directly expands groping of RT-PCR reaction condition
The reaction tube filtering out peak optimization reaction system in step 3 is put in quantitative real time PCR Instrument, to series
Reaction condition is groped, including:
(1) viral RNA release and the temperature (55～60 DEG C) of reverse transcription；
(2) annealing/elongating temperature (60～65 DEG C), annealing/extension of time (30～120s), PCR cycle
Number (35～45).
During PCR, collect fluorescence signal (FAM and CY5) in the annealing/extension stage of each circulation.
Through groping, it is determined that fluorescent quantitation directly expands the response procedures of RT-PCR: 55 ± 1 DEG C are reacted 30
Min, 95 DEG C of reaction 5min；94 DEG C of sex change 30s, 60 ± 1 DEG C of annealing/extension 40s, react 35～45
Circulation.Optimum fluorescent quantitation directly expands RT-PCR reaction condition and is: 55 DEG C of reaction 30min, 95 DEG C
Reaction 5min；94 DEG C of sex change 30s, 60 DEG C of annealing/extension 40s, react 40 circulations.
5, result judges
Using the peak optimization reaction system of step 3, detection sample is carried out directly by the optimum reaction condition of step 4
Amplification RT-PCR, obtains amplification curve and the Ct value of each reaction tube from quantitative fluorescent PCR, obtains every kind
The Ct value of sample.The knot when the Ct value of internal standard testing result is less than 35 or target gene testing result is positive
Fruit is effectively；If result is invalid when internal standard detection and target gene testing result are feminine gender, this sample need to be repeated
Detection.Normal testing result should be: negative control is without detection signal, and positive control has detection signal
(Ct<30)；
Positive sample: target gene has detection signal, and internal standard detection is with or without detection signal；
Negative sample: target gene has detection signal without detection signal, internal standard detection；
The target gene detection of sample, if Ct value ＞ 40.0 or without amplification curve, is then feminine gender；Ct value≤35.0,
And typical amplification curve occurs, the positive can be judged to；The detection of reforming between 35.0-40.0 of the Ct value, reforms
Result Ct value is negative more than 40.0 or without amplification curve person, is otherwise positive.
Carry out directly with bird flu H5 hypotype, H7 hypotype, H9 hypotype inactivation of viruses and deionized water for sample
Meeting amplification RT-PCR, result is as shown in table 1.
Table 1 directly expands RT-PCR testing result
The sensitivity test of the detection method of embodiment 2 embodiment 1
Using the peak optimization reaction system of step 3 in embodiment 1, the optimum reaction condition of step 4 is to throat swab
Liquid sample and allantoic fluid sample directly expand RT-PCR, obtain each reaction tube from quantitative fluorescent PCR
Amplification curve and Ct value, calculate the Ct mean value of every kind of sample.From figures 2 and 3, it will be seen that for
Throat swab liquid sample, the AIV RNA using 1 μ L to purify is template, and the Ct value of quantitative fluorescent PCR is
19.66, and use the directly amplification RT-PCR of embodiment 1 to detect from 1 μ L AIV PBS eluent
Ct value is 18.25 (such as Fig. 2).
For allantoic fluid sample, the AIV RNA using 1 μ L to purify is template, the Ct of quantitative fluorescent PCR
Value is 20.05, and uses the directly amplification RT-PCR of embodiment 1 to detect from 1 μ L AIV allantoic fluid
Ct value be 19.19 (such as Fig. 3), this result explanation the present invention directly amplification RT-PCR method detect AIV
Sensitivity can compare favourably with traditional RT-PCR method with purifying RNA as template, even tradition
RT-PCR sensitivity is higher.
The throat swab liquid tolerance test of the detection method of embodiment 3 embodiment 1
The reactant liquor in addition to throat swab liquid is prepared according to the peak optimization reaction system of embodiment 1 step 3；By 1 μ L
AIV positive throat swab liquid, is diluted to 2.5,5,7.5,10 μ L respectively with the negative throat swab liquid without AIV,
It is then respectively adding in reaction tube, makes the AIV viral copy number in each pipe keep consistent, and throat swab liquid accounts for
The percentage of cumulative volume is respectively 10%, 20%, 30%, 40%.Each reaction tube is put into quantitative fluorescent PCR
Instrument, is reacted by the optimum reaction condition of embodiment 1 step 4.
Result shows, directly amplification RT-PCR still has greater activity in the throat swab liquid of up to 40%, with
10% concentration is compared the change of Ct value and is less than 1 value, has the highest tolerance, such as Fig. 4.
The detection dynamic range of the detection method of embodiment 4 embodiment 1 and the linearity
The reactant liquor in addition to sample is prepared according to the peak optimization reaction system of step 3 in embodiment 1；Take portion
AIV throat swab liquid positive sample, carries out 10 times of gradient dilutions with the negative throat swab liquid without AIV, obtains
100、10-1、10-2、10-3μ L positive throat swab liquid sample, the throat swab liquid of all reaction tubes maintenance 5% is eventually
Concentration.Take various dilution throat swab liquid 1 μ L respectively and add reaction tube, be placed in quantitative real time PCR Instrument,
React by the optimum reaction condition of embodiment 1 step 4.
Result shows, the throat swab liquid of 1000 times of dilutions still obtains positive assay signal, and (Fig. 5 is that each concentration is glimmering
Light PCR amplification curve diagram, Fig. 6 is range of linearity figure)；Coefficient correlation between extension rate and Ct value is
0.996, show that linear relationship is preferable.
Above content is to combine specific embodiment further description made for the present invention, it is impossible to recognize
Determine the present invention be embodied as be confined to these explanations.Ordinary skill for the technical field of the invention
For personnel, without departing from the inventive concept of the premise, it is also possible to make some simple deduction or replace,
All should be considered as belonging to protection scope of the present invention.
Claims (7)
1. the primer for the RT-PCR detection of the universal direct amplified fluorescence of avian influenza virus and probe sequence, it is characterised in that including:
The detection primer of a pair avian influenza virus, its sequence is as shown in SEQ ID NO:1 and SEQ ID NO:2；
Article one, the detection probe of avian influenza virus, its sequence is as shown in SEQ ID NO:3；3 ' end combined with fluorescent quenching groups of this sequence, 5 ' end combined with fluorescent reporter groups；
Article one, the probe of internal standard RNA, its sequence is as shown in SEQ ID NO:4；3 ' end combined with fluorescent quenching groups of this sequence, 5 ' end combined with fluorescent reporter groups.
Primer the most according to claim 1 and probe sequence, it is characterised in that the 3 ' ends of described probe SEQ ID NO:3 combine BHQ1 fluorescent quenching group, 5 ' ends combine FAM fluorescent reporter group；Described probe SEQ
3 ' the ends of ID NO:4 combine BHQ3 fluorescent quenching group, and 5 ' ends combine CY5 fluorescent reporter group.
3. the kit for the RT-PCR detection of the universal direct amplification of avian influenza virus, it is characterized in that, described kit includes detection primer, detection probe and internal standard probe, the sequence of described detection primer is as shown in SEQ ID NO:1 and SEQ ID NO:2, the sequence of described detection probe is as shown in SEQ ID NO:3,3 ' end combined with fluorescent quenching groups of this sequence, 5 ' end combined with fluorescent reporter groups；Described internal standard probe is as shown in SEQ ID NO:4, and 3 ' end combined with fluorescent quenching groups of this sequence, 5 ' hold combined with fluorescent reporter groups.
Kit the most according to claim 3, it is characterised in that the 3 ' ends of described probe SEQ ID NO:3 are combined with BHQ1 fluorescent quenching group, 5 ' ends are combined with FAM fluorescent reporter group；3 ' the ends of described probe SEQ ID NO:4 combine BHQ3 fluorescent quenching group, and 5 ' ends combine CY5 fluorescent reporter group.
5. according to the kit described in any one of claim 3-4, it is characterised in that described kit includes the universal inner mark solution of avian influenza virus；
Preferably, described inner mark solution is by being cloned into universal for avian influenza virus M gene in RNA pseudovirus expression vector pAR and the method that the plasmid pAR-AIV UN obtained is converted by Escherichia coli being prepared.
6. according to the kit described in any one of claim 3-4, it is characterised in that described kit also includes PCR reinforcing agent；
Preferably, one or more in carnitine, glycine betaine, trehalose and nonionic detergent NP-40 of described PCR reinforcing agent.
7. according to the kit described in any one of claim 3-4, it is characterised in that the detection object of described kit is brush,throat liquid, cloacal swab liquid, tissue exudates or allantoic fluid.
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| CN106978501A (en) * | 2017-05-02 | 2017-07-25 | 陈超 | A kind of method of the direct real-time fluorescence PCR of whole blood |
| CN108624714A (en) * | 2018-04-28 | 2018-10-09 | 佛山科学技术学院 | RPA-L FD visual kit for detecting avian influenza virus and application thereof |
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| CN103773896A (en) * | 2014-01-13 | 2014-05-07 | 深圳澳东检验检测科技有限公司 | Fluorescent quantitation RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent of West Nile virus, kit and detection method of West Nile virus |
| CN104561383A (en) * | 2015-01-15 | 2015-04-29 | 深圳澳东检验检测科技有限公司 | Influenza A virus and B virus joint detection primer, probe, kit and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106978501A (en) * | 2017-05-02 | 2017-07-25 | 陈超 | A kind of method of the direct real-time fluorescence PCR of whole blood |
| CN106978501B (en) * | 2017-05-02 | 2020-11-10 | 陈超 | Direct real-time fluorescence PCR method for whole blood |
| CN108624714A (en) * | 2018-04-28 | 2018-10-09 | 佛山科学技术学院 | RPA-L FD visual kit for detecting avian influenza virus and application thereof |
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Inventor after: Di Jianxin Inventor after: Wu Xiaowei Inventor after: Deng Guodong Inventor after: Lu Shousheng Inventor after: Zhang Li Inventor after: Sun Yanwei Inventor after: Lin Lin Inventor after: Pan Yanping Inventor after: Yu Xiyao Inventor after: Zhong Wenyang Inventor after: Gao Huimin Inventor before: Di Jianxin Inventor before: Zhang Li Inventor before: Sun Yanwei Inventor before: Lu Shousheng Inventor before: Lin Lin Inventor before: Pan Yanping Inventor before: Wu Xiaowei Inventor before: Gao Huimin Inventor before: Deng Guodong Inventor before: Yu Xiyao |
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Application publication date: 20160713 |