CN108384899A - A kind of PCR kit for fluorescence quantitative of the novel goose astrovirus of detection and application - Google Patents
A kind of PCR kit for fluorescence quantitative of the novel goose astrovirus of detection and application Download PDFInfo
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Abstract
The invention discloses a kind of PCR kit for fluorescence quantitative of novel goose astrovirus of detection and applications.Contain probe and standard items and quantitative fluorescent PCR reaction reagent shown in primer shown in SEQ ID NO.1 SEQ ID NO.2 and SEQ ID NO.3 in the PCR kit for fluorescence quantitative.The PCR kit for fluorescence quantitative of the present invention can be detected the novel goose astrovirus, the high specificity of the kit, only be specifically bound with novel goose astrovirus, with other goose source viruses all without cross reaction；And the high sensitivity of detection, it is 4 × 10 in standard items0‑4×109There are fabulous linear relationship, detection range that can carry goose up to 10 orders of magnitude with the novel goose astrovirus disease goose tissue of efficient detection and the virus without clinical symptoms, improve detection efficiency in copy range.
Description
Technical field
The present invention relates to avian viruses detection technique fields, and in particular to a kind of fluorescent quantitation of the novel goose astrovirus of detection
PCR kit and application.
Background technology
Astrovirus (AStV) is a kind of single strand plus RNA virus without cyst membrane, 28-30nm spherical in shape, a diameter of, gene
There is group infectivity, the infection of long 6.4-7.9kb, the virus to have certain popularity, can cause enteritis, the abdomen of human or animal
It rushes down, some is with vomiting and abdominal pain.Astroviridae according to infection host difference can be divided into mammal Astrovirus and
Fowl Astrovirus, fowl astrovirus can cause birds that a variety of diseases occur, and clinical manifestation is because of virus stain, virulence or sense
Contaminate the different and variant of host.Goose astrovirus (Goose astrovirus, GAstV), it is main to encroach on young age young bird goose, the disease
Occur in multiple province prevalences, the death rate of goose is made to increase, the sound development of goose industry is supported in high risks China.
2~December in 2017, the ground such as China Shandong, Anhui, Jiangsu, Liaoning, Henan and Guangdong young bird gaggle have broken out one kind
Using gout as the communicable disease of main feature.The disease takes place mostly in the young goose of 5~20 ages in days, and the death rate reaches as high as
50%.Serious uric acid mineralization occurs for illness young bird goose body cavity and joint, and sleepingly tired dynamic, feeding is difficult, causes young goose slow-growing,
Feedstuff-meat ratio increases, and meat goose delivers qualification rate significant decrease for sale, and serious financial consequences are caused to meat goose cultivation industry.
It has been investigated that the outburst of the disease is caused by a kind of novel goose astrovirus infection, it there is no vaccine can at present
To be prevented；Conventional antiviral and antibacterial therapy method is invalid.In the effective measures for currently in this case, controlling the disease
It is to be monitored to the novel goose astrovirus infection, is isolated or slaughters measure to find that infection gaggle takes it early,
It is lost caused by novel goose astrovirus infection with reducing.
Currently, including mainly cytodiagnosis, immunology diagnosis and molecular biology for the detection method of astrovirus
Diagnose three categories, wherein cytodiagnosis technology is mainly separately cultured using cell and is observed with Electronic Speculum.This method operates more
Cumbersome, detection cycle is long, and testing conditions are more demanding.Immunology diagnosis technology includes mainly enzyme linked immunosorbent assay (ELISA)
(ELISA) and immunofluorescence method, such method is sensitive higher, but also high to antibody preparation.Diagnostic technique in molecular biology master
To include conventional polymeric enzyme chain reaction (PCR) method and fluorescence PCR method, and traditional PCR method can only infect virus
It is qualitative, it cannot quantify, sensitivity is relatively low；The advantages that fluorescent quantitative PCR technique is with its high sensitivity, speed is fast, high specificity
It is widely used in gene expression dose analysis, the qualitative and quantitative detection of pathogen etc..But due to the novel goose
Astrovirus system reports for the first time, and technical blank is still fallen within to the fluorescence quantitative PCR detection of the novel goose astrovirus.
Invention content
For the above-mentioned prior art, the object of the present invention is to provide a kind of fluorescent quantitations of the novel goose astrovirus of detection
PCR kit and application.
To achieve the above object, the present invention adopts the following technical scheme that：
The first aspect of the present invention provides one group of primer and probe, the nucleotide sequence such as SEQ ID of the primer
Shown in NO.1-SEQ ID NO.2；The nucleotide sequence of the probe is as shown in SEQ ID NO.3.It is specific as follows：
Forward primer：ORF2-F：5’-CCAGGTCAAGATACAATG-3’；(SEQ ID NO.1)
Reverse primer：ORF2-R；5’-GTGTGTTCCAAGTGTAAA-3’；(SEQ ID NO.2)
Fluorescence probe：ORF2-tag：5’-FAM-AGTCCTTCCACGATAGCCAACAT-BHQ1-3’；(SEQ ID
NO.3)
The second aspect of the present invention provides above-mentioned primer and probe and is preparing the reagent for detecting novel goose astrovirus or examination
Application in agent box.
The novel goose astrovirus is preserved in China typical culture collection center, preservation on April 26th, 2018
Number is CCTCC NO：V201808, address：Wuhan, China, Wuhan University.
The third aspect of the present invention provides a kind of PCR kit for fluorescence quantitative of the novel goose astrovirus of detection, described glimmering
Fluorescent Quantitative PCR kit contains above-mentioned primer and probe.The deposit number of the goose astrovirus is CCTCC NO：
V201808。
Further, further include in the PCR kit for fluorescence quantitative：Standard items and quantitative fluorescent PCR reaction reagent.
Preferably, the standard items are prepared by the following method：
Use primer amplification deposit number shown in SEQ ID NO.1-SEQ ID NO.2 for CCTCC NO：V20180 8
Goose astrovirus genome, obtain amplified production, amplified production be connected on pMD18-T carriers, screening positive clone
And extract Plasmid DNA, that is, standard items are prepared.
The quantitative fluorescent PCR reaction reagent includes：One Step RT-PCR Buffer、TaKaRa Ex Taq HS、
PrimeScript RT Enzyme Mix II and ROX reference dyes.
The fourth aspect of the present invention, provides a kind of detection reagent of the novel goose astrovirus of detection, and the detection reagent contains
There is above-mentioned primer and probe.
The fifth aspect of the present invention provides above-mentioned PCR kit for fluorescence quantitative or above-mentioned detection reagent in following (1)-(3)
In it is any in application：
(1) novel goose astrovirus is infected to goose and carries out epidemiological survey；
(2) the novel goose astrovirus pollution in blood or serum product is monitored；
(3) novel goose astrovirus copy number is accurately detected, the progression of infection of goose astrovirus is specified.
The sixth aspect of the present invention provides a kind of side for detecting goose astrovirus using above-mentioned PCR kit for fluorescence quantitative
Method includes the following steps：
(1) standard items are subjected to gradient dilution, carry out TaqMan Fluorescence PCRs later, according to the concentration of standard items and
Ct values draw fluorescent quantitation standard curve；
(2) sample to be tested total serum IgE is extracted, TaqMan Fluorescence PCRs are carried out, sample to be tested fluorescence signal is collected, to glimmering
Optical signal carries out data processing, obtains Ct values and amplification curve, and qualitative and quantitative detection is carried out to sample to be tested.
Preferably, in step (1) and step (2), the program of TaqMan Fluorescence PCRs is：
42℃5min；95℃10s；95 DEG C of 5s, 53 DEG C of 30s, 72 DEG C of 30s carry out 40 cycles later.
Beneficial effects of the present invention：
(1) it is directed to the newfound novel goose astrovirus that can result in young goose gout, the present invention devises can be to this
The PCR kit for fluorescence quantitative that novel goose astrovirus is detected, the high specificity of the kit, only with the starlike disease of novel goose
Poison specific binding, such as Goose Parvovirus viral with other goose sources, goose tembusu virus, goose influenza virus etc. all do not intersect
Reaction.
(2) high sensitivity detected is 4 × 100-4 × 10 in standard items9There is fabulous linear pass in copy range
System, detection range is up to 10 orders of magnitude, and minimum detectable 4 copies, the high sensitivity of detection can be with the novel goose of efficient detection
Astrovirus disease goose is organized and the virus without clinical symptoms carries goose, improves detection efficiency.
(3) kit using the present invention can be monitored novel goose astrovirus infection, to find to feel early
Dye gaggle takes it isolation or slaughters measure, is lost caused by novel goose astrovirus infection with reducing；It can also be to goose
It infects novel astrovirus and carries out epidemiological survey, and the goose astrovirus pollution in blood or serum product is supervised
Control.
Specific implementation mode
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
As background technology is introduced, from 2017, the ground such as China Shandong, Anhui, Jiangsu, Liaoning, Henan and Guangdong
Young gaggle has been broken out a kind of using gout as the communicable disease of main feature.The astrovirus sense different from the past of the symptom of the disease
Dye, occurs the symptom of gout in gaggle, thus deduces, which may be to be drawn by novel astrovirus for the first time
It rises.It there is no the drug and method that can effectively control the novel astrovirus infection at present.Based on this, the present invention provides one
Kind can detect the PCR kit for fluorescence quantitative of the novel goose astrovirus, to find that infection gaggle takes it early
It is isolated or slaughters measure, is lost caused by novel goose astrovirus infection with reducing.
Since astrovirus is RNA virus, there are multiple segmentations, in antigenic structure, pathogenic, cell culture between different strains
It is had a certain difference on characteristic and host specificity, in genetic evolution, is easy to happen variation.Therefore from different hosts
The astrovirus being separated to its there are larger variability, it is poor that the coding of pathogenic, gene order and major protein all exists
It is different.
Present inventor isolates one plant of new virus N- from the tissues such as liver, spleen and the kidney of the young goose that dies of illness
AStV-SDPY, identified new virus N-AStV-SDPY are goose astrovirus.The present invention carries out strain N-AStV-SDPY
Genome sequencing, and with the astrovirus of existing report homology analysis and phylogenetic analysis have been carried out, as a result, it has been found that,
The homology of each genetic fragments of strain N-AStV-SDPY is respectively less than 70%；Phylogenetic analysis shows strain N-AStV-SDPY
In a variation branch；It these results suggest that：Strain N-AStV-SDPY is different from other astrovirus, is the starlike disease of fowl
The new kind that poison belongs to.And the novel goose astrovirus is preserved in China typical culture collection center, deposit number is
CCTCC NO：V201808.Therefore, the astrovirus of novel goose astrovirus to be detected of the invention and existing report exists
Variability, for its difficulty bigger of the detection of the novel goose astrovirus.
For fluorescence quantitative PCR detection, how design primer and probe sequence are the key that ensure detection validity.
Although having software and design of primers principle of many design of primers etc. in the prior art, to design to obtain high specificity, sensitive
High primer and probe combination is spent, being not can be simply obtained by primer-design software, this needs technical staff sharp
Optimization targeting regions are constantly selected with its professional knowledge, carry out design of primers further according to the targeting regions of optimization, and pair set
The primer of meter carries out screening repeatedly, optimization, redesign.Specifically, due to the present invention novel starlike disease of goose to be detected
Poison be different from existing report fowl astrovirus, how specificity detection the application novel goose astrovirus, and avoid and
Cross reaction occurs for other fowl astrovirus (the goose astrovirus especially reported), is that primer and probe of the present invention designs
Where difficult point.Detection for fowl astrovirus, some are set according to the polymerase 1b protein gene sequence features of goose astrovirus
Primer and probe is counted, some encodes target sequence design primer and the spy of caspid protein with astrovirus open reading frame 2
Therefore how needle, etc. for the detection of astrovirus, selects targeting regions to carry out primer and probe design at present still
Without unified cognition.The present invention is during the test optimized the targeting regions for carrying out primer and probe design, as a result
It was found that carrying out primer and probe design with the sequence conservation of the ORF2 genes of the novel goose astrovirus, may be implemented to this
The specific detection of novel goose astrovirus.
Primer, probe designed by the present invention are used cooperatively, and the two complements each other, in the design process further
The reaction condition for having fully considered quantitative fluorescent PCR, avoid interfering with each other between primer, probe, final design the application
Primer, probe.Wherein：
Forward primer：ORF2-F：5’-CCAGGTCAAGATACAATG-3’；(SEQ ID NO.1)
Reverse primer：ORF2-R；5’-GTGTGTTCCAAGTGTAAA-3’；(SEQ ID NO.2)
Fluorescence probe：ORF2-tag：5’-FAM-AGTCCTTCCACGATAGCCAACAT-BHQ1-3’；(SEQ ID
NO.3)
5 ' end mark fluorescent reporter group FAM of fluorescence probe, 3 ' end mark fluorescent quenching group BHQ1；Amplified fragments are long
Spend 83bp.
The present invention during the test, also passes through other conserved region sequences of goose astrovirus gene and other primers
Design principle devises multigroup different primer, probe, and is respectively combined, and detects its specificity after reacted respectively and expands
Increasing Efficiency is combined with the primer and probe that can be used for clinical detection for screening optimal.Such as：
First group：
Forward primer：F:5’-GGCCAATATTCAACAACA-3’；
Reverse primer：R；5’-CCTTCCTTATTGACACAAG-3’；
Fluorescence probe：5’-FAM-TGTGTAATGTCTGGCTCACCCA-Eclipse-3’.
Second group：
Forward primer：F:5’-GACGCGTGCCACAAGGA-3’；
Reverse primer：R；5’-CCACGCTTGATCTTATCTGTAAGC-3’；
Fluorescence probe：5’-FAM-AGCAAGGCGCGCATTCCCC-BHQ-3’.
As a result, it has been found that new to this with primer using the present invention and probe combinations (SEQ ID NO.1-SEQ ID NO.3)
The specificity and sensitivity that type goose astrovirus is detected are optimal.And other primer and probe combinations then cannot be to novel goose
Astrovirus and other goose sources virus are effectively distinguished, and false positive or false negative are susceptible to.
The present invention also optimizes quantitative fluorescent PCR condition during the test, the maximum limit in the clinical application of kit
Degree reduces operation, not only ensures sensitivity, but also reduce various pollutions to greatest extent, it is ensured that the science of result, accurate.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
The test material that test material is this field routine is not specifically described used in the embodiment of the present invention,
It can be commercially available by commercial channel.Specific experiment condition and method are not specified in the embodiment of the present invention, usually according to routine
Condition, such as J. Pehanorm Brooker chief editors, Science Press, 2002, Molecular Cloning:A Laboratory guide (third edition)；D.L. this peck
Top grade is edited, Science Press, and 2001, cell experiment guide；Or the condition suggested according to manufacturer.
Embodiment 1：The design of fluorescence quantification PCR primer and probe
Obtaining novel goose astrovirus according to two generation sequencing technologies, (deposit number is CCTCC NO：V201808 full base)
Because of a group sequence, whole genome sequence is as shown in SEQ ID NO.4.Drawing for specificity is designed for the sequence conservation of ORF2 genes
Object sequence and fluorescence probe sequence, sequence design are as follows：
Forward primer：ORF2-F：5’-CCAGGTCAAGATACAATG-3’；(SEQ ID NO.1)
Reverse primer：ORF2-R；5’-GTGTGTTCCAAGTGTAAA-3’；(SEQ ID NO.2)
Fluorescence probe：ORF2-tag：5’-FAM-AGTCCTTCCACGATAGCCAACAT-BHQ1-3’；(SEQID NO.3)
5 ' end mark fluorescent reporter group FAM of fluorescence probe, 3 ' end mark fluorescent quenching group BHQ1；Amplified fragments are long
Spend 83bp (shown in SEQ ID NO.5).
Embodiment 2：The foundation of fluorescent quantitative PCR detection method
(1) extraction of viral RNA：
The doubtful illness young bird goose kidneys of 30mg, liver mixing homogenised tissue are taken, is tried using classical Trizol methods or commercialization
Agent box extracts total serum IgE.
(2) Fluorescence PCR Establishing：
One-step method fluorescence quantitative kit used uses the article No. from Takara companies for the One Step of RR064A
PrimerScriptTMRT-PCR Kit sequentially add 2 × One Step RT-PCR Buffer in 200 μ L PCR reaction tubes
III~10 μ L, 1 × TaKaRa Ex Taq HS (5U/ μ L)~0.4 μ L, PrimeScript RT Enzyme Mix, II~0.4 μ
L, PCR Forward Primer (10 μM)~0.4 μ L, PCR Reverse Primer (10 μM)~0.4 μ L, Probe~0.8 μ
The II μ L of (50 ×)~0.4 of L, ROX Reference Dye or Dye, middle RNA~2 sample to be tested Total extracted of step (1)
μ L use RNase Free dH2O is supplemented to 20 μ L systems.It is placed in ABI 7300Fast fluorescence quantitative PCR instruments and is reacted,
Reaction condition is 42 DEG C of 5min；95 DEG C of 5s, 53 DEG C of 30s, 72 DEG C of 30s carry out 40 cycles after 95 DEG C of 10s, collect fluorescence.
(3) preparation of standard curve：
For copy number viral in accurate quantitative analysis sample, prepare the plasmid containing purposeful amplified fragments as standard items with
Draw standard curve.The forward and reverse primer (shown in SEQ ID NO.1 and SEQ ID NO.2) of ORF2 genes is designed first,
It expands novel goose astrovirus genome and obtains the amplified production of 83bp (shown in SEQ ID NO.5).
It according to classical molecular cloning protocols method, is connected on pMD18-T carriers, is named as pMD-ORF2, sieve
It selects positive colony and extracts plasmid, utilize spectrophotometric determination plasmid concentration, as standard items.10 times of ladders are carried out to standard items
TaqMan Fluorescence PCRs are carried out after degree dilution, according to the concentration of standard items and Ct values, it is fixed that instrument software draws out fluorescence automatically
Measure standard curve；Specially：Y=-3.173x+40.059, coefficient R2=0.992, good linear relationship is presented；Its
In, the copy number logarithm of x representative samples, y represents Ct values.
(4) judgement of testing result：
According to quantitative fluorescent PCR react collect fluorescence signal, recycle instrument software handle data, obtain amplification curve,
Ct values.Using the corresponding Ct values of standard items minimum concentration and amplification curve as judgment basis.
Result judgement method：If sample to be tested fluorescence signal is more than threshold value and Ct≤30.0, while amplification curve is smooth
" S " type, then can be determined that in sample to be tested contain novel goose astrovirus.
According to fluorescent quantitation standard curve, novel goose astrovirus in sample can be quantitative determined.
Embodiment 3：The optimization of the composition, experiment parameter of kit and specificity, sensitivity and repeatability are investigated
1. the composition of kit：
The kit of the present embodiment is PCR kit for fluorescence quantitative, and for detecting novel goose astrovirus, (deposit number is
CCTCC NO：V201808).Contain in kit：Forward primer (10 μM) shown in the SEQ ID NO.1 that embodiment 1 designs,
Embodiment 1 design SEQ ID NO.2 shown in reverse primer (10 μM), embodiment 1 design SEQ ID NO.3 shown in visit
Needle, standard items and quantitative fluorescent PCR reaction reagent.
Standard items are prepared by the following method：
Use primer amplification deposit number shown in SEQ ID NO.1-SEQ ID NO.2 for CCTCC NO：V201808's
The genome of goose astrovirus obtains the amplified production (shown in SEQ ID NO.5) of 83bp, amplified production is connected to pMD18-
In carrier T, screening positive clone simultaneously extracts Plasmid DNA, that is, standard items are prepared.
Quantitative fluorescent PCR reaction reagent includes：2 × One Step RT-PCR Buffer III, 1 × TaKaRa Ex Taq
HS (5U/ μ l), PrimeScript RT Enzyme Mix II, ROX Reference Dye or Dye II (50 ×), RNase
Free dH2O。
2. the optimization of experiment parameter：
Exploration is optimized to the quantitative fluorescent PCR reaction condition of the primer (SEQ ID NO.1-2) in kit, is tied
Fruit shows that primer is 10 μM, and reaction condition is 42 DEG C of 5min；95℃10s；95 DEG C of 5s, 53 DEG C of 30s, 72 DEG C of 30s carry out 40 later
A cycle, kit sensibility, detection result are best.
3. the specificity of kit, sensitivity and repeatability are investigated
(1) specific test
Using the kit, the specific test of the kit is carried out, respectively with Goose Parvovirus, goose tembusu virus,
Goose influenza virus, GsFJ2017 plants of goose astrovirus (the GenBank numbers of logging in are MF576430), CastV-8 plants of goose astrovirus
And the deposit number that the present invention is detected is CCTCC NO：The novel goose astrovirus of V201808 is template, using the reagent
Box carries out quantitative fluorescent PCR, as a result, it has been found that, Goose Parvovirus, goose tembusu virus, goose influenza virus, goose astrovirus
GsFJ2017 plants of (the GenBank numbers of logging in are MF576430), CastV-8 plants of goose astrovirus cannot be expanded effectively；Only protect
It is CCTCC NO to hide number：The novel goose astrovirus of V201808 can be expanded effectively.
The above test results show that the specificity of kit of the present invention is 100%, there is stronger specificity, kit
Middle primer and probe is specifically bound with novel goose astrovirus, with other goose source viruses all without cross reaction.
(2) repetitive test
Using the kit, the repetitive test of the kit is carried out.Take that clinical acquisitions arrive using gout as cardinal symptom
Young goose renal tissue sample, carry out repeated detection using kit, three repeat samples are corresponding in primary experiment
The coefficient of variation (CV) is less than 0.5%, illustrates it with high repeatability.
(3) sensitivity is tested
Standard items are diluted to various concentration, are detected using the kit, at the same using regular-PCR detection as pair
Than.As a result, it has been found that fluorescent quantitative PCR result is shown, kit of the invention can detect the standard items of 4 copy numbers, and
100-109There is fabulous linear relationship in copy range, detection range is up to 10 orders of magnitude；Show that the kit has
High sensitivity.
Embodiment 4：The clinical application experiment 1 of kit of the present invention
(1) extraction of viral RNA：
The doubtful illness young bird goose kidneys of 30mg, liver mixing homogenised tissue are taken, is tried using classical Trizol methods or commercialization
Agent box extracts total serum IgE.
(2) Fluorescence PCR Establishing：
2 × One Step RT-PCR Buffer III~10 μ L, 1 × TaKaR are sequentially added in 200 μ L PCR reaction tubes
II~0.4 μ L, PCR Forw ard of a Ex Taq HS (5U/ μ L)~0.4 μ L, PrimeScript RT Enzyme Mix
Primer (10 μM)~0.4 μ L, PCR Reverse Primer (10 μM)~0.4 μ μ of L, Probe~0.8 L, ROX
The II μ L of (50 ×)~0.4 of Reference Dye or Dye, the middle sample to be tested Total μ L of RNA~2 extracted of step (1), make
It is supplemented to 20 μ L systems with RNase Free dH2O.It is placed in ABI 7300Fast fluorescence quantitative PCR instruments and is reacted, reacted
Condition is 42 DEG C of 5min；95℃10s；95 DEG C of 5s, 53 DEG C of 30s, 72 DEG C of 30s carry out 40 cycles later, collect fluorescence.
Result judgement method：If sample to be tested fluorescence signal is more than threshold value and Ct≤30.0, while amplification curve is smooth
" S " type, then can be determined that in sample to be tested contain novel goose astrovirus.It the results are shown in Table 1.
Table 1：Morbidity goose field inspection pathological material of disease fluorescence quantitative PCR detection result
The result shows that kit using the present invention can detect the starlike disease of novel goose in 7 different regions goose field pathological material of diseases
Poison.
Moreover, 40 parts of identified samples of kit pair using the present invention are detected, there are 36 parts of pattern detections to contain
There is novel goose astrovirus, consistent with qualification result, detection accuracy rate is 100%.
Embodiment 5：The clinical application experiment 2 of kit of the present invention
The young goose of gout morbidity for the novel goose astrovirus of infection that clinical acquisitions are arrived, takes kidney, liver line and staff control to add
Enter and organize homogenate after 5 times of physiological saline, after multigelation 3 times, supernatant is taken to be inoculated with GKC cells, after stable cytopathy to appear
Receive poison.
The extraction of above-mentioned viral RNA：
The cell sample for taking 400 μ L virus infection to be measured is extracted total using classical Trizol methods or commercial kit
RNA。
Fluorescence PCR Establishing：
2 × One Step RT-PCR Buffer III~10 μ L, 1 × TaKaR are sequentially added in 200 μ L PCR reaction tubes
II~0.4 μ L, PCR Forw ard of a Ex Taq HS (5U/ μ L)~0.4 μ L, PrimeScript RT Enzyme Mix
Primer (10 μM)~0.4 μ L, PCR Reverse Primer (10 μM)~0.4 μ μ of L, Probe~0.8 L, ROX
The II μ L of (50 ×)~0.4 of Reference Dye or Dye, the middle sample to be tested Total μ L of RNA~2 extracted of step (1), make
It is supplemented to 20 μ L systems with RNase Free dH2O.It is placed in ABI 7300Fast fluorescence quantitative PCR instruments and is reacted, reacted
Condition is 42 DEG C of 5min；95 DEG C of 5s, 53 DEG C of 30s, 72 DEG C of 30s carry out 40 cycles after 95 DEG C of 10s, collect fluorescence.
Result judgement method：If sample to be tested fluorescence signal is more than threshold value and Ct≤30.0, while amplification curve is smooth
" S " type, then can be determined that in sample to be tested contain novel goose astrovirus.It the results are shown in Table 2.
Table 2：Novel goose astrovirus infection cell sample fluorescence quantitative PCR detection result
After testing, novel goose astrovirus is contained in sample, meets expection, show the detection method of the present invention it is accurate, can
It leans on.
The foregoing is merely the preferred embodiments of the application, are not intended to limit this application, for the skill of this field
For art personnel, the application can have various modifications and variations.Within the spirit and principles of this application, any made by repair
Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.
SEQUENCE LISTING
<110>Shandong Agricultural University
<120>A kind of PCR kit for fluorescence quantitative of the novel goose astrovirus of detection and application
<130> 2018
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
ccaggtcaag atacaatg 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
gtgtgttcca agtgtaaa 18
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<400> 3
agtccttcca cgatagccaa cat 23
<210> 4
<211> 7252
<212> DNA
<213>Goose astrovirus（Goose astrovirus）
<400> 4
tgtagcctct gtgttttcgc ttcctgcgca tcaaggcacg ccttctcacc caaattcata 60
aggtttgcac caatatcggg tgaccatgtt ggtttggtga catcatcggc gaaaaagaga 120
ccatacctcc cattgtcaac cccttttcct cacttgacac tcgtgaggaa aggaagactg 180
atgagattca gtcaggcctt gagaaggtct tccacttcaa tggtgttaat gaattataca 240
ccaagatgaa aggcttttat ggtcgcactc cagcctggag tgctctcatg aagtgtaatg 300
ccatctatct taaagattgg aaaacagcta taggggttga caatgggagg tatggtctct 360
ttttcgccga tgatgtcacc aaaccaacat ggtcacccga tattggtgca aaccttatga 420
atttgggtga gaaggcgtgc cttgatgcgc aggaagcgaa aacacagagg ctacaatgct 480
cgctgaaacg atgtggagct cttgtccaac aagttcttga gaagagcaaa caagcaaagg 540
aacaaatgca aaaagccgag gaattacaga agaagattga tcaaattaat gaaagcaaca 600
aggttctaca tcgcaaaatg caggagagaa atgttgagta ttctcagaag gtcataactc 660
gtattcatga gctgcaaaaa gacagagacc tgtggatgtc gcgtgctgct cattacgagc 720
aggagaacgt gcggctacgt attcaactgg ggacatatga tccgaacagg tctaagctga 780
tgactttcct agcatggatg tgtgttgcga tcattgcttt cagcttgatt caattggcgg 840
atgctagtga gattaaggat aacagaacag aaaattgttt cccaacatct ggctgtgttc 900
tctcagacgt tccttggact aagtcgtaca cctttatgga gctaatggac aagtgtgtga 960
ccggtactgc catcatacct aaaaacacat ttaatggtga agggcttgag ctggagtgta 1020
ttaagaattt tggcaagaaa tggtgcaaag gaagggttga gaagctcata ccgcattatt 1080
gtgatagaga taatactttt gatgccctct atcagtatta tgttgaaaca gtagcaattg 1140
ctgttgactt ctttaaagtt gcggtgcagt atcggattga caattggatt gtggctctct 1200
ttagtctagt attgactggg aagatggaga agtttcttaa gacgctaccc tttgtaattt 1260
tggcatggtg gtttaagttg cccatctttg ttatgagcat tgccacgaca atctttcctg 1320
tgcatgctct gccattcttg ttcttccagg tgtgctttcc aaactttgtc atcatcactg 1380
gttttctctt gtggatgacc tctatattag tggcttttta ttggtttgat gggactgcaa 1440
ttttggttga ggtttcttat gcggtattct atactattat cttctttgtg tggtctacag 1500
cattgacaat tatttcaact ttgtcattga cagtccctgt acagatttta ttattctgtg 1560
tttcattaac catttattgt ggcacaaagt ttgcgtgttc cactattaca gtcactaatc 1620
ctgacggtac tgtgtcgaaa tactcaagag ttgccaaaat gaaggatact gttgccatac 1680
agtgcaaaaa gatggtgagc tttgggcagg cccgtggtgt gataccagca actccagtga 1740
aggtgaattc cattgtcgtt gttgaaggaa aaaaaggttc aggggttggc ttccggttca 1800
tgaattatat aattacagca ggacacgtca cattaggctc tgactgggta acagtaaaac 1860
atgctggcac agtagccaaa acaaaagtcc tgaagcagat tgaggtgttt gagtgcgtcg 1920
atacaatagc ggttataaag ctgccccccg aattacagaa tttgaaaccc ataaaactcg 1980
caaaacgtgt tgagtccaca tacttaacat tgacagcttt tgatcctaat tttcaaaatg 2040
ttgtgtcata cactggctgg tgtattattg atggaaattg gatcagtaat gcatttaata 2100
ctcaatttgg aaacagtggt ggcccatacg ttgatgcaga gggtaaattg gttgggattc 2160
atcttggaac tcagggagtt atgtcccaag gtattgttgt ggtggatgcc cttaaaacgc 2220
atttcacgct cgcagaccag tgcaaggatt ttgattatga tggcttcctg gagaaagtta 2280
ttgcaggaac aaagacatca catgaagcaa ttctcaaagg tattgaggaa ttgcgagagg 2340
acgtcgacac gctcaagaac aagtgcaaga attatgatga gtactggctt gttcaaacta 2400
tttttggcca gaagaaaaag ggcaaaacaa agaaaacagc ccgggggaat aaacatcttg 2460
taacaaagag attcctgagt aaaggccact tcatgaaact caaaatgctc accgatgagg 2520
aatataatca tatgattgag caaggtttca ccgctgatga gatacgtgag gcagtcaatg 2580
agctgagaga acaagcatgg ctcaactact gcattgataa tgatattgat gaagaaggtg 2640
cggaagagtg gtatgaagat atgattgaag atgaccaaat taatgaaaag attgatgaag 2700
ccattgagaa agcaatggaa gaaaggggcg agttttatca agtcaaaaaa aggaaaacat 2760
ttgcacaaca agcattatta cacctcatcc gtgtcagtaa agccaggagc caaactgcaa 2820
agcaagaggt tcagaaagaa aacgcagatc agctttacga gatgttcaaa tctgccgtaa 2880
aggatgaaca agttgatgaa ggcacaacct cttacgcact cttcgccaat ggtgatcaga 2940
ttgagatggt cgaaggaaag gaacttgatt ttgaaaaggt aaagcttatc cacgttgatg 3000
gtgaacaaaa aaatgagacc attccgggga cgaaagtcac tgagatttct actggccctg 3060
agaataagaa gaacattttg cgcaagaagg acactcaaat taatccagaa caagctcaac 3120
ctcttgagca gcgcaagagg atctgcaagt ggtgtgggtc gagccaaaag catgactttc 3180
gtgattgcaa gttccagcgt gagaaaaggt tttgtgtctt ctgtgcaaag atgcactcat 3240
tgtttgatgg tcatcaacgg gacgtcttgt gcgacgtttg tgggaagaag ttcaagagtg 3300
tagaggagct tgaacagcac gtaactcgtg aaacgtgtga aaaaaactag ataaggggga 3360
cgatgccatc tgggtccccc gcataccacc tgatgattct cacatttttt cttggcagga 3420
tgatattatt gagtgtgata agaaaatcac tttgcccgac tatatagata taattggttt 3480
tatacctgta gataaattag ttgtcagagc aaagaccatt catgacccct tattaaactt 3540
ggttgagcat tggcaacaag atgaatatgc tcctactaaa tgggactata aagcctatac 3600
caagatgttt gaaaaatttt tctatcatga acctcaggat tttgtaaaca attttccaga 3660
gctggtcatc ttaagtgata gtgttgtgtt agaagaacat tcttatatgg ccaattccaa 3720
tttaacaccc ataatggcaa ctaagaagaa tgtcgatagt acccctggtt atccaaaatt 3780
taagtacttt gacacagaga aggactatct tgaacaatgt ggctggggtg agtacttgaa 3840
ggctgtatca gatattgatg agactgtgca aaataggccc ctctggtggt gttttctcaa 3900
aaatgaggtc ctcaaaaaga agaaaatcat ggataatgat attcgtatga tcttgtgtgc 3960
tgatccagta tatacaagga ttggagccat gtttgagcag gaccagaatg agaaaatgaa 4020
gcaacagaca gaacggcggg ctgcacaagt tggttggaca cccttttttg gtggaataca 4080
tcagcgagta tctaggctca ttggtggtgg tgacaggttt tttgtagaga cggactggac 4140
gcgttatgat ggtacgttgc ccaagcctct cttctggcgg atacgacaga tgcgttactt 4200
tttcctgtcc aaccatcata agacaccaca gcttaagaaa ctctatgatt ggtatgtcaa 4260
aaacttagtt gaaaaaatta tattattacc aactggggag gtttgtacag ttaagaaggg 4320
aaatccaagt ggccaatatt caacaacagt ggacaacaat atgtgtaatg tctggctcac 4380
ccattttgag atagcttatc tttattggaa acagcatggg tcattgccga cgctcagatt 4440
actcagggat aatgtcacca tgatttgcta tggcgatgat aggcttgtgt caataaggaa 4500
gggttttatt aattacgaca cgaatgtcgt catagatatg tataaaaata tttttggtat 4560
gtgggttaag ccagaaaatg tcaaggtctc tgatgatatt gagggtatga ctttctgtgg 4620
tcttacgttg gttaaaagga gtgatggtag atttgttgga gttcccaatg ttgataagat 4680
attgtcaaca cttgaaaatc ctgttagaaa attacccaac ttagaatcac tctggggtaa 4740
attggtttct ttaaggatct tgtgcgaaaa tgcacccaat agtgttagag agtttcttga 4800
taaacaaatc aacacggttg aggagcacgc tgccagtgaa aatattaact tacctgaggt 4860
cgggcccgac ttcttttctc aaatctggtg agtggcggac cgaaataaaa tggcagacag 4920
ggcggtggcc ccgcgcgaga aggtgaccaa gaaggttaca aaagtagtca ccgttaagaa 4980
aaaacaccca aaaaagaaac caaagcagaa agtatataag ccccaaaaat tacccatgaa 5040
ggccgagagg aagcttgaga aagaagtgaa aggtttgaag aaaagagtag ctggaccacc 5100
cgttaatgac aaaatgacta ccacgataac acttggtcag atcactggga attcaacaga 5160
cacactcgac cggaagcata aatacttcac aaatccactc atgatgaaaa accaggaaaa 5220
tgggcaaaca gcaactcctc taagtataag ggcctcacaa tataacttgt ggaggatcag 5280
aaagctgcat atccgccttg ttccacttgc tggtagggcc aatattcttg gctcggttgt 5340
cttcctagat atagaacagg aagctaacac agctgggcca gagtccatag ataccataaa 5400
agctcggccg catcttgaac tcccgattgg ctcaaaacat ctttggaggg ttcaacccag 5460
gctgatgcag ggaccccggc aaggatggtg gaatgtagac cccggggatt cacctactga 5520
ctcacttggt ccagcaatca atatgtggac atatttgaaa acagtaaatg cactttcaac 5580
acgggcgcag gcacaacaag ttccctatac ttctgccctc ttcctggttg aagcaacggt 5640
cacttatgag ttctcgaact atggtccaaa gccagggctc tcactcatga ccagtgaaac 5700
actttcagca tcagggaaaa cggcaaccct tgtaaatacc caggatggtg cacttgcttt 5760
aacagttagt ggcgcattgc agagattcct cgatgagaag gagcaacaca ggcgcgtttc 5820
aaatgcgcag acctctggtg tcggtgaggt gttttgggct gtttccacag aggtggtcga 5880
gaccgtagcg agtgcacttg gaggctgggg ttggctcttg aaaggaggat ggttcgtgat 5940
cagaaaattg tttggtgctg cttcaaattc tggttccact tatctgatct attcctcagt 6000
ttcagacgcc cagatagaca gcaggattta tcagacagtt ccaccgaaca cgccactaca 6060
gctggctgca aatacagtta agcttgtaca gctaacacag ccaaatgtga atacaactgg 6120
acaaggtacc actgtccttt cacgggatgc cgattacctg cctctgccag tggcaccaat 6180
gcaggttact ccctcgcttg tgtacaactt ccagggtgaa aggcagagca ctacagaatc 6240
gtgctcattc ctggtgtttg gaataccaca ggcagaatcc aggtcaagat acaatgcaaa 6300
tataaccttc aatgttggct atcgtggaag gacttcaaca tcatttacac ttggaacaca 6360
caattggtgg gctgttatga cactttcaca aacaggagta atttttgcac caccggctgt 6420
gggcacaggg gtctgtaata cattggctac agccatacaa cacttaaacc ctgagcttga 6480
aacagcagtc ctgcgtgtga ataccagtac aacatctact ggtgggctaa taacggaact 6540
caggaatcgg ctcaacatcg ctgatgggga ctatgtgatt tcaatgggtg atccgcaagg 6600
aaataggtca gcactgtact ttaggaattc agaccagaaa tgggtgtggc tctgggctgg 6660
cgactctaac cctggtgaaa ctttccaaag ttttaagatg ccagtcctaa ttaattggtc 6720
agtgtcagat tcccaggaac aatataatgc tagagtcagg atggtacagt atgctaatgc 6780
acaacagcag actttgacag accctgagga agatgatgat cccctctctg atgtcacttc 6840
gctttttgat ccaacagcgg aagatgagac tgacttccac ctagcagtct cactcaagac 6900
ctctgactac ttaaaagaag aggctgagta ctggaaagcg aaggcgcagg ccttgcttat 6960
ggagaaggca ctaagtgcac cacaagcagg gacagtccgc tttgagaagg gcggacatga 7020
gtgatctctt cactagcagc cgcggccacg ccgagtagga tcgagggtac agctgcacct 7080
tctcattagg cttgagggag aatcggccct ctctgcctta aattaacccg gtgacagtca 7140
ggtggtcacc cacacaaatc gcgtgcgggt acccctgacc gggtttttgt ttaaagaatc 7200
aaatgtgatt tttaagcatt taaaatcttt aaaaaaaaaa aaaaaaaaaa aa 7252
<210> 5
<211> 83
<212> DNA
<213>Artificial sequence
<400> 5
ccaggtcaag atacaatgca aatataacct tcaatgttgg ctatcgtgga aggacttcaa 60
catcatttac acttggaaca cac 83
Claims (10)
1. one group of primer and probe, which is characterized in that the nucleotide sequence of the primer such as SEQ ID NO.1-SEQ ID NO.2
It is shown；The nucleotide sequence of the probe is as shown in SEQ ID NO.3.
2. primer and probe described in claim 1 answering in preparing the reagent for detecting novel goose astrovirus or kit
With；The deposit number of the novel goose astrovirus is CCTCC NO：V201808.
3. a kind of PCR kit for fluorescence quantitative of the novel goose astrovirus of detection, which is characterized in that the quantitative fluorescent PCR examination
Agent box contains primer and probe described in claim 1.
4. PCR kit for fluorescence quantitative according to claim 3, which is characterized in that in the PCR kit for fluorescence quantitative
Further include：Standard items and quantitative fluorescent PCR reaction reagent.
5. PCR kit for fluorescence quantitative according to claim 4, which is characterized in that the standard items are made by the following method
It is standby to form：
Use primer amplification deposit number shown in SEQ ID NO.1-SEQ ID NO.2 for CCTCC NO：The goose star of V201808
The genome of shape virus, obtains amplified production, and amplified production is connected on pMD18-T carriers, and screening positive clone simultaneously extracts
Standard items are prepared in Plasmid DNA.
6. PCR kit for fluorescence quantitative according to claim 4, which is characterized in that the quantitative fluorescent PCR reaction reagent
Including：One Step RT-PCR Buffer, TaKaRa Ex Taq HS, PrimeScript RT Enzyme Mix II and ROX
Reference dye.
7. a kind of detection reagent of the novel goose astrovirus of detection, which is characterized in that the detection reagent contains claim 1 institute
The primer and probe stated；The deposit number of the novel goose astrovirus is CCTCC NO：V201808.
8. the detection reagent described in claim 3-6 any one of them PCR kit for fluorescence quantitative or claim 7 is as follows
(1) in-(3) it is any in application：
(1) novel goose astrovirus is infected to goose and carries out epidemiological survey；
(2) the novel goose astrovirus pollution in blood or serum product is monitored；
(3) novel goose astrovirus copy number is accurately detected, the progression of infection of goose astrovirus is specified.
9. a kind of side for detecting novel goose astrovirus using claim 3-6 any one of them PCR kit for fluorescence quantitative
Method includes the following steps：
(1) standard items are subjected to gradient dilution, carry out TaqMan Fluorescence PCRs later, according to the concentration of standard items and Ct values,
Draw fluorescent quantitation standard curve；
(2) sample to be tested total serum IgE is extracted, TaqMan Fluorescence PCRs are carried out, collects sample to be tested fluorescence signal, fluorescence is believed
Number data processing is carried out, obtains Ct values and amplification curve, qualitative and quantitative detection is carried out to sample to be tested；
Preferably；In step (1) and step (2), the program of TaqMan Fluorescence PCRs is：
42℃5min；95℃10s；95 DEG C of 5s, 53 DEG C of 30s, 72 DEG C of 30s carry out 40 cycles later.
10. deposit number is CCTCC NO：The goose astrovirus of V201808 leads to the starlike disease of goose of young goose gout in preparation detection
Application in the PCR kit for fluorescence quantitative of poison.
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| CN201810495163.XA CN108384899B (en) | 2018-05-22 | 2018-05-22 | Fluorescent quantitative PCR kit for detecting novel goose astrovirus and application thereof |
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| CN201810495163.XA CN108384899B (en) | 2018-05-22 | 2018-05-22 | Fluorescent quantitative PCR kit for detecting novel goose astrovirus and application thereof |
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| CN109457055A (en) * | 2019-01-03 | 2019-03-12 | 山东省农业科学院家禽研究所 | Detect the primer sets and kit of goose source astrovirus |
| CN109762062A (en) * | 2018-09-19 | 2019-05-17 | 天津瑞普生物技术股份有限公司 | A kind of preparation method of goose goat Yolk antibody |
| CN111088402A (en) * | 2020-01-13 | 2020-05-01 | 华南农业大学 | Novel goose astrovirus detection primer group and kit |
| CN111826473A (en) * | 2020-09-03 | 2020-10-27 | 佛山科学技术学院 | Primer pair for fluorescence quantitative PCR detection of goose type 2 astrovirus and application thereof |
| CN112662814A (en) * | 2021-01-22 | 2021-04-16 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Goose-origin astrovirus nucleic acid CRISPR-Cas13a detection system, RPA primer pair and crRNA |
| CN114214466A (en) * | 2022-02-09 | 2022-03-22 | 江西省农业科学院畜牧兽医研究所 | Novel goose astrovirus and duck tembusu virus multiplex fluorescence quantitative PCR detection primer probe set, kit and application |
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| CN109762062A (en) * | 2018-09-19 | 2019-05-17 | 天津瑞普生物技术股份有限公司 | A kind of preparation method of goose goat Yolk antibody |
| CN109762062B (en) * | 2018-09-19 | 2022-02-25 | 天津瑞普生物技术股份有限公司 | Preparation method of goose gout egg yolk antibody |
| CN109457055A (en) * | 2019-01-03 | 2019-03-12 | 山东省农业科学院家禽研究所 | Detect the primer sets and kit of goose source astrovirus |
| CN111088402A (en) * | 2020-01-13 | 2020-05-01 | 华南农业大学 | Novel goose astrovirus detection primer group and kit |
| CN111826473A (en) * | 2020-09-03 | 2020-10-27 | 佛山科学技术学院 | Primer pair for fluorescence quantitative PCR detection of goose type 2 astrovirus and application thereof |
| CN111826473B (en) * | 2020-09-03 | 2023-11-07 | 佛山科学技术学院 | Primer pair for fluorescent quantitative PCR detection of goose type 2 astrovirus and application thereof |
| CN112662814A (en) * | 2021-01-22 | 2021-04-16 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Goose-origin astrovirus nucleic acid CRISPR-Cas13a detection system, RPA primer pair and crRNA |
| CN112662814B (en) * | 2021-01-22 | 2023-12-22 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Goose astrovirus nucleic acid CRISPR-Cas13a detection system, RPA primer pair and crRNA |
| CN114214466A (en) * | 2022-02-09 | 2022-03-22 | 江西省农业科学院畜牧兽医研究所 | Novel goose astrovirus and duck tembusu virus multiplex fluorescence quantitative PCR detection primer probe set, kit and application |
| CN114214466B (en) * | 2022-02-09 | 2022-07-22 | 江西省农业科学院畜牧兽医研究所 | Novel goose astrovirus and duck tembusu virus multiplex fluorescence quantitative PCR detection primer probe set, kit and application |
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