CN107447054A - Bird flu and the dual droplet digital pcr absolute quantitation detection kit of ewcastle disease - Google Patents
Bird flu and the dual droplet digital pcr absolute quantitation detection kit of ewcastle disease Download PDFInfo
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- CN107447054A CN107447054A CN201710845520.6A CN201710845520A CN107447054A CN 107447054 A CN107447054 A CN 107447054A CN 201710845520 A CN201710845520 A CN 201710845520A CN 107447054 A CN107447054 A CN 107447054A
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Abstract
The invention discloses a kind of bird flu and the dual droplet digital pcr absolute quantitation detection kit of ewcastle disease.The invention provides primer combination of probe, is made up of primed probe group AIV F1R1P1 and primed probe group NDV F1R1P1；Primed probe group AIV F1R1P1 are made up of primer AIV F1, primer AIV R1 and probe AIV P1；Primer AIV F1 are as shown in sequence 1, and primer AIV R1 are as shown in sequence 2；Probe AIV P1 nucleotides sequence is classified as sequence 3；Primed probe group NDV F1R1P1 are made up of primer NDV F1, primer NDV R1 and probe NDV P1；Primer NDV F1 are as shown in sequence 4, and for primer NDV R1 as shown in sequence 5, probe NDV P1 nucleotides sequence is classified as sequence 6.Prevention and control of the present invention for avian influenza virus and/or NDV have great application value, help from Sources controlling epidemic situation, effectively to prevent the large-scale outbreak of birds epidemic disease.
Description
Technical field
The invention belongs to field of virus detection, and in particular to a kind of bird flu and the dual droplet digital pcr of ewcastle disease are absolute
Immue quantitative detection reagent box.
Background technology
Avian influenza virus (AIV) belongs to influenza A virus.Influenza virus belongs to the orthomyxoviridae family of RNA virus, divide first,
Second, the third 3 types.Wherein influenza A virus is mainly in birds, is to cause a kind of from respiratory system of birds by influenza A
To the infectious disease of a variety of symptoms such as severe total septicemia, World Organization for Animal Health is located category A infectious disease.Bird flu 1994,
Broken out respectively on Australia, Italy, Hong-Kong, Holland and other places within 1997,1999 and 2003,2005 then main
Broken out in Southeast Asia and Europe.Mainly through respiratory infectious, the birds infected by close contact and its secretion, excreta,
Water by virus pollution etc., and directly contact virus stain are infected.The disease containing high concentration in the excrement of infection aquatic bird
Poison, and influenza virus is propagated by excrement-mouth approach by the water source spring of pollution.
Position of the NDV (NDV) in viral taxonomy belongs to molecule strand RNA mesh
(Mononegavirales), paramyxovirus section (Paramyxoviridae), paramyxovirus subfamily (Paramyxovirinae),
Fowl mumps virus or avian paramyxovirus category (Avulavirus).Viral main harm chicken, galeeny and the turkey, what is attacked
Propagated rapidly in chicken group, velogen strain can destroy the full group of chicken group.Low virulent strain only causes chicken group respiratory tract infection and egg production to decline,
But can rapid rehabilitation.The mankind can cause conjunctivitis or adenolymphitis because contacting sick fowl and malicious vaccine living, but just rehabilitation quickly.This
Sick major source of infection is diseased chicken and excrement and mouth mucus with malicious chicken.By feed, drinking-water and the dust of virus pollution through digestion
It is main circulation way that road, respiratory tract or conjunctiva, which infect susceptible chicken,.
At present, mainly there are virus neutralization tests, immunofluorescence technique, immuno-electron microscope to the diagnostic method of both infectious diseases
Method, ELISA method and RT-PCR methods.With the continuous development of molecular biology, ELISA method and RT-PCR methods with its sensitiveness it is high,
High specificity is increasingly valued by people.Traditional detection method, such as virus neutralization tests (VNT), immunofluorescence technique, exempt from
Epidemic disease electron microscopy, EUSA etc., existing needs time length, dependent on veteran testing staff, test operation
It is complicated, be related to live virus operation, be higher to detection environmental requirement, and sensitiveness is relatively low and many deficiencies such as repeatability is poor.
The nucleic acid detection methods such as RT-PCR, although having than conventional method, specificity is stronger, sensitivity is higher, automaticity is higher
With the advantage such as result repeatability is more preferable, but it is only capable of realizing that the quantitative and semi-quantitative of bird flu and ewcastle disease detects, can not be to fowl
Influenza carries out accurate absolute quantitation detection with ewcastle disease nucleic acid, so as to which testing result can not reflect that virus infects true in sample
Truth condition.RT-PCR method still suffers from certain limitation in sensitivity and specificity simultaneously, is extracted for viral nucleic acid
Some chemical substances in journey are more sensitive, and then suppression PCR reacts, and " false negative " result occur.
The concept for digitizing PCR (Digital PCR, dPCR) was just used simultaneously early in 1999 by Bert Vogelstein
Pertinent literature is delivered, its original intention is to be able to substantial amounts of normal from clinical sample (such as urine, lymph, blood plasma, excrement)
Micro mutant cell is detected in body cell, but because the consumptive material that can be used for dilute sample at that time is 384 orifice plates, therefore also
The core concept of digital pcr can not very well be embodied --- " infinite dilution " (terminal dilution).Bio-Rad is public
A sample can be divided into the droplet of 20,000 nanoliter level by the droplet technology of the QX200 system cores of department, substantially will
A traditional quantitative PCR test becomes 20,000 test, substantially increases sensitivity and the precision of nucleotide sequence detection,
It is the perfection deduction to " infinite dilution " this concept, its Method And Principle can be described as droplet type digitlization PCR (Droplet
Digital PCR, ddPCR).QX200ddPCR systems include two instruments：Drop generator and droplet analyzer, and its it is related
Consumptive material.Each sample is divided into 20,000 uniform nanoliter level droplet by drop generator, wherein each droplet or without treating
Nucleic acid target molecule is examined, or contains one to several nucleic acid target molecules to be checked.The PCR reaction independent as one of each droplet
Device.Subsequent droplet is transferred in 96 hole PCR plates, carries out terminal PCR amplifications.Using droplet analyzer (droplet reader) by
Individual that each droplet is detected, the droplet interpretation for having fluorescence signal is 1, and the droplet interpretation without fluorescence signal is 0, final root
According to Poisson distribution principle and the ratio of positive droplet, analysis software can calculate the concentration or copy number for providing target molecule to be checked.
Compared with traditional quantitative PCR, the accuracy of digital pcr and sensitivity are more preferably.Using droplet type digital pcr technology, people is studied
Member can detect rare mutation, accurate measure copy number variation, and carry out absolute quantitation to gene expression.By QX200 systems
High sensitivity, researcher nowadays can detect concentration as little as 1/1,000,000 target molecule.
The content of the invention
It is an object of the invention to provide a kind of bird flu and the dual droplet digital pcr absolute quantitation detection reagent of ewcastle disease
Box.
The invention provides a kind of primer combination of probe, by primed probe group AIV-F1R1P1 and primed probe group NDV-
F1R1P1 is formed；
The primed probe group AIV-F1R1P1 is made up of primer AIV-F1, primer AIV-R1 and probe AIV-P1；
The primer AIV-F1 is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) sequence 1 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 1
The DNA molecular of identical function；
The primer AIV-R1 is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) sequence 2 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 2
The DNA molecular of identical function；
The probe AIV-P1, an end have fluorescent reporter group, and another end has fluorescent quenching group, core
Nucleotide sequence is following (a5) or (a6)：
(a5) as shown in the sequence 3 of sequence table；
(a6) as shown in by sequence 3 by the substitution of one or several nucleotides and/or missing and/or addition；
The primed probe group NDV-F1R1P1 is made up of primer NDV-F1, primer NDV-R1 and probe NDV-P1；
The primer NDV-F1 is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 4 of sequence table；
(a2) sequence 4 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 4
The DNA molecular of identical function；
The primer NDV-R1 is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 5 of sequence table；
(a4) sequence 5 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 5
The DNA molecular of identical function；
The probe NDV-P1, an end have fluorescent reporter group, and another end has fluorescent quenching group, core
Nucleotide sequence is following (a5) or (a6)：
(a5) as shown in the sequence 6 of sequence table；
(a6) as shown in by sequence 6 by the substitution of one or several nucleotides and/or missing and/or addition；
The probe AIV-P1 and probe NDV-P1 has the fluorescent reporter group of different colours.
Specifically, probe AIV-P1 5 ' end mark fluorophor FAM, 3 ' end mark fluorescent quenching groups
BHQ1。
Specifically, probe NDV-P1 5 ' end mark fluorophor HEX, 3 ' end mark fluorescent quenching groups
BHQ1。
The purposes of the primer combination of probe is following (b1), (b2), (b3), (b4), (b5) or (b6)：
(b1) avian influenza virus and/or NDV are identified；
(b2) kit for identifying avian influenza virus and/or NDV is prepared；
(b3) whether detection sample to be tested contains avian influenza virus and/or NDV；
(b4) prepare for detect sample to be tested whether the kit containing avian influenza virus and/or NDV；
(b5) content of avian influenza virus and/or NDV in sample to be tested is detected；
(b6) kit for detecting the content of avian influenza virus and/or NDV in sample to be tested is prepared.
The present invention also protects the application of the primer combination of probe, for following (b1), (b2), (b3), (b4), (b5) or
(b6)：
(b1) avian influenza virus and/or NDV are identified；
(b2) kit for identifying avian influenza virus and/or NDV is prepared；
(b3) whether detection sample to be tested contains avian influenza virus and/or NDV；
(b4) prepare for detect sample to be tested whether the kit containing avian influenza virus and/or NDV；
(b5) content of avian influenza virus and/or NDV in sample to be tested is detected；
(b6) kit for detecting the content of avian influenza virus and/or NDV in sample to be tested is prepared.
The present invention also protects a kind of kit, including the primer combination of probe；The function of the kit is as follows
(c1), (c2) or (c3)：(c1) avian influenza virus and/or NDV are identified；(c2) whether detection sample to be tested contains fowl
Influenza virus and/or NDV；(c3) content of avian influenza virus and/or NDV in sample to be tested is detected.
The kit also includes recording method I and/or method II and/or method III carrier.
The present invention also protect it is a kind of detect virus to be measured whether be avian influenza virus and/or NDV method (side
Method I), comprise the following steps：Using viral total serum IgE to be measured as template, digital RT-PCR is carried out using the primer combination of probe；
Testing result if based on the corresponding fluorophors of probe AIV-P1 is that virus positive, to be measured is or candidate is avian flu
Poison, the testing result if based on the corresponding fluorophors of probe AIV-P1 are that virus negative, to be measured is or candidate is non-fowl stream
Influenza Virus；Testing result if based on the corresponding fluorophors of probe NDV-P1 is that virus positive, to be measured is or candidate is new
City epidemic disease poison, the testing result if based on the corresponding fluorophors of probe NDV-P1 is that virus negative, to be measured is or candidate is
Non- NDV.
The present invention also protect it is a kind of detection sample to be tested whether the method containing avian influenza virus and/or NDV
(method II), comprises the following steps：Using the total serum IgE of sample to be tested as template, digital RT- is carried out using the primer combination of probe
PCR；Contain avian influenza virus if based on the testing result of the corresponding fluorophors of probe AIV-P1 for positive, sample to be tested,
If based on the testing result of the corresponding fluorophors of probe AIV-P1 avian influenza virus is not contained for negative, sample to be tested；Such as
Testing result of the fruit based on the corresponding fluorophors of probe NDV-P1 contains NDV for positive, sample to be tested, if base
In the testing result of the corresponding fluorophors of probe NDV-P1 NDV is not contained for negative, sample to be tested.
The present invention also protects a kind of method for detecting the content of avian influenza virus and/or NDV in sample to be tested
(method III), comprises the following steps：Using the total serum IgE of sample to be tested as template, digital RT- is carried out using the primer combination of probe
PCR；The content of avian influenza virus in sample to be tested is obtained according to the quantity of avian influenza virus positive droplet, avian influenza virus is positive
Droplet is that the testing result based on the corresponding fluorophors of probe AIV-P1 is positive droplet；It is micro- according to the NDV positive
The quantity of drop obtains the content of NDV in sample to be tested, and NDV positive droplet is corresponding based on probe NDV-P1
The testing result of fluorophor be positive droplet.
In methods described I or method II or method III, in the reaction system of the digital RT-PCR, primer AIV-F1's is dense
Spend for 0.9 μM, primer AIV-R1 concentration is 0.9 μM, and probe AIV-P1 concentration is 0.2 μM, and primer NDV-F1 concentration is
0.9 μM, primer NDV-R1 concentration is 0.9 μM, and probe NDV-P1 concentration is 0.2 μM.
In methods described I or method II or method III, the reaction system of the digital RT-PCR can be by 2 × One-step
RT-ddPCR Supermix for probes, primer AIV-F1, primer AIV-R1, probe AIV-P1, primer NDV-F1, primer
NDV-R1, probe NDV-P1 and template composition.
In methods described I or method II or method III, the reaction system of the digital RT-PCR can be by 2 × One-step
RT-ddPCR Supermix for probes, primer AIV-F1, primer AIV-R1, probe AIV-P1, primer NDV-F1, primer
NDV-R1, probe NDV-P1, template and RNase Free dH2O is formed.
In methods described I or method II or method III, the response procedures of the digital RT-PCR can be：50 DEG C of reverse transcriptions
10min；95 DEG C of pre-degeneration 10min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 60sec, totally 40 circulations；98℃10min.
In methods described I or method II or method III, in the response procedures of the digital RT-PCR, warming and cooling rate is set
For 2.5 DEG C/sec.
The present invention also protects a kind of premixed liquid, wherein containing primer AIV-F1, primer AIV-R1, probe AIV-P1, primer
NDV-F1, primer NDV-R1 and probe NDV-P1；In the premixed liquid, primer AIV-F1 concentration is 1 μM, primer AIV-R1's
Concentration is 1 μM, and probe AIV-P1 concentration is 2/9 μM, and primer NDV-F1 concentration is 1 μM, and primer NDV-R1 concentration is 1 μ
M, probe NDV-P1 concentration are 2/9 μM.The function of the premixed liquid is following (c1), (c2) or (c3)：(c1) fowl stream is identified
Influenza Virus and/or NDV；(c2) whether detection sample to be tested contains avian influenza virus and/or NDV；(c3)
Detect the content of avian influenza virus and/or NDV in sample to be tested.
The present invention also protects a kind of kit, including the premixing and droplet that oil occurs.The kit also includes the moon
Property control and positive control；The negative control is RNase Free dH2O.The positive control is to contain avian influenza virus
The solution of the total serum IgE of total serum IgE and NDV.The function of the kit is following (c1), (c2) or (c3)：(c1) identify
Avian influenza virus and/or NDV；(c2) whether detection sample to be tested contains avian influenza virus and/or NDV；
(c3) content of avian influenza virus and/or NDV in sample to be tested is detected.
The kit also includes recording method IV and/or the carrier of method V and/or method VI.
Methods described IV is that one kind detects whether virus to be measured is avian influenza virus and/or ewcastle disease by digital RT-PCR
The method of virus, comprises the following steps：Using viral total serum IgE to be measured or its dilution as template solution, by 2 μ L template solutions
Droplet is formed with same be placed in droplet generation instrument of oil occurs after premixed liquid mixing described in 18 μ L with 70 μ L droplets, then carries out RT-
PCR is expanded, and is then detected in droplet analyzer；If based on the testing result of the corresponding fluorophors of probe AIV-P1
It is for virus positive, to be measured or candidate is avian influenza virus, if based on the detection knot of the corresponding fluorophors of probe AIV-P1
Fruit is that virus negative, to be measured is or candidate is non-avian influenza virus；If based on the inspection of the corresponding fluorophors of probe NDV-P1
It is that virus positive, to be measured is or candidate is NDV to survey result, if based on the corresponding fluorophors of probe NDV-P1
Testing result is that virus negative, to be measured is or candidate is non-NDV.
Methods described V detects whether sample to be tested contains avian influenza virus and/or new city to be a kind of by digital RT-PCR
The method of epidemic disease poison, comprises the following steps：It is using the total serum IgE of sample to be tested or its dilution as template solution, 2 μ L templates are molten
Oil occurs with 70 μ L droplets after premixed liquid mixing described in liquid and 18 μ L and forms droplet with being placed in droplet generation instrument, then carries out
RT-PCR is expanded, and is then detected in droplet analyzer；If based on the detection of the corresponding fluorophors of probe AIV-P1
As a result avian influenza virus is contained for positive, sample to be tested, if based on the testing result of the corresponding fluorophors of probe AIV-P1
Avian influenza virus is not contained for negative, sample to be tested；Testing result if based on the corresponding fluorophors of probe NDV-P1 is
Positive, sample to be tested contains NDV, if based on the corresponding fluorophors of probe NDV-P1 testing result for it is negative,
Sample to be tested does not contain NDV.
Methods described VI detects avian influenza virus and/or NDV in sample to be tested to be a kind of by digital RT-PCR
Content method, comprise the following steps：It is using the total serum IgE of sample to be tested or its dilution as template solution, 2 μ L templates are molten
Oil occurs with 70 μ L droplets after premixed liquid mixing described in liquid and 18 μ L and forms droplet with being placed in droplet generation instrument, then carries out
RT-PCR is expanded, and is then detected in droplet analyzer；Obtain treating test sample according to the quantity of avian influenza virus positive droplet
The content of avian influenza virus in this, avian influenza virus positive droplet is the detection knot based on the corresponding fluorophors of probe AIV-P1
Fruit is positive droplet；The content of NDV in sample to be tested is obtained according to the quantity of NDV positive droplet, newly
City epidemic disease virus-positive droplet is that the testing result based on the corresponding fluorophors of probe NDV-P1 is positive droplet.
In methods described IV or method V or method VI, the response procedures of the digital RT-PCR can be：50 DEG C of reverse transcriptions
10min；95 DEG C of pre-degeneration 10min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 60sec, totally 40 circulations；98℃10min.
In methods described I or method II or method III, in the response procedures of the digital RT-PCR, warming and cooling rate is set
For 2.5 DEG C/sec.
The present invention also protects the application of the premixed liquid or the kit, for following (c1), (c2) or (c3)：(c1) reflect
Determine avian influenza virus and/or NDV；(c2) whether detection sample to be tested contains avian influenza virus and/or Newcastle Disease
Poison；(c3) content of avian influenza virus and/or NDV in sample to be tested is detected.
Concretely droplet type digitizes RT-PCR to digital RT-PCR described in any of the above.
The concretely droplet numeral RT-PCR absolute quantitation detection kits of kit described in any of the above.
Digital RT-PCR described in any of the above specifically uses QX200Droplet Digital PCR systems.
Sample to be tested described in any of the above can be birds tissue samples to be measured, such as chicken tissues sample.
Virus to be measured described in any of the above can be avian influenza virus, NDV, infectious bursa of Fabricius virus or infection
Property bronchitis virus.
Avian influenza virus described in any of the above can be H1N1 subtype avian influenza virus, H5N6 subtype avian influenza virus, H5N1
Subtype avian influenza virus, H7N9 subtype avian influenza virus or H9N2 subtype avian influenza virus.
Avian influenza virus described in any of the above can be A/duck/Hubei/3/2005 strains, MD/San-jiang/390/
2007 strains, A/Hero/Guangdong/C1/2013 strains, A/chicken/Yunnan/1/2014, A/Anhui/1/2013
Strain or A/chicken/Shanghai/10/01 strains.
NDV described in any of the above can be JSD0812 strains.
Infectious bursa of Fabricius virus described in any of the above can be HQ-b strains.
Infectious bronchitis virus described in any of the above can be LX4 strains.
The present inventor designs and screens to have obtained the primer that can identify avian influenza virus and/or NDV
Probe combinations, and further groped the concentration of primer and probe, the working concentration of most suitable primer and probe is have found, with
Improve ddPCR amplification efficiency.
The invention provides with high sensitivity, high specific, high accuracy, pinpoint accuracy, accurate quantification can be achieved
Droplet type digitizes RT-PCR kit, for the detection of avian influenza virus and/or NDV, can direct quantitative, without
Standard curve, easy to operate, result accurately and reliably, are particularly suitable for Site Detection.The present invention is for avian influenza virus and/or new city
The prevention and control of epidemic disease poison have great application value, contribute to from Sources controlling epidemic situation, effectively prevention birds epidemic disease is extensive
Outburst.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result make even
Average.
Digital pcr in embodiment uses Bio-Rad Laboratories, Inc. QX200Droplet
DigitalPCR systems (including two instruments：QX200 droplets generate instrument and QX200 droplets analyzer).Used in embodiment
Heat-sealing instrument is PX1TMPCR seals instrument, and it is DG8 that card, which occurs, for droplet used in embodimentTMCard (8 × 3), embodiment occur for droplet
In used base to occur card base, droplet used occurs oil and oily (being used for probe) occurs for droplet in embodiment, is
Bio-Rad Laboratories, Inc. product.2 × One-step RT-ddPCR Supermix for probes, it is
Bio-Rad Laboratories, Inc. product.
Fowl influenza virus strain used is that (strain is H5N1 to A/duck/Hubei/3/2005 in embodiment 1 to 3
Hypotype), the Serial No. DQ520844.1 in GENBANK.
Newcastle Disease poison strain used is JSD0812 in embodiment, the Serial No. in GENBANK
GQ849007.1。
The design and screening of embodiment 1, primer and probe
First, for the design and screening of the primer and probe for detecting avian influenza virus
By a large amount of retrievals, analysis, comparison and preliminary experiment, design and preliminary screening obtains four primers and two
Probe, nucleotide sequence are as follows：
AIV-F1 (sequence 1 of sequence table)：5’-ACYGAGGTCGARACGTACGT-3’；
AIV-R1 (sequence 2 of sequence table)：5’-CTTCAAGTYTCTGCGCGATCT-3’；
AIV-P1 (sequence 3 of sequence table)：5’-CTCTCTATCATCHCGTCAGGCMC；
AIV-F2：5’-TAACCGAGGTCGAMACGTACGT-3’；
AIV-R2：5’-CGCAGARACTTGAAGATGTCTTTG-3’；
AIV-P2：5’-TATCATCSCGTCAGGCVCCTCAA；
In above nucleotide sequence, Y represents C or T, R represent A or G, H represent A, T or C, and M represents A or C, S represent G or C,
V represents G, A or C.
AIV-F1 and AIV-F2 is sense primer, and AIV-R1 and AIV-R2 are anti-sense primer, AIV-P1 and AIV-P2
It is probe.AIV-P1 5 ' end mark fluorophor FAM, 3 ' end mark fluorescent quenching group BHQ1.The 5 ' of AIV-P2
End mark fluorophor FAM, 3 ' end mark fluorescent quenching group BHQ1.Fluorophor FAM shows blue-fluorescence.
2nd, for the screening for the primed probe group for detecting avian influenza virus
Four primers and two probes progress random combines that step 1 is screened to obtain, form eight primed probe groups：
The primed probe group AIV-F1R1P1 being made up of AIV-F1, AIV-R1 and AIV-P1, is made up of AIV-F1, AIV-R1 and AIV-P2
Primed probe group AIV-F1R1P2, the primed probe group AIV-F1R2P1 being made up of AIV-F1, AIV-R2 and AIV-P1, by
The primed probe group AIV-F1R2P2 of AIV-F1, AIV-R2 and AIV-P2 composition, is made up of AIV-F2, AIV-R1 and AIV-P1
Primed probe group AIV-F2R1P1, the primed probe group AIV-F2R1P2 being made up of AIV-F2, AIV-R1 and AIV-P2, by AIV-
The primed probe group AIV-F2R2P1 of F2, AIV-R2 and AIV-P1 composition, the primer being made up of AIV-F2, AIV-R2 and AIV-P2
Probe groups AIV-F2R2P2.
1st, avian influenza virus is taken, extracts total serum IgE.
2nd, using the total serum IgE that step 1 obtains as template, each primed probe group is respectively adopted and carries out real time fluorescent quantitative RT-
PCR。
RT-qPCR reaction system (20 μ L)：2×Probe qPCR dUTP Master Mix 10μL,
GoScript RT Mix for 1-step RT-QPCR 0.5 μ L, 10 μM of μ L of sense primer solution 1.2,10 μM of anti-sense primers are molten
The μ L of liquid 1.2,10 μM of μ L of probe solution 0.6, the μ L of template 2, surplus is water.
RT-qPCR response procedures：45℃10min；95℃2min；95 DEG C of 15s, 60 DEG C of 1min, 40 circulations.
Real-time fluorescence quantitative RT-PCR is carried out using primed probe group AIV-F1R1P1, △ Rn peak values are 1000000.Using
Other seven primed probe groups carry out real-time fluorescence quantitative RT-PCR, and △ Rn peak values are 300000-500000.Using primed probe
The signal value of group AIV-F1R1P1 amplification curve is most strong.
3rd, for the design and screening of the primer and probe for detecting NDV
By a large amount of retrievals, analysis, comparison and preliminary experiment, design and preliminary screening obtains four primers and two
Probe, nucleotide sequence are as follows：
NDV-F1 (sequence 4 of sequence table)：5’-AGTGDGACAGCCTGCTATCCT-3’；
NDV-R1 (sequence 5 of sequence table)：5’-CGCAGTWTGGCTCCAGAGTAT-3’；
NDV-P1 (sequence 6 of sequence table)：5’-TAGCARATGCCTCTCCBCAGGTTGC；
NDV-F2：5’-AGCAGTRGGACAGCCTGCTAT-3’；
NDV-R2：5’-CTCTGGAGCCAAHCTGCGCAC-3’；
NDV-P2：5’-CAAATGCCTCTCVCCAGGTTGCC；
In above nucleotide sequence, D is represented or G, A or T, and W represents A or T, R represent A or G, B represent G, T or C, and H is represented
A, T or C, V represent G, A or C.
NDV-F1 and NDV-F2 is sense primer, and NDV-R1 and NDV-R2 are anti-sense primer, NDV-P1 and NDV-P2
It is probe.NDV-P1 5 ' end mark fluorophor HEX, 3 ' end mark fluorescent quenching group BHQ1.The 5 ' of NDV-P2
End mark fluorophor HEX, 3 ' end mark fluorescent quenching group BHQ1.Fluorophor HEX shows green fluorescence.
4th, for the screening for the primed probe group for detecting NDV
Four primers and two probes progress random combines that step 3 is screened to obtain, form eight primed probe groups：
The primed probe group NDV-F1R1P1 being made up of NDV-F1, NDV-R1 and NDV-P1, is made up of NDV-F1, NDV-R1 and NDV-P2
Primed probe group NDV-F1R1P2, the primed probe group NDV-F1R2P1 being made up of NDV-F1, NDV-R2 and NDV-P1, by
The primed probe group NDV-F1R2P2 of NDV-F1, NDV-R2 and NDV-P2 composition, is made up of NDV-F2, NDV-R1 and NDV-P1
Primed probe group NDV-F2R1P1, the primed probe group NDV-F2R1P2 being made up of NDV-F2, NDV-R1 and NDV-P2, by NDV-
The primed probe group NDV-F2R2P1 of F2, NDV-R2 and NDV-P1 composition, the primer being made up of NDV-F2, NDV-R2 and NDV-P2
Probe groups NDV-F2R2P2.
1st, NDV is taken, extracts total serum IgE.
2nd, using the total serum IgE that step 1 obtains as template, each primed probe group is respectively adopted and carries out real time fluorescent quantitative RT-
PCR。
RT-qPCR reaction system (20 μ L)：2×Probe qPCR dUTP Master Mix 10μL,
GoScript RT Mix for 1-step RT-QPCR 0.5 μ L, 10 μM of μ L of sense primer solution 1.2,10 μM of anti-sense primers are molten
The μ L of liquid 1.2,10 μM of μ L of probe solution 0.6, the μ L of template 2, surplus is water.
RT-qPCR response procedures：45℃10min；95℃2min；95 DEG C of 15s, 60 DEG C of 1min, 40 circulations.
Real-time fluorescence quantitative RT-PCR is carried out using primed probe group NDV-F1R1P1, △ Rn peak values are 1000000.Using
Other seven primed probe groups carry out real-time fluorescence quantitative RT-PCR, and △ Rn peak values are 300000-500000.Using primed probe
The signal value of group NDV-F1R1P1 amplification curve is most strong.
The optimization of correlated response parameter in embodiment 2, digital RT-PCR
First, the optimization of annealing temperature
1st, avian influenza virus is taken, extracts total serum IgE.
2nd, NDV is taken, extracts total serum IgE.
The mass mixings such as the total serum IgE that the total serum IgE and step 2 for the 3, obtaining step 1 obtain, obtain RNA mixtures.
4th, the RNA mixtures obtained using step 3 is templates, using primed probe group AIV-F1R1P1 and primed probe group
The primer combination of NDV-F1R1P1 compositions carries out digital RT-PCR.
(1) it is formulated as follows system (20 μ L)：10 μ L 2 × One-step RT-ddPCR Supermix for probes, 1
μ L AIV-F1,1 μ L AIV-R1,0.2 μ L AIV-P1,1 μ L NDV-F1,1 μ L NDV-R1,0.2 μ L NDV-P1,2 μ L templates,
3.6μL RNase Free dH2O.In system, the concentration of sense primer is 0.5 μM, and the concentration of anti-sense primer is 0.5 μM, probe
Concentration be 0.1 μM.
(2) droplet is taken to occur to fix due on base, every hole adds 20 μ L steps (1) and prepared in 8 holes of a middle row
System, add 70 μ L droplets per hole in 8 holes of bottom row and oil occur, then put the base for being fixed with droplet card
Be placed in droplet generation instrument and form droplet (droplet is created on droplet and occurred in 8 holes of one row of card top).
(3) 96 orifice plates (being named as 96 orifice plates I) are taken, after completing step (2), occur the 8 of one row of card top from droplet
In individual hole, each hole takes 40 μ L, is added in a manner of one-to-one in 8 holes of 96 orifice plates I, and sealer is carried out with heat-sealing instrument.Take
One 96 orifice plate (being named as 96 orifice plates II), after completing step (2), from 8 holes that one row of card top occurs for droplet, Mei Gekong
40 μ L are taken, are added in a manner of one-to-one in 8 holes of 96 orifice plates II, sealer is carried out with heat-sealing instrument.Take 96 orifice plates (life
Entitled 96 orifice plate III), after completing step (2), from 8 holes that one row of card top occurs for droplet, each hole takes 40 μ L, with one by one
Corresponding mode is added in 8 holes of 96 orifice plates III, and sealer is carried out with heat-sealing instrument.96 orifice plates are taken (to be named as 96 orifice plates
IV) after, completing step (2), from 8 holes that one row of card top occurs for droplet, each hole takes 40 μ L, in a manner of one-to-one
Add in 8 holes of 96 orifice plates IV, sealer is carried out with heat-sealing instrument.96 orifice plates (being named as 96 orifice plates V) are taken, complete step
(2) after, from 8 holes that one row of card top occurs for droplet, each hole takes 40 μ L, and 96 orifice plates V are added in a manner of one-to-one
8 holes in, with heat-sealing instrument carry out sealer.96 orifice plates (being named as 96 orifice plates VI) are taken, after completing step (2), from droplet
In 8 holes that one row of card top occurs, each hole takes 40 μ L, is added in a manner of one-to-one in 8 holes of 96 orifice plates VI, uses
Seal instrument and carry out sealer.
(4) each 96 orifice plate for completing step (3) is placed in different PCR instruments, carries out RT-PCR amplifications.
RT-PCR amplification programs：50 DEG C of reverse transcription 10min；95 DEG C of pre-degeneration 10min；94 DEG C of denaturation 30sec, annealing
60sec, totally 40 circulations；98℃10min.The warming and cooling rate of use is arranged to 2.5 DEG C/sec.
PCR instrument where 96 orifice plate, I to 96 orifice plate VI, sets gradually following annealing temperature：52.0℃、54.0℃、55.0
℃、56.0℃、58.0℃、60.0℃。
(5) after completing step (4), 96 orifice plates is taken, are detected in droplet analyzer, the positive droplet of avian influenza virus
Blue-fluorescence is shown, the positive droplet of NDV shows green fluorescence.
Using 52.0 DEG C of annealing temperature, in every microlitre of template, the detected value of avian influenza virus is 1420copies, new city
The detected value of epidemic disease poison is 1340copies.Using 54.0 DEG C of annealing temperature, in every microlitre of template, the detection of avian influenza virus
It is worth for 1360copies, the detected value of NDV is 1320copies.Using 55.0 DEG C of annealing temperature, every microlitre of template
In, the detected value of avian influenza virus is 1680copies, and the detected value of NDV is 1640copies.Using 56.0 DEG C
Annealing temperature, in every microlitre of template, the detected value of avian influenza virus is 1400copies, and the detected value of NDV is
1570copies.Using 58.0 DEG C of annealing temperature, in every microlitre of template, the detected value of avian influenza virus is 1520copies,
The detected value of NDV is 1550copies.Using 60.0 DEG C of annealing temperature, in every microlitre of template, avian influenza virus
Detected value is 1390copies, and the detected value of NDV is 1440copies.
It is closest with actual value using 55.0 DEG C of annealing temperature, detected value.
As a result show, annealing temperature can select 55-58 DEG C, and optimal annealing temperature is 55 DEG C.
2nd, the optimization of primer, concentration and probe concentration
1st, avian influenza virus is taken, extracts total serum IgE.
2nd, NDV is taken, extracts total serum IgE.
The mass mixings such as the total serum IgE that the total serum IgE and step 2 for the 3, obtaining step 1 obtain, obtain RNA mixtures.
4th, the RNA mixtures obtained using step 3 is templates, using primed probe group AIV-F1R1P1 and primed probe group
The primer combination of NDV-F1R1P1 compositions carries out digital RT-PCR.
(1) different systems are prepared, are specifically shown in Table 1 (numeral in form is the addition volume of each component, and unit is μ L).
Sense primer concentration, anti-sense primer concentration and concentration and probe concentration for preparing system are 10 μM.
Table 1
| System 1 | System 2 | System 3 | System 4 | |
| AIV-F1 | 1.0 | 1.4 | 1.6 | 1.8 |
| AIV-R1 | 1.0 | 1.4 | 1.6 | 1.8 |
| AIV-P1 | 0.2 | 0.3 | 0.35 | 0.4 |
| NDV-F1 | 1.0 | 1.4 | 1.6 | 1.8 |
| NDV-R1 | 1.0 | 1.4 | 1.6 | 1.8 |
| NDV-P1 | 0.2 | 0.3 | 0.35 | 0.4 |
| 2×One-step RT-ddPCR Supermix for probes | 10 | 10 | 10 | 10 |
| RNAse Free dH2O | 3.6 | 1.8 | 0.9 | 0 |
| Template | 2 | 2 | 2 | 2 |
| Cumulative volume | 20 | 20 | 20 | 20 |
(2) droplet is taken to occur to fix due on base, every hole adds 20 μ L steps (1) and prepared in 8 holes of a middle row
System, add 70 μ L droplets per hole in 8 holes of bottom row and oil occur, then put the base for being fixed with droplet card
Be placed in droplet generation instrument and form droplet (droplet is created on droplet and occurred in 8 holes of one row of card top).
(3) 96 orifice plates are taken, after completing step (2), from 8 holes that one row of card top occurs for droplet, each hole takes 40 μ L,
Added in a manner of one-to-one in 8 holes of 96 orifice plates, sealer is carried out with heat-sealing instrument.
(4) 96 orifice plates that will complete step (3) are placed in PCR instrument, carry out RT-PCR amplifications.
RT-PCR amplification programs：50 DEG C of reverse transcription 10min；95 DEG C of pre-degeneration 10min；94 DEG C of denaturation 30sec, 55 DEG C of annealing
60sec, totally 40 circulations；98℃10min.The warming and cooling rate of use is arranged to 2.5 DEG C/sec.
(5) after completing step (4), 96 orifice plates is taken, are detected in droplet analyzer, the positive droplet of avian influenza virus
Blue-fluorescence is shown, the positive droplet of NDV shows green fluorescence.
Using system 1, in every microlitre of template, the detected value of avian influenza virus is 1430copies, the inspection of NDV
Measured value is 1120copies.Using system 2, in every microlitre of template, the detected value of avian influenza virus is 1520copies, ewcastle disease
The detected value of virus is 1350copies.Using system 3, in every microlitre of template, the detected value of avian influenza virus is
1580copies, the detected value of NDV is 1260copies.Using system 4, in every microlitre of template, avian influenza virus
Detected value is 1680copies, and the detected value of NDV is 1520copies.
Using system 4, detected value is closest with actual value.
As a result show, in reaction system, optimal primed probe concentration is as follows：AIV-F1 0.9μM、AIV-R1 0.9μM、
0.9 μM of 0.2 μM of AIV-P1, NDV-F1,0.9 μM of NDV-R1,0.2 μM of NDV-P1.
The preparation of embodiment 3, kit
First, the preparation of each reagent
Solution A is one-step method ddPCR sonde method premixed liquids.The composition of every 900 μ L solution As is as follows：500μL 2×One-
Step RT-ddPCR Supermix for probes, (AIV-F1 concentration is 90 μ L AIV-F1 solution in AIV-F1 solution
10 μM), 90 μ L AIV-R1 solution (AIV-R1 concentration is 10 μM in AIV-R1 solution), (AIV-P1 is molten for 20 μ LAIV-P1 solution
AIV-P1 concentration is 10 μM in liquid), 90 μ L NDV-F1 solution (upper NDV-F1 concentration is 10 μM in NDV-F1 solution), 90 μ
L NDV-R1 solution (in NDV-R1 solution NDV-R1 concentration be 10 μM), 20 μ L NDV-P1 solution (NDV- in NDV-P1 solution
P1 concentration is 10 μM).
Solution B is that oil occurs for droplet.
Solution C is positive control.The preparation method of solution C：The total of avian influenza virus and NDV is extracted respectively
RNA, then mix, diluted using Tris-EDTA buffer solutions (pH8.0,0.01M), make avian influenza virus RNA and NDV
RNA concentration be 10000 copy numbers/microlitre.
Solution D is negative control.Solution D is RNase Free dH2O。
2nd, the assembling of droplet digital pcr absolute quantitation detection kit
Kit forms are as follows：Solution A, solution B, solution C, solution D, are independently packed.
Sample to be tested is birds tissue to be measured viral or to be measured.
1st, the total serum IgE of sample to be tested is extracted, using total serum IgE or its dilution as template solution.
2nd, 18 μ L solution As are taken, add 2 μ L template solutions.Template solution is replaced as at negative control by the use of isometric solution D
Reason.Template solution is replaced as positive control processing by the use of isometric solution C.
3rd, take droplet to occur to fix due on base, the body that 20 μ L steps 2 are prepared is added per hole in 8 holes of a middle row
It is to add 70 μ L solution Bs in 8 holes of bottom row per hole, the base for being fixed with droplet card is then positioned over droplet
Droplet is formed in generation instrument (droplet is created on droplet and occurred in 8 holes of one row of card top).
4th, 96 orifice plates are taken, after completing step 3, from 8 holes that one row of card top occurs for droplet, each hole takes 40 μ L, with
One-to-one mode is added in 8 holes of 96 orifice plates, and sealer is carried out with heat-sealing instrument.
5th, 96 orifice plates for completing step 4 are placed in PCR instrument, carry out RT-PCR amplifications.
RT-PCR amplification programs：50 DEG C of reverse transcription 10min；95 DEG C of pre-degeneration 10min；94 DEG C of denaturation 30sec, 55 DEG C of annealing
60sec, totally 40 circulations；98℃10min.The warming and cooling rate of use is arranged to 2.5 DEG C/sec.
(5) after completing step (4), 96 orifice plates is taken, are detected in droplet analyzer, the positive droplet of avian influenza virus
Blue-fluorescence is shown, the positive droplet of NDV shows green fluorescence.
Meet following four condition stub credible result simultaneously：1. in every microlitre of positive control, the detection of avian influenza virus
It is worth for 10000 ± 100 copy numbers；2. in every microlitre of positive control, the detected value of NDV is 10000 ± 100 copy numbers；
3. in every microlitre of negative control, the detected value of avian influenza virus is 0；4. in every microlitre of negative control, the detection of NDV
It is worth for 0.If detecting the positive droplet of avian influenza virus in template, illustrate to contain avian influenza virus in sample to be tested, can root
The copy number of avian influenza virus is obtained according to positive number of droplets；If the micro- of the avian influenza virus positive is not detected in template
Drop, illustrates not containing avian influenza virus in sample to be tested.If detecting the positive droplet of NDV in template, explanation is treated
Contain NDV in test sample sheet, the copy number of NDV can be obtained according to positive number of droplets；If do not have in template
There is the droplet for detecting that NDV is positive, illustrate not containing NDV in sample to be tested.
Embodiment 4, universality detection and specific assay
Sample to be tested is respectively：
H1N1 subtype avian influenza virus MD/San-jiang/390/2007 strains；
H5N6 subtype avian influenza virus A/Hero/Guangdong/C1/2013 strains；
H5N1 subtype avian influenza virus A/chicken/Yunnan/1/2014；
H7N9 subtype avian influenza virus A/Anhui/1/2013 strains；
H9N2 subtype avian influenza virus A/chicken/Shanghai/10/01 strains；
Infectious bursa of Fabricius virus HQ-b strains；
Infectious bronchitis virus LX4 strains；
DF-1 cells (chicken fibroblasts system).
The kit prepared using embodiment 3, is detected according to the application method of kit.
H1N1 subtype avian influenza virus, H5N6 subtype avian influenza virus, H5N1 subtype avian influenza virus, H7N9 hypotypes fowl stream
After Influenza Virus, H9N2 subtype avian influenza virus are detected, the positive droplet of avian influenza virus can be detected；Other are to be measured
After sample is detected, the positive droplet of avian influenza virus is not detected by.After all samples to be tested are detected, do not detect
The droplet positive to NDV.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, some improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
SEQUENCE LISTING
<110>China Animal Disease Control And Prevention Center
<120>Bird flu and the dual droplet digital pcr absolute quantitation detection kit of ewcastle disease
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Claims (10)
1. a kind of primer combination of probe, it is made up of primed probe group AIV-F1R1P1 and primed probe group NDV-F1R1P1；
The primed probe group AIV-F1R1P1 is made up of primer AIV-F1, primer AIV-R1 and probe AIV-P1；
The primer AIV-F1 is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) have by sequence 1 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 1 identical
The DNA molecular of function；
The primer AIV-R1 is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) have by sequence 2 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 2 identical
The DNA molecular of function；
The probe AIV-P1, an end have fluorescent reporter group, and another end has fluorescent quenching group, nucleotides
Sequence is following (a5) or (a6)：
(a5) as shown in the sequence 3 of sequence table；
(a6) as shown in by sequence 3 by the substitution of one or several nucleotides and/or missing and/or addition；
The primed probe group NDV-F1R1P1 is made up of primer NDV-F1, primer NDV-R1 and probe NDV-P1；
The primer NDV-F1 is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 4 of sequence table；
(a2) have by sequence 4 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 4 identical
The DNA molecular of function；
The primer NDV-R1 is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 5 of sequence table；
(a4) have by sequence 5 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 5 identical
The DNA molecular of function；
The probe NDV-P1, an end have fluorescent reporter group, and another end has fluorescent quenching group, nucleotides
Sequence is following (a5) or (a6)：
(a5) as shown in the sequence 6 of sequence table；
(a6) as shown in by sequence 6 by the substitution of one or several nucleotides and/or missing and/or addition；
The probe AIV-P1 and probe NDV-P1 has the fluorescent reporter group of different colours.
2. the application of primer combination of probe described in claim 1, for following (b1), (b2), (b3), (b4), (b5) or (b6)：
(b1) avian influenza virus and/or NDV are identified；
(b2) kit for identifying avian influenza virus and/or NDV is prepared；
(b3) whether detection sample to be tested contains avian influenza virus and/or NDV；
(b4) prepare for detect sample to be tested whether the kit containing avian influenza virus and/or NDV；
(b5) content of avian influenza virus and/or NDV in sample to be tested is detected；
(b6) prepare for detecting avian influenza virus and/or the kit of NDV content in sample to be tested.
3. a kind of kit, including primer combination of probe described in claim 1；The function of the kit be following (c1),
Or (c3) (c2)：
(c1) avian influenza virus and/or NDV are identified；
(c2) whether detection sample to be tested contains avian influenza virus and/or NDV；
(c3) content of avian influenza virus and/or NDV in sample to be tested is detected.
4. it is a kind of detect it is to be measured virus whether be avian influenza virus and/or NDV method, comprise the following steps：To treat
The total serum IgE for surveying virus is template, and digital RT-PCR is carried out using primer combination of probe described in claim 1；If based on probe
The testing result of the corresponding fluorophors of AIV-P1 is that virus positive, to be measured is or candidate is avian influenza virus, if based on spy
The testing result of the corresponding fluorophors of pin AIV-P1 is that virus negative, to be measured is or candidate is non-avian influenza virus；If base
It is that virus positive, to be measured is or candidate is NDV in the testing result of the corresponding fluorophors of probe NDV-P1, if
Testing result based on the corresponding fluorophors of probe NDV-P1 is that virus negative, to be measured is or candidate is non-NDV.
5. it is a kind of detection sample to be tested whether the method containing avian influenza virus and/or NDV, comprise the following steps：With
The total serum IgE of sample to be tested is template, and digital RT-PCR is carried out using primer combination of probe described in claim 1；If based on spy
The testing result of the corresponding fluorophors of pin AIV-P1 contains avian influenza virus for positive, sample to be tested, if based on probe
The testing result of the corresponding fluorophors of AIV-P1 does not contain avian influenza virus for negative, sample to be tested；If based on probe
The testing result of the corresponding fluorophors of NDV-P1 contains NDV for positive, sample to be tested, if based on probe NDV-
The testing result of the corresponding fluorophors of P1 does not contain NDV for negative, sample to be tested.
6. a kind of method for detecting the content of avian influenza virus and/or NDV in sample to be tested, comprises the following steps：With
The total serum IgE of sample to be tested is template, and digital RT-PCR is carried out using primer combination of probe described in claim 1；According to bird flu
The quantity of virus-positive droplet obtains the content of avian influenza virus in sample to be tested, and avian influenza virus positive droplet is based on probe
The testing result of the corresponding fluorophors of AIV-P1 is positive droplet；Treated according to the quantity of NDV positive droplet
The content of NDV in test sample sheet, NDV positive droplet are the inspection based on the corresponding fluorophors of probe NDV-P1
Result is surveyed as positive droplet.
7. a kind of premixed liquid for being used to detect avian influenza virus and/or NDV by digital RT-PCR, has the right wherein containing
Profit requires primer AIV-F1, primer AIV-R1, probe AIV-P1, primer NDV-F1, primer NDV-R1 and the probe described in 1
NDV-P1；In the premixed liquid, primer AIV-F1 concentration is 1 μM, and primer AIV-R1 concentration is 1 μM, probe AIV-P1's
Concentration is 2/9 μM, and primer NDV-F1 concentration is 1 μM, and primer NDV-R1 concentration is 1 μM, and probe NDV-P1 concentration is 2/9
μM。
8. a kind of kit for being used to detect avian influenza virus and/or NDV by digital RT-PCR, including right will
Ask 7 premixings and droplet that oil occurs.
9. kit as claimed in claim 8, it is characterised in that：The kit also includes negative control and positive control；
The negative control is RNase Free dH2O；The positive control is total serum IgE and NDV containing avian influenza virus
Total serum IgE solution.
10. the application of kit described in kit described in premixed liquid described in claim 7 or claim 8 or claim 9, is
(c1), (c2) or (c3) as follows：
(c1) avian influenza virus and/or NDV are identified；
(c2) whether detection sample to be tested contains avian influenza virus and/or NDV；
(c3) content of avian influenza virus and/or NDV in sample to be tested is detected.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531650A (en) * | 2018-04-12 | 2018-09-14 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | Probe primer group, kit and the detection of the rapid differential diagnosis of APMV-1 and APMV-4 |
| CN112831596A (en) * | 2019-11-22 | 2021-05-25 | 华农（肇庆）生物产业技术研究院有限公司 | Method for detecting content of H9N2 subtype avian influenza virus by using digital PCR |
| CN112852988A (en) * | 2019-11-12 | 2021-05-28 | 中国检验检疫科学研究院 | Microdroplet digital PCR detection method for simultaneously detecting penicillium and fusarium |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108531650A (en) * | 2018-04-12 | 2018-09-14 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | Probe primer group, kit and the detection of the rapid differential diagnosis of APMV-1 and APMV-4 |
| CN112852988A (en) * | 2019-11-12 | 2021-05-28 | 中国检验检疫科学研究院 | Microdroplet digital PCR detection method for simultaneously detecting penicillium and fusarium |
| CN112831596A (en) * | 2019-11-22 | 2021-05-25 | 华农（肇庆）生物产业技术研究院有限公司 | Method for detecting content of H9N2 subtype avian influenza virus by using digital PCR |
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Application publication date: 20171208 |