CN105200049B - Differentiate the GeXP rapid detection kit of H5 subtype avian influenza virus and its four kinds of difference NA hypotypes simultaneously - Google Patents
Differentiate the GeXP rapid detection kit of H5 subtype avian influenza virus and its four kinds of difference NA hypotypes simultaneously Download PDFInfo
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Abstract
The invention belongs to avian viral detection technique field, the invention discloses a kind of while differentiating GeXP primer sets, the kit of H5 subtype avian influenza virus and its four kinds of difference NA hypotypes.Inventor devises 6 pairs of specific primers and 1 pair of universal primer based on GeXP system researches, establishes while differentiating the GeXP kits of H5 subtype avian influenza virus and its four kinds of difference NA hypotypes accordingly.It can differentiate H5 subtype avian influenza virus and its four kinds of difference NA hypotypes, sensitivity 10 simultaneously using the present invention2Copy/μ L.The present invention has many advantages, such as high throughput, high specificity, high sensitivity and quick, is of great significance to the epidemiological survey and its antidiastole of H5 subtype avian influenza virus.
Description
Technical field
The invention belongs to avian viral detection technique field, more particularly to it is a kind of differentiate simultaneously H5 subtype avian influenza virus and
GeXP primer sets, kit and its application of its four kinds difference NA hypotypes.
Background technology
Bird flu is a kind of birds acute infectious disease caused by avian influenza virus, while certain subtype avian influenza virus
The mankind can be infected.Avian influenza virus is poor according to the antigenic relationship of its surface glycoprotein hemagglutinin (HA) and neuraminidase (NA)
It is different, 16 kinds of HA hypotypes and 9 kinds of NA hypotypes can be classified as.Pathogenic it is divided into highly pathogenic bird flu and low pathogenicity according to its
Bird flu, highly pathogenic bird flu include H5 and H7 hypotypes.The H5 subtype avian influenzas epidemic situation especially frequently broken out in recent years to
The aviculture band of many countries and regions causes huge economic loss.Since 2003, H5N1 subtype avian influenza virus epidemic diseases
Feelings gradually spread to total 56 countries and regions including Europe and Africa from Asia.Since 2014, Chinese is black
The epidemic situation of poultry infection H5N6 subtype avian influenza virus, South Korea, Japan, Holland, English successively occur for the provinces such as Longjiang, Jiangsu and Hunan
There is H5N8 bird flu epidemic situation, also 2015 H5N2 in U.S.'s outburst in succession in the area such as state, Germany, the U.S. and TaiWan, China
Subtype avian influenza epidemic situation, above-mentioned epidemic situation cause a large amount of poultry dead and are slaughtered.In addition, H5N1 hypotypes and H5N6 hypotypes AIV
With direct infection people and dead event prompt H5 hypotypes AIV can be caused not only to cause huge economic losses to aviculture, may be used also
To jeopardize human security in turn across host species barrier direct infection people.
Currently, avian influenza virus laboratory detection technology is mainly including viral isolation and culture, serological test, molecule
The methods of biological experiment and immunofluorescence, and the separation of virus and culture are diagnosis avian flu virus infections " goldstandard ".
Since the numerous and new variant of avian influenza subtypes continuously emerges, parting and diagnosis are carried out to it using above-mentioned detection method not only
There is operating method complexity, and detection cycle it is long and be easy to be influenced by factors such as antibody so that testing result from accurate and
In time.Although multiplex PCR has can amplify multiple purposes because of segment and then realize the quick diagnosis of multiple pathogens simultaneously simultaneously
The advantages of reducing inspection cost, but common multiplex PCR is easy the characteristic by target gene template during amplification, draws
The influence of the interaction between reagent used in object concentration and ratio and PCR etc., and then cause sample amplification efficiency inconsistent
So that the accuracy of testing result substantially reduces.The multiple reverse transcription polymerase chain of GeXP multiple-factor inheritance expression analysis systems is anti-
It is by general to answer (Multiplex reverse transcription-polymerase chainreaction, mRT-PCR)
Primer (upstream adds fluorescent marker) and specific chimeric primer (ends gene-specific primer 5' connection universal primer sequence) phase
In conjunction with multiple reverse transcription polymerase chain reaction system is established, the strategy caused using universal primer can overcome common multiplex PCR
The problem of existing amplification efficiency differs.In addition, this method carries out the separation of product using Capillary Electrophoresis, inspection is substantially increased
Survey sensitivity and the reliability of result.Currently, GeXP technologies have applied to new H1N1virus and seasonal influenza
Virus, hand-foot-and-mouth disease cause of disease, common respiratory diseases cause of disease and HPV viruse etc. infect detection and the parting of human pathogen, this experiment
Room has also been successfully established while having detected the GeXP methods of nine kinds of breathing problem pathogen of chicken.
Since the outburst of the H5AI of different NA hypotypes all has, morbidity is anxious, spread speed is fast, clinical symptoms are similar and dead
The high common feature of rate, existing diagnostic techniques are difficult to make accurate judgement to it within a short period of time again, are unfavorable for it
Prevention and control and the formulation of emergency measure.Currently, the nucleic acid detection method of detection H5 hypotypes AIV is mostly to be directed to its HA gene, examine simultaneously
It surveys H5, N1, N2, N6 and N8 gene and carries out the technology of parting there is not yet report.
Being disclosed in the information of the background technology part, it is only intended to increase understanding of the overall background of the invention, without answering
It has been the prior art well known to persons skilled in the art when being considered as recognizing or imply that the information is constituted in any form.
Invention content
For overcome the deficiencies in the prior art, the GeXP multiple PCR techniques that the present invention establishes can be simultaneously to H5 hypotype fowl
Four kinds of difference NA hypotypes of influenza virus are detected and parting, and it is maximum to aviculture harm at present to reach quickly discriminating
H5N1, H5N2, H5N6 and H5N84 kind combine the purpose of avian influenza virus, epidemiological survey to H5 subtype avian influenzas and its
Prevention and control ensure that the healthy and sustainable development of aviculture is of great significance.
Differentiating H5 subtype avian influenza virus and its four kinds of differences simultaneously the technical problem to be solved in the present invention is to provide a kind of
GeXP primer sets, kit and its application of NA hypotypes.
In order to solve the above technical problems, the present invention uses following technical scheme：
GeXP primer sets that are a kind of while differentiating H5 subtype avian influenza virus and its four kinds of difference NA hypotypes, including 6 couples of spies
Specific primer, be respectively primer pair A1 and A2, primer pair B1 and B2, primer pair C1 and C2, primer pair D1 and D2, primer pair E1 and
E1, primer pair F1 and F2 are respectively provided with the sequence as shown in SEQ ID No.1 to SEQ ID No.12.
GeXP kits that are a kind of while differentiating H5 subtype avian influenza virus and its four kinds of difference NA hypotypes, including cDNA,
10×PCR buffer、MgCl2, dNTP, Jumpstart Taq polymerase, 6 pairs of specific primers, 1 pair of universal primer
With RNA-free water, described 6 pairs of specific primers, 1 pair of universal primer are respectively provided with such as SEQ ID No.1 to SEQ ID
Sequence shown in No.14.
Preferably, 6 couples of specific primers A1 and A2, primer pair B1 and B2, primer pair C1 and C2, primer pair D1
With D2, primer pair E1 and E2, primer pair F1 and F2 molar concentration corresponding in PCR reaction systems be respectively 100nmol/L,
50nmol/L,50nmol/L,150nmol/L,100nmol/L,100nmol/L；Described universal primer Cy5-Tag-F, the Tag-R
Molar concentration in PCR reaction systems is 500nmol/L.
Preferably, the kit total volume is 25 μ L/ pipes, wherein including：CDNA 2.0 μ L, 10 × PCR
Buffer 2.5 μ L, MgCl22.5 1 μ L, Jumpstart Taq polymerase of μ L, dNTP, 1.2 μ L, 6 pairs of specificity are drawn
1.25 μ L of object mixture, 1.25 μ L, RNA-free water of upstream and downstream universal primer mixture complement to 25 μ L.
The present invention also provides it is described while differentiate the GeXP of H5 subtype avian influenza virus and its four kinds of difference NA hypotypes
Primer sets or it is described while differentiate that the GeXP kits of H5 subtype avian influenza virus and its four kinds of difference NA hypotypes are differentiating
Application in H5N1, H5N2, H5N6 and H5N8 subtype avian influenza virus.
Compared with prior art, the present invention has the advantages that：
1. the present invention using GenomeLab (tm) GeXP genetic analysis systems establish it is a kind of can detect simultaneously M, H5, N1,
Avian influenza virus Multiplex RT-PCR (mRT-PCR) method of 6 genes of N2, N6 and N8.First to reaction
Condition and multiple reaction system optimize, and then carry out specificity to multiplex PCR system with the positive template having verified that respectively
Verification.Multiple detection system can detect 4 kinds of difference NA hypotypes of H5 subtype avian influenza virus, sensitivity 10 simultaneously2Copy/
μL.This method has many advantages, such as high throughput, high specificity, high sensitivity and quick, to the epidemic disease of H5 subtype avian influenza virus
It learns investigation and its antidiastole is of great significance.
2. the GeXP multiple PCR techniques that the present invention establishes can be sub- to four kinds of difference NA of H5 subtype avian influenza virus simultaneously
Type is detected and parting, reaches and quickly differentiates at present to maximum H5N1, H5N2, H5N6 and H5N84 kind group of aviculture harm
The purpose of avian influenza virus is closed, epidemiological survey and its prevention and control to H5 subtype avian influenzas ensure that the health of aviculture continues
Development is of great significance.
Description of the drawings
Fig. 1 is the capillary electrophoresis analysis figure of GeXP multiplex PCRs；
Fig. 2 is the testing result to clinical Simple infection H5N1 subtype avian influenza virus；
Explanation in relation to reference numeral：
The base number of abscissa-pcr amplification product；Ordinate-fluorescence signal value.
Specific implementation mode
With reference to specific embodiment, further details of elaboration is made to the present invention, but embodiments of the present invention are not
It is confined to the range of embodiment expression.These embodiments are merely to illustrate the present invention, range and is not intended to limit the present invention.This
Outside, after reading present disclosure, those skilled in the art can various modifications may be made to the present invention, these equivalent variations are same
Sample falls within the appended claims limited range of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.Institute in following embodiments
Material, reagent for using etc., are commercially available unless otherwise specified.Avian influenza strain used in embodiment
And other avian viral Reference Strains are preserved by Veterinary Institute of Guangxi Zhuang Autonomous Region.
Embodiment 1：Avian influenza virus multiple RT-PCR design of primers
6 genes of avian influenza virus M, H5, N1, N2, N6 and N8 are downloaded from GenBank databases with reference to pertinent literature
Sequence, each gene nucleotide series are analyzed and are compared respectively using DNAStar, search out be suitble to design specificity
The conservative region of primer is directed to the specificity of 6 genes of avian influenza virus using GeXP express profiler tool designs
Primer (is shown in Table 1), and designed primer is carried out using Primer Premier 5.0, NCBI PrimerBlast and Oligo7.0
Analysis and screening, are then separately added into one section of non-homology unique sequences conduct at the ends 5' of whole forward primers and reverse primer
Universal primer (Uni-Primer), the upstream ends universal primer 5' mark fluorescent dyestuff Cy5, i.e. Cy5-Tag-F, by Shanghai
Invitrogen companies synthesize, HPLC purifying.
1 primer information of table
In table 1, annex base code R=A/G, D=A/G/T, Y=C/T, underscore indicate to be that upstream and downstream are general draw
Object sequence；Fluorescent dye Cy5 marks upstream universal primer label, downstream primer not to mark.According to actually detected pathogen
Strain difference and GeXP systems (such as GenomeLabTM GeXP Genetic Analysis System capillary electrophoresis)
Error, detected practical amplified production length can be expanded estimated using above-mentioned primer pair A-F and GeXP universal primers
Increase production and fluctuates 3bp on the length of object up and down.
Embodiment2：The foundation of multiplex PCR detection architecture
The preparation of 2.1 templates and monoclonal plasmid standard containing target gene
According to TaKaRa companies MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 (catalog number (Cat.No.)s
DV819A) specification obtains 50 from the nucleic acid of extraction different subtype (H1-16 and N1-9) avian influenza virus and other avian virals
The nucleic acid samples of μ L, packing are placed in -80 DEG C of preservations.RT reaction systems are with reference to TaKaRa companies reverse transcriptase (catalog number
D2639A) specification carries out, and the RNA sample of acquisition is carried out reverse transcription according to following reaction system and reaction condition respectively, is obtained
To cDNA；Using DEPC water as the control of total serum IgE.
Reaction system (25 μ L)：5×Reverse Transcriptase Buffer 5μL,50mmol/L Random
Primer(9mer)1μL、dNTP Mixture(10mM/L)2μL,40U Ribonuclease Inhibitor 0.5μL,5U/μ
0.5 μ L of L MLV Reverse Transcriptase, 1 μ g, RNA-free water of template ribonucleic acid complement to 25 μ L.
42 DEG C of 1.5h of reverse transcription temperature, are placed in -20 DEG C of preservations.It is expanded respectively with PCR and contains M, H5, N1, N2, N6 and N8 6
The segment in a target gene region, the PCR positive products obtained after amplification, which are cloned into T-easy carriers, is built into plasmid, warp
Sequencing confirms that this 6 recombinant plasmids are that T-easy carriers insert a kind of recombinant plasmid of said gene respectively, and on being respectively
State the target gene of 6 couples of primer pair A-F.
2.2 verify primer using substance RT-PCR methods
2.2.1 substance specific primer (SP-Primer) is diluted to working concentration 1 μm of ol/L, Cy5-Tag-F and Tag-R
It is diluted to 10 μm of ol/L of working concentration.
Reaction total volume is 25 μ L, and system component is：2.0 μ L, 10 × PCR buffer of cDNA 2.5 μ L, MgCl2
(25mM/L) 2.5 μ L, Singa Jumpstart Taq polymerase 1.2 μ L (2.5U/ μ L) (Singa companies, catalog number (Cat.No.)：
D4184), 1 μ L (TaKaRa companies, catalog number (Cat.No.) of dNTP (10mM/L):D4030RA), specific primer mixture takes 1.25 μ L,
Upstream and downstream universal primer mixture takes 1.25 μ L, finally supplies 25 μ L with RNA-free water.
Reaction condition is：94 DEG C of 5min, then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 10 cycles；94 DEG C of 30s, 65 DEG C
30s, 10 cycles；94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 30s, 20 cycles；72 DEG C of extension 3min, are placed in 4 DEG C.
2.2.2 Capillary Electrophoresis
It is carried out at the same time capillary electrophoresis detection using each PCR product of GenomeLab GeXP genetic analysis systems, is operated
Steps are as follows：It is sample-loading buffer (Beckman Coulter Inc. of the U.S., catalog number 608082), DNA size with formamide
Standard Kit-400Base Pairs (Beckman Coulter Inc. of the U.S., catalog number 608098) are pressed with sample-loading buffer
Volume ratio 1:(80-160) thorough mixing is added the good liquid of 39 μ L mixings per hole in sample panel, 10- is carried out with PCR product
100 times of dilutions, take the 1 μ L of product after dilution to add to sample panel, blow and beat mixing, finally instill dropstone wax oil closing in every hole,
In order to avoid formamide oxidation and sample evaporation.2/3 buffer solution is added in every hole on buffer solution plate, carries out Capillary Electrophoresis.Capillary
The condition of electrophoresis tube is as follows：Capillary heats up：Temperature 50 C；Denaturation：90℃,120s；Inject sample：2.0KV, 30s；Separation：
6.0KV, 35min.The actually detected of various special primer amplified fragments is determined using GenomeLab GeXP genetic analysis systems
Size.
2.2.3 the substance specific detection of primer pair A-F
Expand the cDNA samples obtained in embodiment 1 respectively with primer pair A-F：H5N1, primer pair B are expanded with primer pair A
H5N1 is expanded, primer pair C expands H5N1, and primer pair D expands H9N2, and primer pair E expands H6N6, and primer pair F expands H6N8.PCR
The result of amplification and Capillary Electrophoresis shows that substance specific primer only has target gene and well expands and there are no miscellaneous peak.It is different
Target fragment amplification after magnitude range be：AIV M,210-213bp；AIV-H5,223-226bp；AIV-N1,160-
163bp；AIV-N2,188-191bp；AIV-N6,240-243bp；AIV-N8,280-283bp.
The foundation of 2.3 GeXP multiplex PCR systems
Primer pair A, B, C, D, E and F are mixed into multiple mix primer (Mix-Primer) working solution, by dense to primer
The optimization of degree, the molar concentration for keeping the primer of target gene M, H5, N1, N2, N6 and N8 corresponding in PCR reaction systems are respectively
100nmol/L, 50nmol/L, 50nmol/L, 150nmol/L, 100nmol/L, 100nmol/L；Universal primer Cy5-Tag-F and
Molar concentrations of the Tag-R in PCR reaction systems is 500nmol/L.Remaining ingredient is verified identical with primer.With known to more plants
The single positive sample of viral nucleic acid, as template, carries out more primer PCRs as the mixing cDNA samples of template or a variety of cause of diseases
Reaction, response procedures and electrophoresis are verified identical with primer.
2.4 result
Single cDNA or mixing cDNA are detected respectively after 6 pairs of primer pair A-I mixing.
As shown in Figure 1, the results showed that more primer single mode versions detected respectively 210bp, 223bp, 162bp, 188bp,
The amplified production of 240bp, 280bp, negative control is without any amplified production；More primer multi-templates are the result shows that 6 target genes can
It is detected, through GeXP system Multiple detection system anlysis, can be detected with 6 purpose peaks being actually consistent and without other simultaneously
Miscellaneous peak, while four difference NA hypotypes of detected H5 subtype avian influenza virus can be distinguished by product clip size.Knot
Fruit also demonstrates the primer specificity in Multiple detection detection architecture.
Embodiment3：GeXP system Multiple detection system specific detections
Distinguish from allantoic fluid according to MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 specifications
HA (1-16) and NA (1-9) subtype avian influenza virus nucleic acid is extracted, while extracting the common disease poultry and livestocks such as IBV, NDV and ILTV poison
Nucleic acid is added separately in the GeXP Multiple detection systems of the foundation of embodiment 2, detects the specificity of this method.Multiplex PCR is completed
Afterwards, machine carries out GeXP capillary electrophoresis analysis in PCR product, and nonspecific signal only occurs in each reaction of as a result display, and no intersection is anti-
It answers.HA genes remove H5 hypotypes, other NA hypotypes and NDV, IBV, ILTV and sky of NA genes in addition to N1, N2, N6 and N8 hypotype
The white reactionless signal of control, prompts established method high specificity, with other detection object no cross reactions.
Embodiment4：The analysis of the sensitivity test and detection clinical sample ability of GeXP system Multiple detection systems
The sensitivity test of 4.1 GeXP system Multiple detection systems
With Promega companies RiboMAXTM Large Scale RNA Production System-T7 kit (mesh
Record P1300) by its specification respectively use SpeI and PvuII to built in real-time example 2 contain M, H5, N1, N2, N6 and N8
The plasmid of gene carries out digestion, and carrying out in-vitro transcription containing different target gene linearization plasmids by above-mentioned 6 synthesizes RNA.Profit
In-vitro transcription RNA concentration is measured and is quantified with DU 800UV/Vis spectrophotometers.According to nucleic acid concentration and molecular weight
Calculate the copy number of RNA.10 times are carried out after the in-vitro transcription RNA equal proportions of M, H5, N1, N2, N6 and N8 gene are mixed continuously
It is diluted to 106- 10 copies/μ L.Using cut back as detected sample, the multiple inspections of GeXP are carried out in the method that embodiment 2 is established
The detection and analysis of survey system sensitivity.Experiment be not repeated 3 times on the same day.
Non- 3 repetitions on the same day experiments have shown that, this method for H5, M, N1, N2, N6 and N8 gene it is minimum can detect to
The sample of nucleic acid of 100 copies/μ L.
4.2 GeXP system Multiple detection systems detect the ability of clinical sample
The cDNA that H5N1 subtype avian influenza virus is randomly choosed from embodiment 1, the method through step 2 in embodiment 2 carry out
Detection.The testing result of H5N1 subtype avian influenza virus such as Fig. 2 can detect 162.46,210.60 and 224.86 3 simultaneously
A purpose peak and without other miscellaneous peaks, shows the template that H5N1 subtype avian influenza virus is contained only in sample, and is actually consistent.
The description of the aforementioned specific exemplary embodiment to the present invention is in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed
And variation.The purpose of selecting and describing the exemplary embodiment is that explaining the specific principle of the present invention and its actually answering
With so that those skilled in the art can realize and utilize the present invention a variety of different exemplary implementation schemes and
Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (4)
1. GeXP primer sets that are a kind of while differentiating H5 subtype avian influenza virus and its four kinds of difference NA hypotypes, it is characterised in that：
Including 6 pairs of specific primers, be respectively primer pair A1 and A2, primer pair B1 and B2, primer pair C1 and C2, primer pair D1 and D2,
Primer pair E1 and E1, primer pair F1 and F2 are the sequence as shown in SEQ ID No.1 to SEQ ID No.12 respectively.
2. GeXP kits that are a kind of while differentiating H5 subtype avian influenza virus and its four kinds of difference NA hypotypes, including cDNA, 10
×PCR buffer、MgCl2, dNTP, Jumpstart Taq polymerase, 6 pairs of specific primers, 1 pair of universal primer and
RNA-free water, it is characterised in that：6 pairs of specific primers, 1 pair of universal primer are such as SEQ ID No.1 to SEQ respectively
Sequence shown in ID No.14.
3. according to claim 2 while differentiating H5 subtype avian influenza virus and its GeXP reagents of four kinds of difference NA hypotypes
Box, it is characterised in that：6 couples of specific primers A1 and A2, primer pair B1 and B2, primer pair C1 and C2, primer pair D1 and
D2, primer pair E1 and E2, primer pair F1 and F2 corresponding molar concentrations in PCR reaction systems be respectively 100nmol/L,
50nmol/L,50nmol/L,150nmol/L,100nmol/L,100nmol/L；Described universal primer Cy5-Tag-F, the Tag-R
Molar concentration in PCR reaction systems is 500nmol/L.
4. according to claim 3 while differentiating H5 subtype avian influenza virus and its GeXP reagents of four kinds of difference NA hypotypes
Box, it is characterised in that：The total volume of the kit is 25 μ L/ pipes, wherein including：CDNA 2.0 μ L, 10 × PCR
Buffer 2.5 μ L, MgCl22.5 1 μ L, Jumpstart Taq polymerase of μ L, dNTP, 1.2 μ L, 6 pairs of specificity are drawn
1.25 μ L of object mixture, 1.25 μ L, RNA-free water of upstream and downstream universal primer mixture complement to 25 μ L.
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| CN105349697B (en) | 2015-11-27 | 2020-01-14 | 广西壮族自治区兽医研究所 | GeXP rapid detection primer group and kit for simultaneously identifying HA genes of 8 avian influenza viruses infecting different subtypes of human and application of primer group and kit |
| CN105441586A (en) * | 2015-11-28 | 2016-03-30 | 长沙市疾病预防控制中心 | A-type H5N6 subtype avian influenza virus dual-channel real-time fluorescence PCR (polymerase chain reaction) detection kit and detection method |
| CN106191306A (en) * | 2016-07-14 | 2016-12-07 | 广西壮族自治区兽医研究所 | A kind of primer combination identifying H5 hypotype AIV and N6 hypotype AIV and application thereof |
| CN107312872A (en) * | 2017-06-22 | 2017-11-03 | 广州市华南农大生物药品有限公司 | Differentiate multiple PCR primer group, kit and the detection method of H5 subtype avian influenza virus and its NA hypotypes simultaneously |
| CN109487011B (en) * | 2019-01-07 | 2022-03-01 | 广西壮族自治区兽医研究所 | Primer combination for detecting H5, H7, H9 and 9 NA subtypes of AIV and application thereof |
| CN109628641B (en) * | 2019-01-07 | 2022-03-01 | 广西壮族自治区兽医研究所 | Primer combination for detecting H7 and 5 NA subtype AIV and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102899424A (en) * | 2012-10-23 | 2013-01-30 | 广西壮族自治区兽医研究所 | GeXP rapid detection kit capable of simultaneously identifying nine pathogens of chicken respiratory tract diseases |
| CN102899423A (en) * | 2012-10-23 | 2013-01-30 | 广西壮族自治区兽医研究所 | GeXP (Gene Expression) rapid detection kit capable of simultaneously identifying six virus of chicken respiratory disease |
| CN103981286A (en) * | 2014-05-19 | 2014-08-13 | 广西壮族自治区兽医研究所 | GeXP rapid detection kit for identifying eight types of porcine viral diseases and primer group thereof |
-
2015
- 2015-10-21 CN CN201510694288.1A patent/CN105200049B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102899424A (en) * | 2012-10-23 | 2013-01-30 | 广西壮族自治区兽医研究所 | GeXP rapid detection kit capable of simultaneously identifying nine pathogens of chicken respiratory tract diseases |
| CN102899423A (en) * | 2012-10-23 | 2013-01-30 | 广西壮族自治区兽医研究所 | GeXP (Gene Expression) rapid detection kit capable of simultaneously identifying six virus of chicken respiratory disease |
| CN103981286A (en) * | 2014-05-19 | 2014-08-13 | 广西壮族自治区兽医研究所 | GeXP rapid detection kit for identifying eight types of porcine viral diseases and primer group thereof |
Non-Patent Citations (1)
| Title |
|---|
| Simultaneous detection of four different neuraminidase types of avian influenza A H5 viruses by multiplex reverse transcription PCR using a GeXP analyser;Meng Li et al;《Influenza and Other Respiratory Viruses》;20161231;第10卷(第2期);141-149页 * |
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