CN108929919A - II type astrovirus ring mediated isothermal amplification detection primer group of chicken, kit and its detection method - Google Patents
II type astrovirus ring mediated isothermal amplification detection primer group of chicken, kit and its detection method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention belongs to field of biotechnology, and the invention discloses II type astrovirus ring mediated isothermal amplification detection primer groups of chicken, and according to II type astrovirus genome conserved sequence design primer of chicken: outer primer F3 and B3, sequence are shown in SEQ ID NO.1, SEQ ID NO.2；Inner primer FIP and BIP, sequence are shown in SEQ ID NO.3, SEQ ID NO.4.The invention also discloses II type astrovirus detection kits of chicken, including above-mentioned primer sets.The present invention discloses II type astrovirus loop-mediated isothermal amplification detection method of chicken again: establishing a kind of loop-mediated isothermal amplification method for detecting II type astrovirus of chicken according to designed primer.Detection method of the invention have quickly, efficiently, high specificity, accuracy be good, high sensitivity, without expensive instrument and equipment, used convenient for base scene, fast and easy.
Description
Technical field
The present invention relates to a kind of chicken astrovirus ring mediated isothermal amplification detection primer group, kit and its detection methods, belong to
Field of biotechnology.
Background technique
Astroviridae (Astroviridae) is divided into mammal Astrovirus (Mamastrovirus) and fowl is starlike
Tobamovirus (Avastrovirus), wherein fowl Astrovirus is divided into 3 kinds, and respectively fowl astrovirus GI.A type, fowl is starlike
Viral GI.B type and fowl astrovirus GII.A type.According to separate sources and genotype, identified in poultry come 6 kinds it is different
Astrovirus, including two breeder source astrovirus (fowl ephritis viral (ANV) and chicken astrovirus (CAstV)), two kinds of turkey sources
Astrovirus (TAstV-1 and TAstV-2) and two kinds of duck source astrovirus (DAstV-1 and DAstV-2).Astrovirus is sub-thread
Normal chain, the picornavirus without cyst membrane, genome length 6.5-7.5kb have 3 open reading frame (ORF), including read
The RNA polymerase (ORF1b) and capsid protein (ORF2) of frame coding relied on without envelope protein (ORF1a), viral RNA.It is multiple
Mode processed and other enteroviruses be when synthesizing time genome it is completely different, there are one between ORF1 and ORF2 to contain
The reverse transcription of serine protease moves frame signal sequence, there is a mobile mount structure, ORF1 coding one between ORF1a and ORF1b
The RNA polymerase that kind protease and RNA are relied on, ORF2 express subgenomic RNA, encode the capsid protein of virus, different serum
Structure between type is different in size.
Chicken astrovirus (CAstV) is a kind of enterovirus popular easily in 1-5 week old poult, often with other enteron aisles
Virus is mixed infection.Chicken group's enterovirus infection situation is continued to monitor, finds the first positive sample detected
It is often exactly the mixed infection of astrovirus or astrovirus and other viruses.The infection conditions of the virus are detected for disease control
System and clinical diagnosis all have significance.Therefore, it is a set of it is easy quickly, the astrovirus detection method of high specificity, can be with
Strong technical support is provided for the prevention and control and clinical diagnosis of chicken diarrhea disease.
Ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) method can wait
Efficient, quick, high specific amplification target sequence is carried out under the conditions of temperature, LAMP has the advantages that many uniquenesses: 1. LAMP technology
Amplification can be realized under isothermal conditions, does not need the initial denaturation for carrying out template, reduce the influence of round pcr heating and cooling bring
And the requirement to expensive, accurate laboratory apparatus, while amplification efficiency is high.2. the specificity of LAMP technology amplification is very high, make
Specific primer can have the selectivity of height to aim sequence, reduce with region specific in identifying purpose sequence
The influence of non-target sequence.3. a large amount of magnesium pyrophosphate white precipitate, the inspection of amplified production can be generated in positive amplification reaction
Survey method multiplicity, can be with detected through gel electrophoresis, directly with the naked eye detect or be added in the reaction system dyestuff, according to the change of color
Change and ultraviolet light detection is carried out by fluorescence irradiation unit；Transmissometer can also be utilized, not according to amplified production turbidity
Real-time quantitative analysis is carried out with to original nucleic acid molecule.In addition, LAMP method also has high specificity, sensitivity is compared with PCR method
Height, without expensive instruments such as PCR instruments, it is only necessary to which common water-bath adjusts temperature (60 DEG C~66 DEG C), greatly reduces detection
Expense, be particularly suitable for base scene use.Currently, yet there are no the correlative study report for II type astrovirus LAMP method of chicken
Road, foundation of the invention can fill up domestic and international related fields blank.
Summary of the invention
The purpose of the present invention is to provide II type astrovirus ring mediated isothermal amplification detection primer groups of a breeder, kit
And its detection method, a kind of simple, fast II starlike disease of type of detection chicken can be provided for base's on-site test using the primer sets
The method of poison.
The first purpose of this invention is achieved through the following technical solutions:
The core of the loop-mediated isothermal amplification method technology of the detection II type astrovirus of chicken is the design of primer, designs
The primer of high specificity is the key that LAMP.Primer sets are made of a pair of of outer primer, a pair of of inner primer, outer are drawn wherein described
Object is made of F3 and B3, inner primer FIP and BIP.According to the II type astrovirus genome sequence of all chickens logged in GenBank
Column feature carries out analysis comparison using molecular biology software, selects II type astrovirus genome conservative region segment of chicken, if
Count primer, including outer primer 1 to (II-B3 of CAstV II-F3 and CAstV) and inner primer 1 to (CAstV II-FIP and CAstV
II-BIP), particular sequence is shown in SEQ ID
The primer sequence of design are as follows:
II-F3:5 '-CTGTGGCATGACGATTACGA-3 ' of CAstV；
II-B3:5 '-TCGACGCAATTGATCTGCTT-3 ' of CAstV；
II-FIP:5 '-GAGCGCTTTGTAGGACCTCGCATGGTCTTATGTCGGA-3 ' of CAstV；
II-BIP:5 '-ATCGAGCACTGGGGGTAAGTAGTCCTTTACGTCGGATCT-3 of CAstV
Second object of the present invention is achieved through the following technical solutions: a kind of detection reagent containing the primer sets
Box, the kit further include: I fluorescent dye of SYBR Green, 2 × reaction buffer, the primer sets, BstDNA polymerization
Enzyme, ultrapure water.
Third object of the present invention is achieved through the following technical solutions: II type astrovirus detection kit of chicken
LAMP detection method, which is characterized in that the corresponding LAMP reaction system of LAMP detection method be 25 μ L:2 × reaction buffer
12.5 μ L, primer CAstV II-F3 (25pmol) 1.0 μ L, primer CAstV II-B3 (25pmol) 1.0 μ L, primer
0.5 μ L, Bst archaeal dna polymerase of CAstV II-FIP (50pmol) II-BIP of 0.5 μ L, primer CAstV (50pmol), 1 μ
I fluorescence of L, measuring samples cDNA template 1.0 μ L, final concentration of 4 mol/L of glycine betaine, fluorescence visual detection reagent SYBR Green
1.0 μ L of dyestuff, remaining complements to 25.0 μ L with ultrapure water, then in 62 DEG C of reaction 40min.
The detection reagent uses I fluorescent dye of SYBR Green, and fluorescent reagent is added before the reaction.
2 × the reaction buffer, including it is 20 mM, MgSO that pH8.8Tris-HCl, which is 40mM, KCl,4For 16 mM,
(NH4)2SO4For 20mM, Tween20 0.2wt.%, Betaine be 1.6 M, every kind of dNTPs mixture be 2.8mM.
Illustrate: being covered in PCR pipe before reaction and 1 μ L SYBR Green, I fluorescent dye is added, LAMP wink after reaction
When be centrifuged, observed under ultraviolet lamp.If there is the massive amplification of purpose DNA in reaction tube, intense green is presented in reaction tube color
Fluorescence, otherwise unstressed configuration.
It is fast in preparation II type astrovirus of chicken that the present invention also provides the primer sets of the loop-mediated isothermal amplification
Purposes in fast diagnostic reagent.
The present invention has the advantages that
1. high sensitivity, high specificity: LAMP method has high specific and high amplification efficiency.Since LAMP method uses 4 kinds
The primer of upper 6 different zones of recognition template DNA, specificity are high.
2. easy to operate, cost is relatively low: several aspects of LAMP reaction are different from other amplification methods.LAMP method
It is mainly characterized by only needing a kind of enzyme, makes its amplification of nucleic acid under the isothermy within the scope of 62 DEG C.Therefore, it allows using letter
Consersion unit that is single and having inexpensive benefit, can be with better implementation in clinical detection.
3. high-efficient, the time is short: the separative efficiency of LAMP method is very high, and isothermal does not need the time wave of alternating temperature process yet
Take.LAMP method synthesizes 10~20 μ L specific DNAs to 25 μ L reaction mixtures in 30~60min.
4. high specificity, high sensitivity: the LAMP method that the present invention establishes detects II type astrovirus of chicken, is detected
Negative control sample (such as newcastle disease virus, family's aviadenovirus, avian leukosis virus, chook MDV and chicken are infected
Property bronchitis virus) and the no positive result of water control sample come out it is consistent with PCR testing result.But LAMP method compared with
100 times of Standard PCR high sensitivity or so, lowest detection is limited to 105 copy/ μ L.
Detailed description of the invention
Fig. 1 is II type astrovirus LAMP specificity experiments of chicken (directly observing), wherein 1: II type astrovirus of chicken；Control
4 kinds of gal virus, it is respectively as follows: 2: newcastle disease virus；3: aviadenovirus；4: avian leukosis virus；5: chook MDV；
6: avian infectious bronchitis virus
Fig. 2 is II type astrovirus LAMP method sensitivity tests (agarose electrophoresis) of chicken.Wherein M: DNA molecular amount standard
2000；1:1.05 × 104copy/μL；2:1.05 × 103copy/μL；3:1.05 × 102copy/μL；4:1.05 × 101
copy/μL；5:1.05 × 100copy/μL；6: negative control.
Specific embodiment
Be described further below the present invention, the case study on implementation introduced in this description be only it is exemplary, not to the present invention
Range be construed as limiting.Those skilled in the art should be understood that without departing from the principle of the invention and method, right
The details and form of technical solution of the present invention carry out part modifications or substitutions, but are belonged to based on this modifications or substitutions of the invention
In protection scope.
Embodiment 1
One, experimental method
1. relevant strains and Reference Strains involved in test
Test II type astrovirus of chicken, newcastle disease virus, family's aviadenovirus, avian leukosis virus, chook MDV
It is identified and is saved by birds preventive medicine key lab of the Ministry of Education of Yangzhou University with avian infectious bronchitis virus.
1. the preparation of nucleic acid extraction and cDNA template
With AxyPrep DNA/RNA extracts kit, method carries out the RNA that II type astrovirus of chicken is extracted in operation to specifications.
And extract nucleic acid (newcastle disease virus, avian leukosis virus and the chicken biography of experimental control strain simultaneously according to the method for kit
Metachromia bronchitis virus extracts RNA；Family's aviadenovirus, chook MDV extract nucleic acid DNA, and the DNA of extraction can be direct
It is detected for LAMP, it is that cDNA just can be used for LAMP detection that the RNA of extraction, which needs reverse transcription,
The reverse transcription system of cDNA template preparation is as follows: 5 μ L of RNA, random primer (0.1 μ g/ μ L) 1 μ L are put into 70 DEG C of PCR instrument
5 × buffer 4 μ L, 10 × dNTP3 μ L, 0.5 μ L of RNase inhibitor, 0.5 μ L of reverse transcriptase is added in 10min ice bath 2min afterwards,
6 μ L of sterile deionized water supplies 20 μ L systems.Response procedures are as follows: 42 DEG C of 1h, 70 DEG C of 10min, 4 DEG C of circulations.
The foundation of 3.LAMP detection method
The design of primers of 3.1 LAMP
According to the gene order of the II type astrovirus polymerase 1b encoding histone of chicken logged in GenBank and this laboratory
The voluntarily sequence characteristic that amplification sequencing obtains, carries out analysis comparison using molecular biology software, utilizes LAMP primer design
Online website (https: //primerexplorer .jp/lampv5/index .html) design primer, including outer primer 1 are right
To (FIP and BIP), particular sequence is shown in SEQ ID NO .1, SEQ ID NO .2, SEQ ID NO .3 for (F3 and B3), inner primer 1
With SEQ ID NO .4.By designed relevant primer, Primer-BLAST analysis, Primer- are carried out through ncbi database
BLAST analysis shows, relevant primer high specificity that the present invention designs, without cross jamming, Pass Test expected design.
The primer sequence of design are as follows:
II-F3:5 '-CTGTGGCATGACGATTACGA-3 ' of CAstV；
II-B3:5 '-TCGACGCAATTGATCTGCTT-3 ' of CAstV；
II-FIP:5 '-GAGCGCTTTGTAGGACCTCGCATGGTCTTATGTCGGA-3 ' of CAstV；
II-BIP:5 '-ATCGAGCACTGGGGGTAAGTAGTCCTTTACGTCGGATCT-3 ' of CAstV
The foundation and optimization of 3.2 LAMP methods
It is configured according to ring mediated isothermal amplification method DNA cloning kit (SLP204 Laoopamp DNA amplification reaction reagent box)
LAMP tests reaction solution, and reaction system is 25 μ L.Contain in every 25 μ L reaction system:
II-B3 of 2 × reaction buffer 12.5 μ L, primer CAstV II-F3 (25pmol) 1.0 μ L, primer CAstV
(25pmol) 1.0 μ L, primer CAstV II-FIP (50pmol) 0.5 μ L, primer CAstV II-BIP (50pmol) 0.5 μ
1 μ L of L, Bst archaeal dna polymerase, 1.0 μ L of measuring samples cDNA template, final concentration of 4 mol/L of glycine betaine, fluorescence visual detection
I fluorescent dye of reagent SYBR Green, 1.0 μ L, remaining complements to 25.0 μ L with ultrapure water.
React optimal conditions: experiment different temperatures (60 DEG C, 62 DEG C, 64 DEG C, 66 DEG C), different time (10min, 20min,
30min, 40min, 50min, 60min) detection primer group reactivity worth.Reaction condition after optimization is 62 DEG C of reaction 40min, 80
DEG C 10min terminates reaction.
3.3 specific test
With the LAMP condition after optimization, related experiment sample template nucleic acid (newcastle disease virus, avian leukosis virus and chicken are detected
Infectious bronchitis virus uses the cDNA after reverse transcription；Family's aviadenovirus, chook MDV use DNA profiling), it comments
The specificity for the LAMP method that valence is established.
The sensitivity test of 3.4 LAMP detection methods
3.4.1 the building of positive criteria product
According to the gene sequence of chicken astrovirus (NJ2017-1 plants of CAstV) polymerase 1b encoding histone of identification laboratory early period
Column feature designs specific primer, primer sequence are as follows: F:5 '-using primer-design software Primer Pirmer 5.0
GGAGACAGCGGTATCGTAG-3 ' and R:5 '-GGTTCATTGACGCTAGGAGGAA -3 ', for expanding about 510 bp's
Polymerase 1b protein gene segment, primer are synthesized by Nanjing Jin Sirui Bioisystech Co., Ltd.
II type astrovirus nucleic acid RNA of chicken is extracted using AxyPrep RNA extracts kit, according to PrimeScript
The 50 μ L systems that One Step RT-PCR Kit Ver .2 (Dye Plus) recommends carry out RT-PCR reaction, wherein
1 Step Enzyme Mix reaction solution of PrimeScript, 2 μ L, 2 × 1 Step Buffer (Dye Plus) reaction solution, 25 μ L,
Each 2 μ L of upstream and downstream primer (F and R, 10 μM), extraction 2 μ L of nucleic acid RNA, supplement sterile deionized water is to 50 μ L of final volume.
Reaction condition are as follows: carry out PCR amplification program after 50 DEG C of 30 min of reverse transcription；94 DEG C of 4 min of initial denaturation；94 DEG C of 50 s, 56 DEG C
30 s, 72 DEG C of 35 s, 35 circulations；72 DEG C of 10 min of extension after circulation terminates.
Ago-Gel QIAquick Gel Extraction Kit carries out gel extraction to specific target fragment after RT-PCR.According to
The specific polymerase 1b egg that pEASY-T1 Simple Cloning Kit clone's connection kit specification expands RT-PCR
On white gene fragment clone to pEASY-T1 cloning vector, blue hickie screening is carried out, digestion correctly clones and send Nanjing Jin Sirui
Bioisystech Co., Ltd is sequenced.Sequencing result is carried out to BLAST analysis verifying, sun expected from Pass Test on NCBI
Positive criteria product (T- CAstV II) of the property recombinant plasmid as PCR, is placed in -20 DEG C after packing and saves backup.
3.4.2 LAMP sensitivity technique
Carrying out serial dilution to the positive criteria product (T- CAstV II) of II type astrovirus of chicken building, (plasmid concentration is respectively
1.05×104, 1.05 × 103, 1.05 × 102, 1.05 × 101With 1.05 × 100Copy/μ L), according to the LAMP condition after optimization
It is detected, obtains its sensitivity tests data.
The detection of 3.5 LAMP method clinical samples
After 41 parts of ground processing of chicken tissues pathological material of disease that this laboratory is collected in the clinical application of the LAMP detection method of foundation, press
According to AxyPrep total serum IgE Miniprep Kit, method carries out the RNA that chicken astrovirus is extracted in operation to specifications, according to biography
The nucleic acid RNA reverse transcription of extraction is cDNA by system method.Reverse transcription system is same as above, and is examined according to the LAMP condition after optimization
It surveys.
LAMP reaction system is 25.0 μ L:2 × 12.5 μ L of reaction buffer, primers F 3 (25pmol) 1.0 μ L, primer B3
0.5 μ L, Bst archaeal dna polymerase 1 of (25pmol) 1.0 μ L, primers F IP (50pmol) 0.5 μ L, primer BIP (50pmol)
I fluorescent dye of 1.0 μ L of μ L, measuring samples cDNA template, fluorescence visual detection reagent SYBR Green, 1.0 μ L, remaining is with ultrapure
Water complements to 25.0 μ L.
Two, experimental result
The observation of 2.1 results
It irradiates through ultraviolet lamp (350-370nm) as it can be seen that II type astrovirus (CAstV NJ2017-1 of positive sample chicken
Strain) issue emerald fluorescence, control sample (newcastle disease virus, family's aviadenovirus, avian leukosis virus, chicken Marek's
Virus and avian infectious bronchitis virus) it is showed no visible fluorescence (see figure 1).
1.2 LAMP detection sensitivities
Through ultraviolet lamp (350-370nm) irradiation as it can be seen that the concentration of II type astrovirus of chicken is 1.05 × 102 copy/
μ L(, that is, 105copy/ μ L) (see figure 2) when be minimum detection limit.Result is subjected to conventional agarose gel electrophoresis as it can be seen that 1.05
×104 copy/μL、1.05×103 copy/μL、1.05×102Copy/ μ L(i.e. 105 copy/ μ L) it is visible have it is discontinuous
Scalariform electrophoretic band；1.05 × 101 copy/μL、1.05×100Copy/ μ L and negative control are showed no discontinuous scalariform
Electrophoretic band.As it can be seen that the lowest detection of LAMP of the invention is limited to 105 copy/ μ L.
The LAMP testing result of 1.3 clinical samples
After 41 parts of ground processing of chicken tissues pathological material of disease that this laboratory is collected, corresponding gene is extracted according to commercial kit
Group DNA, is detected according to the LAMP condition after optimization.As a result as it can be seen that positive sample is 41 parts, positive rate 70.73%(29/
41).And the positive sample (29 parts) of PCR detection is the positive with the testing result of the LAMP method of the invention established, positive symbol
Conjunction rate is 100%(29/29).Related positive sample carries out specific target fragment using Ago-Gel QIAquick Gel Extraction Kit
The sequencing of gel extraction rear clone.Sequencing result is carried out to BLAST analysis verifying on NCBI, is the starlike disease of corresponding II type of chicken
Virus gene segment, equal Pass Test are expected.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
<110>Yangzhou University
<120>II type astrovirus ring mediated isothermal amplification detection primer group of chicken, kit and its detection method
<160>4
SEQ ID NO.1
<210>1
<211>20
<212>DNA
<213>artificial sequence
<400>1
CTGTGGCATG ACGATTACGA 20
SEQ ID NO.2
<210>2
<211>20
<212>DNA
<213>artificial sequence
<400>2
TCGACGCAAT TGATCTGCTT 20
SEQ ID NO.3
<210>3
<211>37
<212>DNA
<213>artificial sequence
<400>3
GAGCGCTTTG TAGGACCTCG CATGGTCTTA TGTCGGA 37
SEQ ID NO.4
<210>4
<211>39
<212>DNA
<213>artificial sequence
<400>4
ATCGAGCACT GGGGGTAAGT AGTCCTTTAC GTCGGATCT 39
Claims (5)
1. II type astrovirus ring mediated isothermal amplification detection primer group of a breeder, it is characterised in that: the primer sets are by 1 pair
Outer primer F3 and B3, primers F IP and BIP composition in 1 pair, F3, B3, FIP, BIP primer sequence are as follows:
II-F3:5 '-CTGTGGCATGACGATTACGA-3 ' of CAstV；
II-B3:5 '-TCGACGCAATTGATCTGCTT-3 ' of CAstV；
II-FIP:5 '-GAGCGCTTTGTAGGACCTCGCATGGTCTTATGTCGGA-3 ' of CAstV；
II-BIP:5 '-ATCGAGCACTGGGGGTAAGTAGTCCTTTACGTCGGATCT-3 of CAstV.
2. II type astrovirus detection kit of chicken, including II type astrovirus ring mediated isothermal of chicken described in claim 1 expand
Increase detection primer group, I fluorescent dye of SYBR Green, 2 × reaction buffer, BstDNA polymerase, ultrapure water.
3. the LAMP detection method of II type astrovirus detection kit of chicken as claimed in claim 2, which is characterized in that LAMP
The corresponding LAMP reaction system of detection method is 25.0 μ L:2 × reaction buffer, 12.5 μ L, 25pmol primers F, 3 1.0 μ L,
1.0 μ L, 50pmol primers F IP of 25pmol primer B3,0.5 μ L, 50pmol primer BIP, 0.5 μ L, Bst archaeal dna polymerase
1.0 μ L, measuring samples cDNA template 1.0 μ L, final concentration of 4 mol/L of glycine betaine, fluorescence visual detection reagent SYBR Green I
1.0 μ L of fluorescent dye, remaining complements to 25.0 μ L with ultrapure water.
4. the LAMP detection method of II type astrovirus detection kit of chicken as claimed in claim 3, which is characterized in that experiment
Different temperatures, different time detection primer group reactivity worth；Different temperatures is respectively 60 DEG C, 62 DEG C, 64 DEG C, 66 DEG C, when different
Between be respectively 10min, 20min, 30min, 40min, 50min, 60min.
5. the LAMP detection method of II type astrovirus detection kit of chicken as claimed in claim 4, which is characterized in that optimization
Reaction condition afterwards is 62 DEG C of reaction 40min.
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