CN109609688A - Goose astrovirus, goose's paramyxovirus, goose parvovirus multiple PCR detection primer pair and detection method and application - Google Patents
Goose astrovirus, goose's paramyxovirus, goose parvovirus multiple PCR detection primer pair and detection method and application Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The present invention relates to technical field of virus detection, in particular to goose astrovirus, goose's paramyxovirus, goose parvovirus multiple PCR detection primer pair, further relate to the detection method using primer pair, the application of primer pair.The multiplex PCR system that the present invention establishes can accurately detect the single or mixed infection of aquatic bird goose astrovirus, goose's paramyxovirus, goose parvovirus, and the multiplex PCR system is optimized, optimal primer ratio, primer concentration and Premix rTaq enzyme has been determined, so that 3 kinds of viral DNA cloning effects are more preferable, detection accuracy is higher, has considerable advantage in viral identification field.
Description
Technical field
The present invention relates to technical field of virus detection, in particular to goose astrovirus, goose's paramyxovirus, goose parvovirus is more
Weight PCR detection primer pair, further relates to the detection method using primer pair, the application of primer pair.
Background technique
Astrovirus belongs to Astroviridae (astroviridae) Astrovirus (astrovirus), not according to host
It is same to be divided into two categories: mammal Astrovirus (Mamastrovirus) and birds Astrovirus (Avastrovirus).
Existing 3 categories (Avastrovirus 1-3) of fowl Astrovirus, including duck astrovirus (Duck astrovirus), bird kidney
Scorching virus (Avian nephritisa strovirus), turkey astrovirus (Turkey astrovirus) etc., turkey is starlike
Virus is its representative species.Astrovirus is single-stranded positive, the picornavirus without cyst membrane, genome length about 6.8kb, from
5 ' ends to 3 ' ends include 4 parts altogether: the 5 ' end noncoding regions, 3 open reading frame (Open reading of 1 85 nucleotide
Frames, ORFs) (ORF1a, ORF1b, ORF2), the 3 ' end noncoding regions of 1 80 nucleotide, 1 30 nucleotide it is more
Poly A tail.Since 2017, occur successively in the young gaggle on the ground such as China Shandong, Jiangsu, Henan, Guangdong and Anhui with
It is the communicable disease of cardinal symptom that uric acid mineralization and arthragra, which occur, in internal organ, which takes place mostly within 20 ages in days
Young goose, the death rate are up to 30% ~ 50%.Huge economic loss is caused to the feeding goose industry in China.Through Identification of etiology, cause young goose
The cause of disease for gout symptom occur is novel astrovirus.
The cause of disease of goose paramyxovirus is that goose is glutinous viral (Goose paramyxovirus, GPMV), belongs to paramyxovirus section pair
Glutinous Tobamovirus.Virus is widely present in the internal organs of disease goose (spleen, liver, intestinal tube etc.).It observes under an electron microscope,
Virion is (average diameter is 120 nanometers) not of uniform size, and not just, there is intensive fine lug structure on surface to form, and viral internal is by capsule
Film is wrapped in helical symmetry nucleocapsid.Isolated strain can be bred rapidly after being inoculated with 10 age in days development of chick embryo, chicken embryo after inoculation 2
Death in~3 days.Goose sticks the red blood cell that virus can be aggregated chicken, and artificial infection chicken can cause death.According to epidemic characteristic, clinical symptoms
And pathological change, tentative diagnosis can be made.It makes a definite diagnosis, needs to carry out etiological diagnosis.
Goose parvovirus (Goose Parvovirus, GPV) is the pathogen of gosling plague.Since 1965, many states
Family once reported goose parvovirus (GPV) disease.The Clinical symptoms of gosling plague mainly with septic lesion performance based on, according to the course of disease
Length be divided into most acute, acute and subacute three types.The disease is fast with spread speed, morbidity and mortality are higher
Feature.Currently, the popular outburst of gosling blast is also difficult to control, once this infectious disease of happening and prevelence gosling plague, it can provisions goose
Industry causes huge economic loss.
Above-mentioned Viral experiment room diagnostic method has serological method, molecular biological testing.Common serology side
Method has agar gel diffusion test, virus neutralization tests, immuno-enzymatic agar gel diffusion test, immunofluorescence diagnosis, indirect enzyme-linked immunosorbent
Adsorption test, reverse indirect hemagglutination assay and protection test.Molecular biology method has Nucleic Acid Probe Technique, PCR technology etc..
Since rapid onset, the death rate are high after above-mentioned three kinds of virus infections, determination is which kind of virus infection is answered with taking in time as early as possible
It is particularly important to measure, therefore, study that a kind of can to detect above-mentioned three kinds of viral methods simultaneously extremely urgent.Multiplex PCR
(multiplex PCR), also known as Multiplex PCR or composite PCR, it is in same PCR reaction system plus two pairs or more
Primer, while amplifying the PCR reaction of multiple nucleic acid fragments.But in the prior art, do not have also using multiplex PCR to the starlike disease of goose
The relevant report that three kinds of poison, goose's paramyxovirus, goose parvovirus viruses are detected.
Summary of the invention
In order to solve it is above in the prior art without and meanwhile detect goose astrovirus, goose's paramyxovirus, three kinds of goose parvovirus
The problem of method of virus, this application discloses goose astrovirus, goose's paramyxovirus, goose parvovirus multiple PCR detection primers
It is right, three kinds of viruses can be detected simultaneously.
The present invention also provides the detection methods for using above-mentioned primer pair.
The present invention more provides application of the above-mentioned primer pair in viral diagnosis.
What the present invention was obtained through the following steps:
A kind of goose astrovirus, goose's paramyxovirus, goose parvovirus multiple PCR detection primer pair,
The detection primer of goose astrovirus is as follows to base sequence:
GoAst-F:5 '-GCTGCACAAGTTGGTTGGAC-3 ',
GoAst-R:5 '-GAGTAATCTGAGCGTCGGCA-3 '；
The detection primer of goose's paramyxovirus is as follows to base sequence:
GPMV-F:5 '-CATCCTCCGACTTAAAGAGAGCA-3 ',
GPMV-R:5 '-TACAGGTTGAGCTCTACACCAAC-3 '；
The detection primer of goose parvovirus is as follows to base sequence:
GPV-F:5 '-TGCACGATTCAATGACCGGA-3 ',
GPV-R:5 '-GTGCAGACCAAATCCTCCGA-3 '.
The goose astrovirus, goose's paramyxovirus, goose parvovirus multiple PCR detection primer are in detection aquatic bird goose star
Shape virus, goose's paramyxovirus and the Simple infection of goose parvovirus or the application in mixed infection.
Viral DNA and RNA in sample to be tested tissue are extracted in the application, are cDNA by the RNA reverse transcription,
The viral DNA and cDNA are expanded using the multi-PRC reaction system of the multiple PCR detection primer pair.
The application, the multi-PRC reaction system total volume are 25 μ L, in which: 10 μ L of Premix rTaq enzyme；
For the 1 μ L of primer of goose's paramyxovirus, wherein each 0.5 μ L of GPMV-F and GPMV-R, (primer concentration is 10 μm of oL/L)；
For the 3 μ L of primer of goose astrovirus, wherein each 1.5 μ L(primer concentration of GoAst-F and GoAst-R is 10 μm of oL/L)；Needle
To the primer 2 μ L of goose parvovirus, wherein each 1 μ L(primer concentration of GPV-F and GPV-R is 10 μm of oL/L)；DNA profiling 3
μL ；Adding aqua sterilisa to total volume is 25 μ L；The concentration of solute is 10 μm of oL/L in the Premix rTaq enzyme.
The application, amplification reaction condition are as follows: reaction total volume is to add in L: Xiang Suoshu multi-PRC reaction system of 25 μ
Enter the viral DNA and each 1 μ L of cDNA in the tissue, adding aqua sterilisa to total volume is 20 μ L, and amplification program is 95 DEG C 3
Min, 95 DEG C of 30 s, 53 DEG C of 30 s, 72 DEG C of 50 s, 35 circulations；Then 72 DEG C of 5 min, then cool to 4 DEG C reaction was completed.
Whether the application includes corresponding viral corresponding expected band by observation amplified production electrophoretogram, to sentence
Whether disconnected clinical sample is three kinds of viral Simple infections or mixed infection, and wherein the corresponding expected band of goose's paramyxovirus is
200bp, the corresponding expected band of goose astrovirus are 400bp, and the corresponding expected band of goose parvovirus is 760bp.
The application, the extracting method are specially to acquire the viscera tissue of dead aquatic bird as clinical sample to be detected
Product are centrifuged after being ground viscera tissue and physiological saline according to the ratio of mass ratio 1:3, using centrifugal column type viral DNA/
RNA extracts kit extracts the viral DNA and RNA in tissue.
The application, reverse transcription reaction condition are as follows: with HiScript cDNA the first chain synthetic agent box to the RNA
It carries out reverse transcription: using 40 μ L systems, 16 μ L of RNA template, 2 μ L of random primer, 2 × RTMIX are sequentially added in EP pipe
20 μ L, 2 μ L of HiScript Enzyme Mix, total volume totally 40 μ L are placed in progress cDNA synthesis in PCR instrument, with 25 DEG C 5
The program progress one of min, 50 DEG C of 45 min, 85 DEG C of 5 min, 4 DEG C of 5min recycle.
Beneficial effects of the present invention:
The multiplex PCR system that the present invention establishes can accurately detect aquatic bird goose astrovirus, goose's paramyxovirus, goose parvovirus list
One or mixed infection, and the multiplex PCR system is optimized, it is determined that optimal primer ratio, primer concentration and
Premix rTaq enzyme, so that 3 kinds of viral DNA cloning effects are more preferable, detection accuracy is higher, has in viral identification field
Considerable advantage.
Detailed description of the invention
Fig. 1 is the electrophoretogram that detects after multi-PRC reaction, in which: M is DNA molecular amount standard, 1: being respectively from top to bottom
Goose parvovirus, goose astrovirus, goose's paramyxovirus；
Fig. 2 is the electrophoretogram of multiplex PCR specific test, in which: M is DNA molecular amount standard, and 1: goose astrovirus, goose pair are glutinous
Virus and goose parvovirus mixture, 2: goose parvovirus, 3: goose astrovirus, 4: goose's paramyxovirus, 5:H9 subtype avian influenza
Virus, 6:H5 subtype avian influenza disease, 7: negative control；
Fig. 3 is the electrophoretogram of multiplex PCR sensitivity tests, in which: M is DNA molecular amount standard (DNA Marker), the glutinous disease of goose pair
Malicious (GPMV) initial concentration is 9.64 × 104Copy/μ L, goose astrovirus (GoAst) initial concentration are 6.78 × 104Copy/μ
L, goose parvovirus (GPV) initial concentration are 8.26 × 104Copy/μ L；
1 is 9.64 × 104Copy/μ L goose's paramyxovirus (GPMV), 6.78 × 104Copy/μ L goose astrovirus (GoAst),
8.26×104Copy/μ L goose parvovirus (GPV)；
2 be 4.82 × 104Copy/μ L goose's paramyxovirus (GPMV), 3.39 × 104Copy/μ L goose astrovirus (GoAst),
4.13×104Copy/μ L goose parvovirus (GPV)；
3 be 2.41 × 104Copy/μ L goose's paramyxovirus (GPMV), 1.70 × 104Copy/μ L goose astrovirus (GoAst),
2.07×104Copy/μ L goose parvovirus (GPV)；
4 be 1.21 × 104Copy/μ L goose's paramyxovirus (GPMV), 8.50 × 103Copy/μ L goose astrovirus (GoAst),
1.04×104Copy/μ L goose parvovirus (GPV)；
5 be 6.05 × 103Copy/μ L goose's paramyxovirus (GPMV), 4.25 × 103Copy/μ L goose astrovirus (GoAst),
5.20×103Copy/μ L goose parvovirus (GPV)；
6 be negative control (Negative control).
Specific embodiment
Invention is further explained combined with specific embodiments below:
Embodiment 1
1 materials and methods
1.1 Strain goose's paramyxovirus (GPMV), goose astrovirus (GoAst), goose parvovirus (GPV), H9 hypotype fowl stream
Influenza Virus (H9 AIV) is provided by poultry diease research department, Domestic Fowls Inst., Shandong Academy of Agricultural Sciences.H5 subtype avian influenza virus (H5
AIV) positive plasmid is given by poultry diease research department, animal medicine institute, Agricultural University Of South China.
1.2 main agents centrifugal column type viral DNAs/RNA extracts kit is limited purchased from Beijing Quan Shijin biotechnology
Company；It is limited that HiScript cDNA the first chain synthetic agent box and Premix rTaq enzyme purchased from Nanjing promise only praise biotechnology
Company；DL2000 DNA Marker is purchased from the special biological Co., Ltd of English.
1.3 test method
1.3.1 design of primers can expand the specific primer of 3 kinds of goose cause of diseases with 3 pairs of compounding design, set -20 DEG C and save backup.3
It is as follows to primer sequence:
The detection primer of goose astrovirus is as follows to base sequence:
GoAst-F:5 '-GCTGCACAAGTTGGTTGGAC-3 ',
GoAst-R:5 '-GAGTAATCTGAGCGTCGGCA-3 '；
The detection primer of goose's paramyxovirus is as follows to base sequence:
GPMV-F:5 '-CATCCTCCGACTTAAAGAGAGCA-3 ',
GPMV-R:5 '-TACAGGTTGAGCTCTACACCAAC-3 '；
The detection primer of goose parvovirus is as follows to base sequence:
GPV-F:5 '-TGCACGATTCAATGACCGGA-3 ',
GPV-R:5 '-GTGCAGACCAAATCCTCCGA-3 '.
1.3.2 viral nucleic acid extracts and cDNA is synthesized
Utilize Easypure Viral DNA/RNA Kit(centrifugal column type viral DNA/RNA extracts kit) three are extracted respectively
Three kinds of viral RNAs are carried out reverse transcription with HiScript cDNA the first chain synthetic agent box by kind viral DNA/RNA: using 40 μ
L system sequentially adds 16 μ L of RNA template, 2 μ L of random primer, 2 × RTMIX, 20 μ L, HiScript in EP pipe
2 μ L of Enzyme Mix, total volume totally 40 μ L are placed in progress cDNA synthesis in PCR instrument, with 25 DEG C of 5min, 50 DEG C of 45 min,
The program progress one of 85 DEG C of 5 min, 4 DEG C of 5min recycle, and save backup for -20 DEG C after cDNA synthesis.
The determination of 1 .3 .3 multi-PRC reaction condition
Multi-PRC reaction carries out in 25 μ L reaction systems.In order to determine optimal PCR reaction condition, we are to each primer
Additive amount has carried out multiple adjustment, and the optium concentration of each primer has finally been determined.Wherein reaction condition are as follows: Premix rTaq
Enzyme (concentration of solute is 10 μm of oL/L) 10 μ L, wherein being directed to primer 1 μ L (GPMV-F and the GPMV-of goose's paramyxovirus
Each 0.5 μ L of R, primer concentration are 10 μm of oL/L), for the 3 μ L of primer of goose astrovirus, (GoAst-F and GoAst-R are each
1.5 μ L, primer concentration are 10 μm of oL/L), for the primer 2 μ L of goose parvovirus, (GPV-F and each 1 μ L of GPV-R, draw
Object concentration is 10 μm of oL/L), each 1 μ L of GPMV cDNA, GoAst cDNA, GPV DNA, adding aqua sterilisa to total volume is 25 μ
L, amplification program are 95 DEG C of 3 min, 95 DEG C of 30 s, 53 DEG C of 30 s, 72 DEG C of 50 s, 35 circulations；72 DEG C of 5 min, 4 DEG C of end
Reaction.
1.3.4 multiplex PCR specific test
Using optimal PCR reaction condition, with GPMV cDNA, GoAst cDNA, GPV DNA, H9 AIV cDNA, H5 AIV
CDNA is detected as sample to be tested, and using sterile water as negative control, detection multiplex PCR specificity.
1.3.5 multiplex PCR sensitivity tests
By three kinds of extraction viral DNA or cDNA under determining best PCR reaction condition, Thermofisher is used
Its concentration of QBUIT3 fluorescent spectrophotometer measuring, and it is incremented by dilution by 2 times, take 1 μ L to carry out PCR detection, respectively with the dilution times of its highest
Number amplification is positive as the susceptibility of its PCR.
1.3.6 the detection of clinical sample
Are collected by 150 parts of clinical samples and is detected for Shandong, Henan, Jiangsu Province gaggle using optimal PCR reaction condition, is adopted
Liver, the renal tissue for collecting dead goose sample, are centrifuged after being ground with physiological saline according to the ratio of 1:3, use centrifugal column
Type viral DNA/RNA extracts kit extracts the DNA/RNA of virus, is reacted using optimal PCR reaction condition, and sun is calculated
Property rate.
2 results
The foundation of 2.1 best multi-PRC reaction conditions
Best multi-PRC reaction condition is 25 μ L:Premix rTaq enzyme (10 μm of oL/L) of total volume, 10 μ L, GPMV-F
With each 1.5 μ L, GPV-F and GPV-R of GPMV-R each 0.5 μ L, GoAst-F and GoAst-R each 1 μ L, GPMV cDNA,
Each 1 μ L of GoAst cDNA, GPV DNA, adding aqua sterilisa to total volume is 25 μ L.Amplification program be 95 DEG C of 3 min, 95 DEG C 30
S, 53 DEG C of 30 s, 72 DEG C of 50 s, 35 circulations；72 DEG C of 5 min, 4 DEG C reaction was completed.The results show that GPMV, GoAst, GPV
Purpose band is consistent with expection, respectively 200bp, 400bp, 760 bp bands, as shown in Figure 1.
2.2 specific test
To detecting containing GPMV, GoAst, GPV nucleic acid samples, can amplify 200bp, 400bp in line,
The product band of 760 bp bands, hybrid template amplification is clear, similar brightness, under the same conditions, and other goose encephalapthy agents
There is not specific band, shows that this method has good specificity, test results are shown in figure 2 for electrophoresis.
2.3 sensitivity tests
The concentration that GPMV, GoAst, GPV are measured with Thermofisher Qubit3 fluophotometer is respectively 9.64 × 104It copies
Shellfish/μ L, 6.78 × 104Copy/μ L, 8.26 × 104Copy/μ L, the multiple PCR method after optimizing application is to 2 times of diluted mixing
DNA/cDNA is detected, the results showed that, which is respectively 6.05 × 10 to the detectable limit of GPMV, GoAst, GPV3
Copy/μ L, 4.25 × 103Copy/μ L and 5.20 × 103Copy/μ L, as shown in Figure 3.
The detection of 2.4 clinical samples
150 parts of clinical samples are collected to Shandong, Henan, Jiangsu Province gaggle using optimal PCR reaction condition and carry out clinical inspection
It surveys, as a result as shown in table 1 below: detect 11 parts of GPMV (positive rate 7.33%), 12 parts of GoAst (positive rate 8%), GPV8
Part (positive rate 5.33%), wherein GPMV and GPV mixed infection case 3；Using conventional single PCR method, same group is faced
Bed sample is detected, and similarly, detects 11 parts of GPMV (positive rate 7.33%), and 12 parts of GoAst (positive rate 8%),
GPV8 parts (positive rate 5.33%), wherein GPMV and GPV mixed infection case 3.Conventional single PCR and multiplex PCR detection pair
Than as a result the coincidence rate of the two is 100%.
Table 1
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention and should not be limited by the examples,
Its any change made without departing from the spirit and principles of the present invention, modification, combination, substitution, simplification should be equivalent
Alternative is included within the scope of the present invention.
Claims (8)
1. a kind of goose astrovirus, goose's paramyxovirus, goose parvovirus multiple PCR detection primer pair, it is characterised in that
The detection primer of goose astrovirus is as follows to base sequence:
GoAst-F:5 '-GCTGCACAAGTTGGTTGGAC-3 ',
GoAst-R:5 '-GAGTAATCTGAGCGTCGGCA-3 '；
The detection primer of goose's paramyxovirus is as follows to base sequence:
GPMV-F:5 '-CATCCTCCGACTTAAAGAGAGCA-3 ',
GPMV-R:5 '-TACAGGTTGAGCTCTACACCAAC-3 '；
The detection primer of goose parvovirus is as follows to base sequence:
GPV-F:5 '-TGCACGATTCAATGACCGGA-3 ',
GPV-R:5 '-GTGCAGACCAAATCCTCCGA-3 '.
2. a kind of goose astrovirus described in claim 1, goose's paramyxovirus, goose parvovirus multiple PCR detection primer to
Detect aquatic bird goose astrovirus, goose's paramyxovirus and the Simple infection of goose parvovirus or the application in mixed infection.
3. application according to claim 2, it is characterised in that the viral DNA and RNA in sample to be tested tissue are extracted, it will
The RNA reverse transcription is cDNA, utilizes the multi-PRC reaction system containing multiple PCR detection primer pair described in claim 1
The viral DNA and cDNA are expanded.
4. application according to claim 3, it is characterised in that the multi-PRC reaction system total volume is 25 μ L,
In: 10 μ L of Premix rTaq enzyme；Each 1 μ L of GPMV-F and GPMV-R, concentration are 10 μm of oL/L；GoAst-F and GoAst-R
Each 1.5 μ L, concentration are 10 μm of oL/L；Each 1 μ L of GPV-F and GPV-R, concentration are 10 μm of oL/L；3 μ L of DNA profiling；Add
Aqua sterilisa to total volume is 25 μ L；The concentration of solute is 10 μm of oL/L in the Premix rTaq enzyme.
5. application according to claim 3, it is characterised in that amplification reaction condition are as follows: reaction total volume is 25 μ L: to institute
The viral DNA being added in the tissue in multi-PRC reaction system and each 1 μ L of cDNA are stated, adding aqua sterilisa to total volume is 25 μ
L, amplification program are 95 DEG C of 3 min, 95 DEG C of 30 s, 53 DEG C of 30 s, 72 DEG C of 50 s, 35 circulations；Then 72 DEG C of 5 min, then drop
Reaction was completed to 4 DEG C for temperature.
6. application according to claim 3, it is characterised in that by observation amplified production electrophoretogram whether include corresponding disease
The corresponding expected band of poison, to judge whether clinical sample is three kinds of viral Simple infections or mixed infection, wherein goose pair is glutinous
The corresponding expected band of virus is 200bp, and the corresponding expected band of goose astrovirus is 400bp, and goose parvovirus is corresponding pre-
Phase band is 760bp.
7. application according to claim 3, which is characterized in that the extracting method is specially the internal organ for acquiring dead aquatic bird
Tissue is used as clinical sample to be detected, is centrifuged after viscera tissue and physiological saline are ground according to the ratio of mass ratio 1:3,
The viral DNA and RNA in tissue are extracted using centrifugal column type viral DNA/RNA extracts kit.
8. application according to claim 3, it is characterised in that reverse transcription reaction condition are as follows: use HiScript cDNA first
Chain synthetic agent box to the RNA carry out reverse transcription: use 40 μ L systems, sequentially added in EP pipe 16 μ L of RNA template, with
Machine primer 2 μ L, 2 × RTMIX, 20 μ L, 2 μ L of HiScript Enzyme Mix, total volume totally 40 μ L, are placed in PCR instrument
CDNA synthesis is carried out, a circulation is carried out with the program of 25 DEG C of 5min, 50 DEG C of 45 min, 85 DEG C of 5 min, 4 DEG C of 5min.
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Cited By (5)
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|---|---|---|---|---|
| CN112941240A (en) * | 2021-04-09 | 2021-06-11 | 福建省农业科学院畜牧兽医研究所 | Primer pair, kit and method for detecting goose astrovirus and goose goblet virus |
| CN113667774A (en) * | 2021-08-23 | 2021-11-19 | 赣州市畜牧水产研究所 | Primer group for detecting gosling plague virus, goose paramyxovirus and duck plague virus, kit and detection method thereof |
| CN113832260A (en) * | 2021-08-28 | 2021-12-24 | 江西省农业科学院畜牧兽医研究所 | Goose astrovirus, goose parvovirus and goose calicivirus multiplex nano PCR (polymerase chain reaction) detection primer pair, kit and application method |
| CN114317835A (en) * | 2022-02-23 | 2022-04-12 | 安徽省农业科学院畜牧兽医研究所 | Multiple PCR detection primer group, kit and detection method for waterfowl parvovirus, duck enteritis virus and goose astrovirus |
| CN114540545A (en) * | 2022-01-26 | 2022-05-27 | 黑龙江省农业科学院畜牧兽医分院 | Nano-PCR detection method for simultaneously detecting GPV, GoAstV and GPMV |
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| CN113832260B (en) * | 2021-08-28 | 2023-07-14 | 江西省农业科学院畜牧兽医研究所 | Multiplex nano PCR detection primer pair, kit and application method for goose astrovirus, goose parvovirus and goose embedded cup virus |
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| CN114317835B (en) * | 2022-02-23 | 2023-11-10 | 安徽省农业科学院畜牧兽医研究所 | Multiplex PCR detection primer set, kit and detection method for waterfowl parvovirus, duck enteritis virus and goose astrovirus |
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