CN108411041A

CN108411041A - A kind of fluorescence quantitative RT-PCR kit of the novel avian reovirus of detection and application

Info

Publication number: CN108411041A
Application number: CN201810494536.1A
Authority: CN
Inventors: 刁有祥; 唐熠; 姜晓宁
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2018-05-22
Filing date: 2018-05-22
Publication date: 2018-08-17
Anticipated expiration: 2038-05-22
Also published as: CN108411041B

Abstract

The invention discloses a kind of PCR kit for fluorescence quantitative of novel avian reovirus 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 avian reovirus, the high specificity of the kit, only be specifically bound with novel avian reovirus, with other chicken source viruses all without cross reaction；And the high sensitivity of detection, it is 10 in standard items¹‑10⁹There is fabulous linear relationship in copy range, for detection range up to 9 orders of magnitude, virus that can be with efficient detection Novel chicken reovirus disease chicken tissues and without clinical symptoms carries chicken, improves detection efficiency.

Description

A kind of fluorescence quantitative RT-PCR kit of the novel avian reovirus of detection and application

Technical field

The present invention relates to avian viruses detection technique fields, and in particular to a kind of fluorescence of the novel avian reovirus of detection is fixed Measure RT-PCR kit and application.

Background technology

Avianreovirus (Avian orthoreovirus, ARV) belongs to Reoviridae, Orthoreovirus, It is the cause of disease for causing Avian viral arthritis, main infection broiler chicken, meat egg dual-purpose type chicken also has infection.ARV mainly encroaches on chicken Gambrel, toe joint, Bock joint and its tendon cause arthrosynovitis or tenosynovitis etc..Illness chicken typically exhibits limping, squats It sits, be reluctant to walk about and the even useless exhausted symptom of anorexia, cause that feed conversion rate is low, extremely washes in a pan residual chicken increases, reduce life Benefit is produced, serious financial consequences are caused to poultry farming.From the mid-term the 1980s disease since China reports for the first time The trend aggravated is presented in the infection conditions of China ARV, and clinical manifestation is also more and more diversified.

2016, the ground such as China Shandong, Jiangsu Breeder hens field and commercial broiler flocks field were in succession with arthroncus and limping The disease of symptom.The disease spread speed is fast, morbidity range is wide, different days, different cultivars chicken can infect, but to commodity The harm of broiler chicken is particularly acute.Early stage is mainly shown as that body temperature increases, spirit is depressed, the useless exhausted and nutrient absorption of appetite is bad Deng, with the development of the course of disease, infected chicken gradually appears arthroncus, inflammation, is reluctant to walk about, walk lamely etc. symptoms；The visible pass of dissect The exudation of intracavitary cellulosic is saved, tenosynovitis, the pathological changes such as sura disinsertion cause serious economy to China's broiler breeding industry Loss.

It has been investigated that the outburst of the disease is caused by a kind of novel avian reovirus infection, vaccine there is no at present It can be prevented, existing commercially available avian reovirus vaccine can not carry out effective prevention and control to the disease；Conventional is antiviral It is invalid with antibacterial therapy method.It is to the novel avian reovirus in the effective measures for currently in this case, controlling the disease Infection is monitored, and isolation is taken to find chicken infected early to it or slaughters measure, and the lonely disease of intestines is exhaled to reduce Novel chicken Loss caused by poison infection.

Currently, including mainly that cytodiagnosis, immunology diagnosis and molecule are given birth to for the detection method of Avianreovirus Object diagnoses three categories, wherein cytodiagnosis technology is mainly separately cultured using cell and is observed with Electronic Speculum.This method operates Relatively complicated, 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..It is current existing using glimmering Fluorescent Quantitative PCR method detects the report of reovirus in cows, but since the novel avian reovirus system reports for the first time, needle It cannot be used for the detection of the novel avian reovirus to the reovirus Diagnostic Strategy of mammal and other birds, so far There are not the effective ways for detecting the novel avian reovirus yet.

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 avian reovirus of detection RT-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：M1-F：5’-ATGGCCTATCTAGCCACACCTG-3’；(SEQ ID NO.1)

Reverse primer：M1-R：5’-CAACGTGATAGCATCAATAGTAC-3’；(SEQ ID NO.2)

Fluorescence probe：5’-FAM-TGCTAGGAGTCGGTTCTCGCA-BHQ1-3’；(SEQ ID NO.3)

The second aspect of the present invention, provide above-mentioned primer and probe prepare the reagent for detecting novel avian reovirus or Application in kit.

The novel avian reovirus is preserved in China typical culture collection center on April 26th, 2018, protects It is CCTCC NO to hide number：V201817, address：Wuhan, China, Wuhan University.

The third aspect of the present invention provides a kind of fluorescence quantitative RT-PCR kit of the novel avian reovirus of detection, The fluorescence quantitative RT-PCR kit contains above-mentioned primer and probe.The deposit number of the avian reovirus is CCT CC NO：V201817.

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：V201817's The genome of avian reovirus, obtains amplified production, and amplified production is connected on carrier, and screening positive clone simultaneously extracts matter Grain DNA, that is, be prepared standard items.

The quantitative fluorescent PCR reaction reagent includes：One Step RT-PCR Buffer, TaKaRa Ex Taq HS and PrimeScript RT Enzyme MixⅡ。

The fourth aspect of the present invention provides a kind of detection reagent of the novel avian reovirus of detection, the detection reagent Contain above-mentioned primer and probe.

The fifth aspect of the present invention provides above-mentioned fluorescence quantitative RT-PCR kit or above-mentioned detection reagent in following (1)- (3) in it is any in application：

(1) novel avian reovirus is infected to chicken and carries out epidemiological survey；

(2) the novel avian reovirus pollution in blood or serum product is monitored；

(3) novel avian reovirus copy number is accurately detected, the progression of infection of avian reovirus is specified.

The sixth aspect of the present invention provides a kind of above-mentioned fluorescence quantitative RT-PCR kit of utilization and detects avian reovirus Method, include the following steps：

(1) standard items are subjected to gradient dilution, carry out Fluorescence PCR later, according to the concentration of standard items and Ct values, painted Fluorescent quantitation standard curve processed；

(2) sample to be tested total serum IgE is extracted, Fluorescence PCR is carried out, sample to be tested fluorescence signal is collected, to fluorescence signal Data processing is carried out, Ct values and amplification curve are obtained, qualitative and quantitative detection is carried out to sample to be tested.

Preferably, in step (1) and step (2), the program of Fluorescence PCR is：42 DEG C of 5min, 95 DEG C of 10sec, Reps：1；95 DEG C of 5sec, 60 DEG C of 34sec, Reps：40.

Beneficial effects of the present invention：

(1) be directed to it is newfound can result in broiler chicken arthrocele, limping novel avian reovirus, the present invention design Can to the fluorescence quantitative RT-PCR kit that the novel avian reovirus is detected, the high specificity of the kit, Only specifically bound with novel avian reovirus, it is viral with other chicken sources, such as avian influenza virus (AIV (H9N2)), fowl pox disease Malicious (APV), newcastle disease virus (NDV), adenovirus (FAV), Egg Drop syndrome virus (EDSV), infectious bursa of Fabricius virus (IBDV) and infectious bronchitis virus (IBV) etc. is all without cross reaction.

(2) high sensitivity detected is 4 × 10 in standard items¹-4×10⁹There is fabulous linear relationship in copy range, Detection range, can be with efficient detection Novel chicken reovirus disease chicken tissues and without the disease of clinical symptoms up to 9 orders of magnitude Poison carries chicken, improves detection efficiency.

(3) kit using the present invention can be monitored Novel chicken reovirus infection, to find early It is chicken infected to take it isolation or slaughter measure, it is lost caused by Novel chicken reovirus infection with reducing；It can be with To chicken infection reovirus carry out epidemiological survey, and to the avian reovirus in blood or serum product pollute into Row monitoring.

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, 2016, the ground such as China Shandong, Jiangsu Breeder hens field and commercial broiler flocks field phase After with the disease of arthroncus and lame behavior symptom.The disease spread speed is fast, morbidity range is wide, different days, different cultivars Chicken can infect, but be particularly acute to the harm of commercial broiler.Early stage be mainly shown as body temperature increase, spirit it is depressed, Appetite is given up absolutely and nutrient absorption is bad etc., and with the development of the course of disease, infected chicken gradually appears arthroncus, inflammation, is reluctant to walk about, The symptoms such as limping；Dissect visible joint intracavitary cellulosic oozes out, tenosynovitis, the pathological changes such as sura disinsertion, gives China's meat Chicken aquaculture causes serious economic loss.Research finds that the prevalence of the disease is caused by novel avian reovirus.At present There is no can effectively control the drug and method of the Novel chicken reovirus infection.Based on this, the present invention proposes a kind of energy The PCR kit for fluorescence quantitative for enough detecting the novel avian reovirus, so as to can find early it is chicken infected it is taken every From or slaughter measure, lost caused by Novel chicken reovirus infection with reducing.

It is known that the host range of Avianreovirus is wider, this virus of generally existing in various birds, from 1954 It has been separated in the birds of a variety of morbidities such as chicken, goose, pigeon, ostrich, duck, turkey and other pheasants or health since year Reovirus, and the reovirus being separated to from different hosts its there are larger variability, pathogenic, gene sequences Row and the coding of major protein all have differences.

Present inventor isolates one plant of N-ARV- from the broiler chicken tendon tissue with arthroncus, limping symptom LY383, birds reovirus gene by segmented 10 genetic fragments (including：L1-L3, M1-M3, S1-S4) composition, this Invention has carried out genome sequencing to the avian reovirus N-ARV-LY383 of above-mentioned new separation, and respectively by 10 genes Segment and the Avianreovirus of existing report have carried out sequence alignment and homology analysis, as a result, it has been found that, new isolated strain N- 10 genetic fragment multidigits of ARV-LY383 illustrate the strain N-ARV-LY383 newly detached in a relatively independent branch It is 1 independent kind of Orthoreovirus different from other Avianreovirus.And the novel avian reovirus is protected It is hidden in China typical culture collection center, deposit number is CCTCC NO：V201817.With existing Avianreovirus phase Than, 10 genetic fragments in avian reovirus N-ARV-LY383 full-length genomes of the invention have occurred genetic recombination and Mutation, therefore, the avian reovirus of novel avian reovirus to be detected of the invention and existing report there are variability, Effective detection method there is no for the avian reovirus reported in the prior art, for the novel avian reovirus Detect its difficulty bigger.

For fluorescence quantitative RT-RCR detection, how design primer and probe sequence are the passes for ensureing detection validity Key.Although having software and design of primers principle of many design of primers etc. in the prior art, to design to obtain high specificity, spirit The high primer and probe combination of sensitivity, being not can be simply obtained by primer-design software, this needs technical staff Optimization targeting regions are constantly selected using its professional knowledge, and design of primers and right is carried out further according to the targeting regions of optimization The primer of design carries out screening repeatedly, optimization, redesign.Specifically, since present invention Novel chicken to be detected exhales intestines Lonely virus is different from the chicken source reovirus of existing report, and how the Novel chicken of detection the application of specificity exhales the lonely disease of intestines Poison, and avoid that cross reaction occurs with other chicken sources virus, it is the difficult point place of primer and probe design of the present invention.For exhaling intestines The detection of lonely virus, some is with the segments S6 conserved regions design primer, and some is with S7 gene conserved regions design specificity amplification primers Therefore for the detection of reovirus, targeting regions how to be selected to carry out primer and probe design with Taqman probes It there is no unified cognition at present.The present invention has during the test carried out the targeting regions for carrying out primer and probe design excellent Change, as a result, it has been found that, primer and probe design, Ke Yishi are carried out with the sequence conservation of the M1 genes of the novel avian reovirus Now to the specific detection of the novel avian reovirus.

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：M1-F：5’-ATGGCCTATCTAGCCACACCTG-3’；(SEQ ID NO.1)

Reverse primer：M1-R；5’-CAACGTGATAGCATCAATAGTAC-3’；(SEQ ID NO.2)

Fluorescence probe：5’-FAM-TGCTAGGAGTCGGTTCTCGCA-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 93bp.

The present invention during the test, also passes through other conserved region sequences of reovirus 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’-GTAAGCAACCTCAGGATATCG-3’；

Reverse primer：R；5’-TCATGTCGCGCGTCGTAT-3’；

Fluorescence probe：5’-FAM-TCAAAATGGTGGACTTCAGTTTCGAT-TMARA-3’.

Second group：

Forward primer：F:5’-TGACAGGCCTGACTACGTTA-3’；

Reverse primer：R；5’-ATGCTTGAAGTGAGACGTAC-3’；

Fluorescence probe：5’-FAM-TGCGAGTCGGTTCAGTTTCGAT-TMARA-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 avian reovirus is detected are optimal.And other primer and probe combinations then cannot be to novel Avian reovirus and other chicken 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 avian reovirus according to two generation sequencing technologies, (deposit number is CCTCC NO：V201817) complete Genome sequence, the sequence point of this 10 genetic fragments of L1, L2, L3, M1, M2, M3, S1, S2, S3, S4 in whole genome sequence Not as shown in SEQ ID NO.4-SEQ ID NO.13.

For the primer sequence and fluorescence probe sequence of the sequence conservation design specificity of M1 genes, sequence design is such as Under：

Forward primer：M1-F：5’-ATGGCCTATCTAGCCACACCTG-3’；(SEQ ID NO.1)

Reverse primer：M1-R；5’-CAACGTGATAGCATCAATAGTAC-3’；(SEQ ID NO.2)

Fluorescence probe：5’-FAM-TGCTAGGAGTCGGTTCTCGCA-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 93bp (shown in SEQ ID NO.14).

Embodiment 2：The foundation of fluorescent quantitative PCR detection method

(1) extraction of viral RNA：

Sample to be tested total serum IgE is extracted using classical Trizol methods or commercial kit；

(2) Fluorescence PCR Establishing：

One-step method fluorescence quantitative kit used uses the article No. from Takara companies for the One Step of RR064A PrimerScript^TMRT-PCR Kit kits sequentially add 2 × One Step RT- in 200 μ l Fluorescence PCR pipes III~10 μ l, 1 × TaKaRa Ex Taq HS (5U/ μ l)~0.4 μ l, PrimeScript RT Enzyme of PCR Buffer Mix II~0.4 μ l, PCR Forward Primer (10 μM)~0.4 μ l, PCR Reverse Primer (10 μM)~0.4 μ l, The II μ l of (50 ×)~0.4 of the μ of Probe~0.8 l, ROX Reference Dye or Dye, the middle sample to be tested extracted of step (1) The μ l of Total RNA~2 use RNase Free dH₂O is supplemented to 20 μ l systems.It is placed in ABI 7300Fast quantitative fluorescent PCRs It is reacted in instrument, reaction condition is 42 DEG C of 5min, 95 DEG C of 10sec, Reps：1；95 DEG C of 5sec, 60 DEG C of 34sec, Reps：40.

(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) for designing M1 genes first, expands Increase Novel chicken reovirus gene group and obtain the amplified production (shown in SEQ ID NO.14) of 93bp, according to classical molecule gram Grand operating method is connected on pMD18-T carriers, is named as pMD-M1, and screening positive clone simultaneously extracts Plasmid DNA, profit With spectrophotometer measurement plasmid concentration, as standard items.10 times of gradient dilutions are carried out to standard items, it is glimmering to carry out TaqMan later Light PCR reactions, according to the concentration of standard items and Ct values, instrument software can draw out fluorescent quantitation standard curve automatically；Specially： Y=-3.3610x+45.655, coefficient R²=0.9948, good linear relationship is presented；Wherein, the copy of x representative samples Number logarithm, y represent 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≤35.0, while amplification curve is smooth " S " type, then can be determined that in sample to be tested contain novel reovirus.

According to fluorescent quantitation standard curve, novel avian reovirus 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, for detecting novel avian reovirus (deposit number For CCTCC NO：V201817).Contain in kit：Forward primer (10 μ shown in the SEQ ID NO.1 that embodiment 1 designs M), reverse primer (10 μM) shown in the SEQ ID NO.2 that embodiment 1 designs, shown in the SEQ ID NO.1 that embodiment 1 designs Probe, 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：V201817's The genome of avian reovirus obtains the amplified production of 93bp, and amplified production is connected on pMD18-T carriers, screening sun Plasmid DNA is cloned and extracted to property, 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 DEG C of 10sec, Reps：1；95 DEG C of 5sec, 60 DEG C of 34sec, Reps：40, 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 avian influenza virus (AIV (H9N2)), fowl Poxvirus (APV), newcastle disease virus (NDV), adenovirus (FAV), Egg Drop syndrome virus (EDSV), infectious bursa of Fabricius virus (IBDV), the deposit number that infectious bronchitis virus (IBV), RS13 plants of avian reovirus and the present invention are detected is CCTCC NO：The novel avian reovirus of V201817 is template, carries out quantitative fluorescent PCR using the kit, as a result sends out It is existing, avian influenza virus, fowlpox virus, newcastle disease virus, adenovirus, Egg Drop syndrome virus, infectious bursa of Fabricius virus, infection Property bronchitis virus, RS13 plants of avian reovirus cannot be expanded effectively；Only deposit number is CCTCC NO： The novel avian reovirus of V201817 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 avian reovirus, with other chicken source viruses all without cross reaction.

(2) repetitive test

Using the kit, the repetitive test of the kit is carried out.Take 1.0 × 10³~1.0 × 10⁸copies/μL 6 The plasmid standard of a dilution carry out batch in batch between repeat to test, take 3 repeated samples every time, three weights in once testing The corresponding coefficient of variation of duplicate sample sheet (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 10 copy numbers, and 10¹-10⁹There is fabulous linear relationship in copy range, detection range is up to 9 orders of magnitude；And regular-PCR is minimum can only Detect 10⁴The standard items of a copy number.Show the kit carry out quantitative fluorescent PCR sensitivity be regular-PCR at least 1000 times, there is higher sensitivity.

Embodiment 4：The clinical application experiment 1 of kit of the present invention

Suffer from from Shandong, Jiangsu and other places and choose 40 parts of samples in arthritic pathological material of disease, extracting RNA is established glimmering with embodiment 2 Light quantitative approach is detected and analysis result.

Result judgement method：If sample to be tested fluorescence signal is more than threshold value and Ct≤35.0, while amplification curve is smooth " S " type, then can be determined that in sample to be tested contain novel avian reovirus.

The result shows that there is 38 parts of samples to contain novel avian reovirus in 40 parts of samples, detection accuracy rate is 100%.

Embodiment 5：The clinical application experiment 2 of kit of the present invention

The arthritic chicken for the novel reovirus of infection that clinical acquisitions are arrived, takes the tendon tissue of swelling to be added 5 It organizes homogenate after times physiological saline, after multigelation 3 times, takes supernatant to be inoculated with LMH cells, received after stable cytopathy to appear Poison.The RNA for extracting virus, the fluorescent quantitation method established with embodiment 2 is detected and analysis result.

After testing, novel avian reovirus is contained in sample, meets expection, show the detection method of the present invention it is accurate, Reliably.

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 fluorescence quantitative RT-PCR kit of the novel avian reovirus of detection and application

<130> 2018

<160> 14

<170> PatentIn version 3.5

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<213>Artificial sequence

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atggcctatc tagccacacc tg 22

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<213>Artificial sequence

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tgctaggagt cggttctcgc a 21

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<213>L1 genetic fragments

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ttccgatctc cgaacgccga aatgagttcg cgcaaagtgg ctagacgtcg tcataaggat 60

gctactgaat ttaaatacac taagaacgtt aacaagtcta agccatcctc tactgacgtt 120

aaagaacctg cggatagtgc cacagataag aaagtcactg ttccatcacc agacaatcca 180

gccgcttcta ctccctcttc cactgatgga gcttctcaaa catccgttgc taagcagacg 240

aatgataatg acgcctcagt taaggaatca gctcccaagc ctaccgtatc tagcgatggg 300

aaagacggaa tgcatggtgc tgtgaagttg caagacgcta aggccactgc agctgtggat 360

agtaataagg atagagatgt ggtatttggt ggcgcaggct ctggtgacaa aaatgctatt 420

acgaagactg gttccgttga caatgatggg ggcgttaaag tcgttccagc caaggatgct 480

acgatatctt cggccaaagc catgatggaa cagaagcagt tagttgctgg tcttccgaag 540

caaccgaagt ctgctaatca tttgtgcacc gtttgtatgg cacaattcgc gtctgctgac 600

gccctaacta tccatcagac tacgcactcc attggctcca atgctgctct gacgagcttc 660

tcgatttcta ctgctgttga agaattcatt caatcatggg ctgctgctac gtccactgcc 720

aataccaaga cggccttaac tgtgtctgac gtggactcgc tgatgatgac tgaaggaata 780

cgtctcataa cttgggattc tgggttatgt acatcttttg agcttgtccc gattgtccat 840

tcaaacactg ttcaagatgt aatttcgtac tcatggttca catcaagtta taatatcaca 900

actcccttcc cacaggcgcc tgtcgtgcga attgttttac gtactaattg ggctgccaaa 960

ttggactctc cctcgtcgtc gcgtgaatgt gatcttcgcc tcgccccacc tacagagagt 1020

aatgctcgat cattctcaat gctgctcaat actggtgcga ctccagaagg tactttcaac 1080

cccaacaccc ttcgtatgaa tgtgctgcag atgtgtcttc agtatgttct ggctaaccta 1140

cacttgaatc gtagcactca gttcaccatg gatttgactg ccgcagctcc caatctgtcg 1200

gcgtctcaac tccgtatcgt tccagatgat aaagatggta aatggttccc tgtcatgtat 1260

ccatctcgcg tgaacatccc actgttcaat aagacagctg attttgttaa tcagtgcatt 1320

cgtgacagaa ttggccgata tgaccgcgcc cagactttcg ctggcgcacc ctctgaatgg 1380

gctgacatgt gggaaacagc ggacgcgtta actctctccg tccgtgaaat gtggatgtca 1440

cgtatttccc aaatgaatat ctctcctgct gatatcgctg acgctatctc tcgatgttct 1500

cagtccctgc tcactgttgc cgcgccaaca gctccttctg tagctcgtct gttaccttgg 1560

cgggttagtt ccgatgagag gcagctcctc caattgttga tgtacttgaa cgttgggact 1620

agtgccgact acgtccaacc tattctgtct gcgttcgctc gaactctatc tcgcgtgtca 1680

ccattgcgta ttaatcccac tttaatcgct aatgccatgt cgacaattgt cgagagcact 1740

actaataccc agagtcctgc ggcagctatt ttgtcgaagc ttaaacctgt tgcctcggac 1800

ttctccgatt ttaggttggc gtgtgccgct tggttgtata atggttgcgt ccagacatac 1860

ttgtctgagg attcatatcc aagcagtggt ggatctgtta ctagcattga tacattggtt 1920

gatatgtttg tgtgcttgtt ggctttgcct cttgttactg atcctaatgc tccttgccaa 1980

gcctttatgg ttgtcgctaa tgccatggtt ggttacgaga atctaccgat ggacgaccct 2040

aattttactc agcaaagatt ggctgcagcg ttcaacaatc ccacgacctg gcctcaatgt 2100

ttcctccacc ctcaaaacat cgatcgacgc caatgtccga ttctctcatg gtgggctcag 2160

cagattcatc gtaattggcc cacaccgtct caaattactt atggggcgcc tgacatcatt 2220

gggtccgcta acttgtttac tcctcctgac gtgctgttgc ttccattaca gcataggccc 2280

atccgtatta ctaatcccac cctgaacttc gataatgagt tgacgacttg gcgtaacacc 2340

gtggttgatc tggttctgcg catcatcgac agtggtcggt accagcccaa ttggaatcag 2400

tccatccgtg cctctatgcg gaatgcgatg acaaatttca gaattattaa gtcctatact 2460

cctgcttaca tagcagaact gctacctgtg gaactggcag ctatcgctcc aactctaccc 2520

ttccagcctt ttcaggtgcc gtttgcccgc ttagatcgcg acgctatcgt cactcacgtc 2580

aatgtatcta gacaagctcc caacaatctt gctcaacctg cattgaacat gtccatgacg 2640

taccagcgca caggggttcc aatctctctt agtgcccgtc ccttggcagt cgctcttctg 2700

tcaggccagt accctactga tccccctctt cagactaatg tttggtacgt aaacactctc 2760

acgcctctat attccaatga tggtctcttt aataacgtcc agcacgctat ggttgcttct 2820

gaagcttacg ctaccttgat caccatgctg gctcagtgca ctgacatgca gtaccctgtg 2880

gatcggccat tgaattggct tcgtcagatt aatttggctg ctaatgaggc gacgattttc 2940

ggtcggtcaa ttaattcact tttccagact gctttcgacc tttcaccttc cactgtattg 3000

cttcaaccgt ttttggaatc tgatccacgt gcgacacagc tagccatttc ttacgttcgt 3060

tataatggtg acagtgaaac cttcgtgcca acagtgcgtc catctatgat ttcagaagcg 3120

acattgctcg ttgagcgtac tctctcgcac gaatacaacc tcttcggttt atgccgtggt 3180

gacatcatcc tgggacagca catgactcca actgcgttca atcctttggc tccgcctcct 3240

tccgtcgttt ttaacagggg tgatactgat gtctatgagt ttggctctcg tagcttcgcc 3300

aacttcggca tgaatgggga ggagatcttg gtcatggatg cgaacggcgt gcgtcgtcca 3360

ttactcggcc ggtgggttat gccactgcag cttttgatgg ttaatattgg cgtatttcct 3420

aagctgttgc tggatcgtat cctgaagggg cgcttgtata tccgacttga agttggcgcg 3480

tatccatata cggtgcagta ttaccaggga cgtgagttta cggatggctt cactctgctt 3540

gagcaatgga tgtctaaggt gtcgcccatg ggtatccctc ccgtcccttt cctcatgccg 3600

cagtccgaag gacacaacat cacttcaggc atggttactc attacatctg gtccactgaa 3660

tataatgatg ggtccctctt cgccacgaac accgacctac cagttactgt gttcggccct 3720

gaccgcacca tcccaatcga gcgttatcgg gcactcgtag atccaggtgc tcttcctgct 3780

accaaccaac tgccgcacac catcgacctt tactgctcac tgagacggta ttatctggaa 3840

acacctccta tcaccgctac tgttaccact tatggcgatg gactccccgc gctgaaccat 3900

tagagcggcg aggctagacg cgagttgatc gcgtcgactc tcgttggaga ttattcatc 3959

<210> 5

<211> 3830

<212> DNA

<213>L2 genetic fragments

<400> 5

gtattttcct caccatgcat gtcaacgggt ttgatgatgc tactctctcc tacgcacagt 60

ccatatcggg ggttatccca atgacaaata agttatttga gcaagcatct acctcaatac 120

gtgccctacc acgctcacat gtttatgcta tattagataa tgttaatttt tcagtttcat 180

gtgtcatccc gaatcgtatt ttccatcatc ctgatcactc tgagtacttt tacattgatg 240

cggttaacag agttagacgg aaacaagtta tcgatcctga tgatgtattt gtgccaaact 300

gcaatttgca gggtcttatt actccaatgg agagattacc aaattatggt caattgtctg 360

agactatttc gtcgaacgct cgggatggct tgccatctgc acgcgtagca gctacttttt 420

ataacacttc ggtgtcacag gctcgccaag ttaaagctcc acttgaatcc tttttgttgc 480

ctttgctatt atctgagact tgctcattat cagacgatcc ttgcggactt gacaccgcag 540

cttctcctcc gatccatacc aatctggcgt tatgggtgtt acgcgaaatt agtcgaacta 600

tttgtggatc ttcgaatgac cgttcacctt ggttactgct cgattcaggg gttgcgtggt 660

tcatgtctcc gttaatgtca tcggccattc cacctcttat ggctgactta acgaatctag 720

cgatctacaa acaaatttgt tctgtgtctg atgagcttca ttcccttgca gttcaagtgg 780

ttttgcaggc cgcggcgtca caatcgtatg gacactacat attgcagacg aagtcagtat 840

tcccccaaaa tacgttacat aacatgtttc gtacgctcac tgatggtatt gttccggtca 900

tagattggtt agaaccgcgt tcaaactatc gcttcatgct tcaaggtgcg cgtaaagtga 960

cttcagatga tgcgaatcag gctccggata acacggaagc cgcggagcaa ctcggtcgca 1020

agatggggtg cctcgatgtc gtacgctcct tacgtaagat gtcctcgtcc atcactgtcc 1080

attcacacga tgccatgacc ttcgtacgtg atgctatgtc gtgcactagt ggcatcttta 1140

tcacgcgtca acctactgag accgttttaa aagagtacac tcaagcgccg accattgaag 1200

tccctattcc tcaatcggac tggtcaccgc ctattggatc tctgcgatat ctctctgatg 1260

cctgttccct ccccgctgtg tatctggcta gggcttggcg cagagctgcc tctgctgtag 1320

tagataatcc gcacacttgg gaccctttat atcaggccat ccttcgctct caatatgtga 1380

cgtcacgcgg tgggtccggt gccgcgttaa gagatgcttt gaaggctgcg gaagttgagc 1440

ttcctcagta tcctggggtc agtgttaaag tggcgaccaa gatttatcaa gcggctcaga 1500

ctgctgacgt gcctttcgat aaattatctc gtgctgttct agctccattg tcgatgggct 1560

tacgtaacca agttcagcga cgtccaagaa ccatcatgcc catgaatgtg gttcaacagc 1620

agatttcagc ggctcatact ctctccgctg actacattaa ttatcacatg aacttgtcga 1680

cgacctcggg tagcgctgtg attgagaagg tggttccatt aggtatgtat gcatcctgtc 1740

ctcccgctca agcggttaat attgatatta aggcttgtga cgcgtccatc acgtaccagt 1800

attttctttc tgttatcgtc ggtgccattc atgagggtgc agcaggccgt cgtgtctcgt 1860

cttcattcat gggcgttcca ccaagcgtgc tgtccgttgt cgatgctagc ggagtgactt 1920

catccatgcc catctcaggt tttcaagtca tgtgtcaatg gttggctaaa ctttaccagc 1980

gaggttttga gtatcaagtg acggatacat tctcacctgg caatattttc acgcatcaca 2040

ctactacttt tccctctggt tcaacagcga cgtctacaga acatactgcc aataatagca 2100

cgatgatgga tggcttcctg cgcgcttgga ttccttcctc cggtgcgtcc gacgtactga 2160

agaagttctg caaatccatc tcaatacaac ggaattacgt ttgtcagggt gacgatggtc 2220

taatggtcgt tgatgggcta tcgacaggta aattatcagg cgagataatc gatgaatttg 2280

ttaaggaatt gagagcctat ggtaaatcat ttgggtggaa ctatgatata gagtttaccg 2340

gaaatgcgga gtatctaaag ttgtatttcc taaacggttg ccgtatacct aatgtttctc 2400

gacatccgat ctgtggcaaa gagcgcgctt caggggacaa gttagaaatg tggccgtcca 2460

ccattgacat cttcaatggc atatttgtga atggtgtgca tgatggtttg ccgtggcgca 2520

gatggttacg ttattgttgg gctcttgctc ttatgtattc tggaaagacc gtgcgtcatg 2580

atgattctga ggtgttgatc caatatccta tgtggtcttt tgtgtattgg ggtttgcctc 2640

ccgtgagcgc gtttgggtct gatccatgga tcttttctcc atatatgccc actggtgatc 2700

atggtttcta ttcaatgttg actttagtgc gccctctgat cactaacttg tccccatcgt 2760

cagacgcttc gggattattt ggtcaatgcg atcacaacgt tttgttcaat tctgagctag 2820

tttatcaggg ctattacatg gctcaatgcc cacggcaacc ttctcgctcg aaccgtagag 2880

atgatcccga ctctgtacag cgcttcgtca aggccttgga gtcttacctt tacatttccc 2940

ctgagctaaa atcgcgagtg cgccttggtc gtgaccgctg gcagaagttg gttgggtata 3000

cggaaaaatc tcccccgtcg cttgatgacg tggcgttcaa atggttccgt agtgcacagg 3060

aagctgatct cccaaccgct acagagattc aaagcatgga tctggccttg ctggcagcca 3120

gacgtaggac atatcagggc ttctccaagt tgttgaatac ctatttaagg gtaacctggg 3180

atttatctga tcctgttgaa cacgctgtag atccccgcgt acccttgtgt gccggtgtct 3240

ctccatcaaa tagcgagccg ttcctcaaat tgtactccgt aggtcctatg atgcaatcca 3300

cgcgtaaata ctttagcaat acgctattta tccatcggac tgtgtctggt cttgacgtcg 3360

atgtcgtcga tcgtgcgctc cttaggttgc gtgcccttaa tgcgcctgat gacgtggttg 3420

tagctcaact tttgatggta gggttgtctg aagccgaagc cgctacatta gcagcgaaaa 3480

tacggacgat ggatatcaat gccgtgcaat tggccagagt tgtcaactta tccatccctg 3540

actcatggat gaccatggat tttgatcgct tgatacgaga tatcgtgtct gtcactcctc 3600

tgaccgttcg atccctaacc accgatctac cctctggcgt accgtgggct cgcgcgatct 3660

tacagttctt aggtgcgggt gtcgccatga cggccgtcgg acccttgcgt cgtccctact 3720

tacactcagt tgccggaggc atgtcttcat tcattaagca gttccgccgg tggatgcgtg 3780

ccgaaacgag gtagcgtccg tgcccggcat ggctcgagga attacccatc 3830

<210> 6

<211> 3876

<212> DNA

<213>L3 genetic fragments

<400> 6

gcttttccac ccatggctca gattcgaggc cttcggttgt ctacgacgct ctcagctcca 60

cctccacgca agattataac atctcacact tatgatgagc tgatctccgc tctgaagtta 120

gcaaccaagc cttggcgccc tttgaagtcg cgaaataatg attctgtcac ggcagtgcag 180

ctcctttttc cccttaatgg ttatattgaa cccatgttca tgttggaaaa ggatatagcc 240

cttagtgatt ttgaggcctg gttgacgcct cttctatctg cactcgctga ccagttgctt 300

agacactacc ctatcgccgc ctatcacggg cgtttgatta atccgctgct atctaatgca 360

attgttgccg ctttcttgtc taacgtgcct tatgcgcatg cattggatca tctcttcctt 420

gttagaggaa acgtcgagga tattatggat gcggggatcg caattcagaa tcacttgtgg 480

ttcgatcgcg gtgcactagt gacccctgct ggacagaaat ttgttcagct gactggctat 540

aacttctcct ctaatgatcc gtgtctattt tctaagcaat tgcgttgtta tggcctcgtt 600

tactactttc tcgacatggc cgaatgtctg gcgtattgtt ggcgtcatct atccaattca 660

actccactga tacactttga ccgtccgtcc aatggagttc attgtttggt gccctctgaa 720

tccacgacgc ctatcgctgg ttcgttacca gtgtcagcac tcagctctat tttgttggaa 780

tcctgtcttc agcaatctac aattaatgcc cttactccca ctggttcgcc cgtcattaga 840

caaatagaag cattgttgcc tatatcatca ccgtttttcg aacgacggaa cactctggaa 900

tattctctct tcgcactgtc aaatgctctg gtaaatggtt atcagcttgt agacttgcgt 960

tccggccacc ctgattgcgc tactgttgct gccatcctag ctagattgat tgatttctct 1020

aaggatatca ccgttattca accgcgccct gctctctttg ctatcaacag cgacagtccg 1080

cttacgtata gtggagaaaa tgctaatttt atttcgcgct tgacttgtgc gtccggtaga 1140

cctattggtc cggtcgttgt cggtaaatcc gttgatcatg ccgttggttg gatgccccag 1200

tttgaccccg ccacgtccta caaccctgat ctctcgatgg actcacttgc tcgtgccacg 1260

acactgcctc tccgtgctaa gtattcgact ttctggtcag gcccagcatt gttttctttc 1320

gcttcatgta ataggcacaa tggtgtatat gacatacagt tcatggctca atttcctcct 1380

acttacttta gtgatgatga cgccttttct agatcacgat tctcttctta tcgtgcagtt 1440

agggaccggt cattgttgaa ggataccgct aatttgatgt acatctcgaa tttgtccagc 1500

tctcacgacc atcgtcttgt cccagattct aaaactatga tttatgtggg ttcctctggt 1560

actcatgtag ataatcaacc ttctatcatt aaacctctct tagctggaac tcttccgggt 1620

gtttttcgcc ccctttctat aaaacaggtt ggttgggagg tcactaatgg aacgatttgt 1680

gatgttgagc ttcctttagc cactggtacg ttcttcttcg tgtacagtga tgtggatcaa 1740

gtgcaatcag gtgattctga tttggacgcc tcctcgcgtc gcttttgctc ccaattggac 1800

atgctaatga agttgacgtt tactggtgga tcgcttgtcg ttaaatgcaa ttttccgact 1860

agtctagtgt ggcgtcacat cttctccact gtttctcctt atttctcggc tattcattta 1920

atgaagcctc tcgtgtcaaa taatttagaa ctgtatctat tgtttgcgga gcgtttgccc 1980

gttcctgacg tcgcgttccg tccttcagcg gacgttgtcg ttttctggcg atcacagcta 2040

caacgctatc gagtgttgcg tgattccttt tctaatgtgc cctccatcgg gtccactctc 2100

actttagatg aacctttgac tgtctctatt ctcaattttg ttgacgtcac ctccctttct 2160

tctcttgagg atcaacgagc cttatctgct ttttcagttt taacttctct agggtcacag 2220

aaactctcgc ttcatcctta ctttgatagc taccgcacgc agctcactgg aataattact 2280

ccacattcac gtaatcttct agatagactg gcgtacgtcc cgcgcgtttt tccttcgacg 2340

attgatgtgc aacatcgtgt catggcctct tcagatccag aaatttttgg ttttcgttct 2400

aattcatgga ctcaactgtc cttcttctac gacgcgacgt taacttctat tgattttact 2460

gatgtaaagc actggttaga tttagggacc gggcctgagg cgcgaccgtt gtcttttctg 2520

ccttccgatc ttcccgtcac gttatgtgac actcgtccat tcatcttccc ttccggctgc 2580

tgggctactt tcactgattt cttaagttat gactaccttg tcacgaatgt cgtgctctca 2640

actggtgccg atgtcgtatc ctgtgttctt tctctgggtg cggcctgtgc tgatgccaac 2700

ataactttac atgaaggcgt gcggcagctg atttcccaat gcgtggatgc caatgtcaag 2760

acattgttcc tgcagcttaa ttgtcctctc ccatctgcgg gtgatgtatc tcgggagatt 2820

cttgagatgg ttcagactaa ttcaacttac gtgtttcata ccttgggtcg tattgaaccg 2880

ttcattccat actccgctct ttcagagata gttgaggact tgtgtcccgg catcgtcatt 2940

gaaattaaga ctatggatcc ctctctctca tggcttgatt acgctgttca atccaatgcg 3000

tcagtgacgt cggatgatat cgtattggca atgcgtctgt ctcacttctg tccacttttc 3060

gtgtttcatt ttgaccgtca gtctgctcaa tttccggatg atgcgcgtgt tgggactcct 3120

tttactgtca cgctgttaga ttatgaagat actcgttcat acgaggtgac gttagataat 3180

gtcactatcg ccaccgttac cgcaggtgct ttggtgggtt tctcatctgg tgtcagcgtc 3240

agttcaacca acaatcagct tattttaact atcgattccg caagtccagg aattctctcg 3300

gtcattcaga ttcttcccgc tcgtatctct ttaggcagtt gtgtgataga agcaccggat 3360

ccatctctct ccttgatctt ccccgccacg ttagatacct ctttgtcggg aaccgatttg 3420

gagctgtatt tgtctgactg gtacgatgtt gcattatttt acgtcgatga aatccactcc 3480

cgcttgctgc cagtgtccga taccaagtat gaaatatatc gcaaggatca ggcgccgaac 3540

agccgggtga tcaactatat tttcgatcgg tctgatgtgt ttttcaagct agtgttatgt 3600

gacgtatctc cctcaggagt aggccgtttc atctaccgtg agttgccaga attaagttca 3660

cctgtttggc cagacaacgc gcgcactttc ttgtccatac cgttcgagtc acccatggtg 3720

attgtctcgc cggacggacc tgttaattac gatggcgcaa actttactcc tccaaactca 3780

tggttgacgg ttgatggcag tacctgcgtt gtagatggcc gtccttcgtt ctacgtgccc 3840

cctggccgat atggtctggt gagagtctaa acgact 3876

<210> 7

<211> 2283

<212> DNA

<213>M1 genetic fragments

<400> 7

gcttttctcg acatggccta tctagccaca cctgtgctag gagtcggttc tcgcattacc 60

gctttagatc gtactattga tgctatcacg ttgaaacctc gaatcgactt acaagatgta 120

tacacaattg atcccacttt gactctgcgt cagatagagt taatctcttc gggaacttca 180

atggacgata tcgctcgtgg actgttgcac cgagactggc gtcgtcaatc catcatcgtt 240

ttgcttccct cgcgtcgctc tctccttgag tatctattgt ctaacccttc tgtctgtcca 300

gacggtttag atcgttctcg acttaaagga tttcagaagc gtccaaatga ttttcgtgtt 360

caagatttct tctctccact gatcacggac tcgacgtcaa ttgctacata ctctcgatgg 420

cttaatgccc accctgttgt gtactcaact actcataagg tcgctggtgc tcgggtgcgt 480

ctctttggac ctgccaaatt atacattctg tcacctgacg ttcttcgcga attatccatt 540

ttgagatcca cggatcgtgt cctcgttgta cctacagcac gtgtatatgt tggttgcttt 600

cctagcgctt ccactagtaa ttgtgtgctc actgcacgcg accgctggaa tgctcctgac 660

gttcatcccg ttgtcaaggc aatccaatta gcatatgacc atcaatatcg tgtcaccgct 720

cgctatcttt cggatcccct tgtctccgcc cttcttgttg ggaatcggtc ggtgaagacc 780

ttgaaggtac agccagtaga ggccagagca gcacgatcag tcggcattcg cgttcaagcg 840

atgacgcccc ctcgtggtat caacacctct atcatccaag tcgttgatct caggctgcaa 900

tgtcgacatt ctctcattcc caccgaaagg cccttcccgc tgacatttat cggcctccca 960

tcctgtttgc tccagcattt ggatttgacg ctatctgacg attgggtgcc tattcgtgat 1020

cccacgggca tgtttgaaat gtggttcatg attcttacgc tcacttgtga taagattctt 1080

gatggacggg gcaacgctgt ttttctcatc cccagttcta ctaatgcatt gtcgattaat 1140

tatgtacagc ttacatcgac cgcgtctcaa cgccctcagt cattagcggc aaatgcatct 1200

ggacggatag attctatcgg actatgtatg cctaaggggt cttttaagtc aactatgatt 1260

aaatttctca ctggcttgga gatttgcggc acacgagtga tgtactcgga cgtcgtgatg 1320

gacagtgatg acgtgggtga cgctttggat cctacttttg aaacggcttt gtatgatgca 1380

ctggtagcac ttgatccgcc ttttgacgtt gataagttgg ctagccccac tgatctagtt 1440

aatcaggagt acgttgcgtc tcatatgtac ccgacattct tacggcttgt caatgagctg 1500

ctgactccta aggcttcaga gttgtactct gagcgtagcg ttgaattccg atctcttact 1560

tacgcgcacg ctgattctga atttcttaac tcatgctgga ccgctcgctt aatgcgttgc 1620

tttatcaact atcatgaaga gcagaatatc ttacttcgtc ctggacgcgt tggtggggtg 1680

ttatttcaag tcgcgttgag ccgttgctat aagatgttcg ctacttccac tcctgcttcc 1740

cctctgtcat tgttcctcaa gtcgttgttc gttccttgga ttgagtctgc cccactgtta 1800

gcgaatctaa cgccaaatga gtcttctcgt gtgttagcat ggtatattcc ttcctcgtac 1860

tggagcgaca atggttggtg cgtttgtgac actcatcgtc acgtcacctt ctccttcatc 1920

cgcggtcttc ccgccgacct gtcggtgtta gatctgtttg attggtctcg attccgcgcg 1980

actataaacg tggacacgtc tctcgtggag ctaggcgcag acattcgtgc ggtcaaagta 2040

tcagtccatt ggacatctca gaagcccact gtggacgttt ttgacaatcg tgcgcttttc 2100

acccccttcc agcactacca tttgagtctc cactgtaatt gcgcacctgg tcgacctttc 2160

ttcgcgaaga acatgaagct atatttgtcg acggtaggag gcgagcactg acgggccgtg 2220

gggcggtgac acccagggag ggtatgctgg taaccctggg ttagtcgtct tgagatactc 2280

agt 2283

<210> 8

<211> 2157

<212> DNA

<213>M2 genetic fragments

<400> 8

gctttttcag tgccagtctt tctcacaaaa tgggaaacgc gacgtctgtt gtgcagaact 60

tcaatatcca aggtgatggt aatcattttg ctccatccgc tgagactgct tcatccgccg 120

taccgtcatt atctttgaat cccggactgt taaatccagg tggtaaggcg tgggtcctga 180

ttgatccatc tctaaatgct tccgatcctt catcactacg tctgatgact tcggctgatc 240

tatcaacact tcctcgatct gctactagta actctaccgg gtttctcccc acttctggca 300

tgtatgccat tgctactaag gagacgttga gtgtaattac tgagcacgcg atttcccagt 360

ttgataagtt acagatggct tgtgagttgg accgcgatta tctggatgct agaggtgttt 420

ctcctgagtc tgtgaatatt catagttata tagcctacgt tgattgcttc gtgggtgtat 480

ctgcaaggca ggctgcgtca aattttaagc ggcatgtgcc agttatcacc aaatctcgta 540

tgacacaatt tatgacatcc gcgcagaata tgttgcaagt gcttgggccc tgggaacgtg 600

atgttcgtga gttactcact attcttccta cttccactac cgctggtaaa attacgtgcg 660

acatgaagtc tgttgtcgct ttcattgatg atcagctctc tgataccagt ttgtgtcgtc 720

tgtaccccga ctgtgctgct gcggcggtgg ctagacgtaa tggtggcatt cgatggaaga 780

cacctgatac tgacgaggct ccttcacttg caactaacga tattgctgct tcaactatgg 840

gtacgcttgc gaatactaca ccactggctg agaagtcgaa ctcgggcgag gagtcgatgc 900

gcttggttag tgatgttggc gtggacatcg tttgttctcg tggccccatc agttcttcag 960

tttggtcccg cacggttgaa cccaaatcgt acaatattag aacccttcgt gtagaagaag 1020

cgctttggct acgcgagtgc caagcgacta ctggttttga tgtacagtac acgctgcccg 1080

accagactac acataagcat ttctggcttc agaaggggtc agtcgtcata aatcttgagc 1140

aaacgggtag tatgatgttc gatgtgaaca tagcgggtaa agattacaag aagggcacct 1200

ttaatcctga taatcataaa ttggtcctct tggttatgca gtcaaagatc cctttcgagt 1260

cttggaccgt cgcttctcaa attactggta tcgctcaagt ggctgaggtc actgtgcatg 1320

ctgctgatag ttcgactcct aaccaaaaga taatcggtga aacttcgctg tcttatttat 1380

ttgagaggga gacggtgacc acatccaata ctgaagtcaa tacatatctg ttgtgcactt 1440

ggcagcttga cgacgcgcag agcaatgacg caaacgcctg gccagatgct tgggacggga 1500

tcacaacatt gaccccactt acgtccggta ctgtaaccat caaggggact tcggtggatt 1560

ctgtcgtacc gtctgattta gttggtgctt atacacctga ggctttggct gccgcgcttc 1620

ctaacgacgc tgggttaatt ttggctaata aggcaactaa attggctgac gccatcaaga 1680

aggaggatga ttctgtgatt gatgagtctt ctccctttag cacccccatt caaggagttc 1740

tggctgttca acaacttgat accgtgggga cacgcggtac acgtgcactc cagcctccat 1800

ccattctgaa acgcatcgcc tcacgagctc ttcacatgtt tcttggtgat ccaaagtcta 1860

ttctaaaaca ggcgacgccc gtattgaggg accctgacgt ttggaccggc tttgttcaag 1920

gtgttagaga cggcatccgg actaagtcgc tatccgctgg agtacggtcc gtgtataata 1980

acgttaccgc cacacagtct gtacaaacgt ggaaacaggg gttcctgacg aaaatacaga 2040

cgttgttcaa gccatcgtga ggtgctaagg cctctctctg cggcgggtcg gtgggcacgt 2100

cgtagtgacg ctgaatgcac ggggaggtga cgctccctgg attggcacgt tattcat 2157

<210> 9

<211> 1999

<212> DNA

<213>M3 genetic fragments

<400> 9

gctttttgag tcctagcgtg gatcatggcg tcaaccaagt ggggagacaa gccgatgtcg 60

ctctcaatgt ctcacgatgg atcatctatc cgcagtgcgg cctcacaatt tctgtctgat 120

cccctgtctc attcaacgcc gatcccacct caacggaaga ccgtactgct gaaattcatg 180

attggtgatg acctggttac cgttcagggc gccctcgctc cttttgatga gtactggtac 240

gataatcaac cgctattgtc tcaggctgtt gagctgctcg cttctgagga tcgtctacgt 300

caatttgagc attatgagaa atttctgctt aagaagggcc accaaatcgc tgagatcatg 360

aacaggctac gtcttttctt cactgacgtt ctcaaagtga agatggaagc tgatgctctt 420

ccttctctgg ctcaatacct aatggctggt acgttggatg ccgtctccaa cgttcacgaa 480

cctgatgctt gtgttccagt cacttcaaaa atcatagcta agcagcagac tgtgtccaag 540

tcccctggac gtcttgatga agaggagtat aatgttatta gatcacgttt cctcactcat 600

gagatctttg acttaacgtc tgacttgccc ggtgttcaac cattcatgga tatgtactat 660

gccaccgttc ctcgtgccga ttccaccggt tggtgtgtct accgcagaaa aggtctattg 720

attcatgccc ctgatgagca atactcggat ctgactattt tcaccacccg tcttacggca 780

gcgcgtgagt tacagcttgt ggctggggag gtcgttgtgg cttgcttcga tcttatggat 840

atctctgata ttgctccatc tcatcatgca tcggttcaag aggaacgtac tctcggcact 900

agcaagtatt ccaacgttac agctaatgag catccgttgg tgttcttttc acccaatgca 960

ttacgctggg caatagatca tgcctgtact gattccttga tttccactag gaatattcgg 1020

gtctgcgtcg gtattgaccc cttagtgacc agatggactc gcgatggcgt gcaggaggct 1080

gcaattctta tggatgacaa gctaccctca gcaggacgtg ctcggatggc tctacgaacc 1140

ttgcttctag cgcgtcgctc accaatgacg tccttcttac taggtgctct caagcagtcc 1200

ggtggtcagc taatggaaca ttatcgatgt gatgcggcta ataggtatgg atctcccacc 1260

attccagttt ctcaccctcc accgtgtcca aaatgtcctg agctgaaaga acagatcacc 1320

aaactttcgt cagctcctgc gcctaaagtt gactcgtccg ctggtcctgc cgtgctgttg 1380

tcgaagatcg ctgagctcca acgtgctaac cgagaactgt ccttgaagtt agtggacgtt 1440

cagccagccc gagaagacca ccttctagct tacctcaacg agcacgtatg tgttaacgct 1500

aaagatcacg agaaaggtct actagcccgt tgtaatgtct cgagtgattc aatcgccgct 1560

atccttggtc aacgtttgaa aaatcgagaa cggtttgaaa cgagactacg gcacgaggct 1620

ggtgcggagt gggagccacg agtggaagcg ttgaatcaag agttggctaa ggcgcgtatt 1680

gagcaacaag atatgatgac tcagtcctta cagtacctga atgaacgtga tgaactgctg 1740

cgcgaggtgg atgagctcaa acgcgaactg gctaccctac ggtttgctaa tgtgagacta 1800

aatgccgata accaccgcat gagtcgtgcg acccgtgttg gagatgtatt cgtcagtgat 1860

gttgatccct taccacccgg tcttcctggt gaatcgaaac catccattga agaactggta 1920

gatgatctgt gagctttgcc ttgtgactcg acttctctct gattccatgt acccacggcg 1980

gactcggcta ttcatctac 1999

<210> 10

<211> 1644

<212> DNA

<213>S1 genetic fragments

<400> 10

gctttttcag tcccttgtat cgatgttccg tatgtcttcc ggttcatgca acggcgcgac 60

gtcaatattt ggtaacgtgc attgtcaagc ggcccaaaat accgcaggcg gagatcttca 120

agctacctcg tcattaattg cttattggcc ttatctcgct gctggtggtg gtctcattat 180

aattttaatt attgttatag gtataatctg ttgttgtaag gccaaagtta aggctgacgc 240

taccagaagt gtgttctatc gggagttgct tgctctgaac tcgggcaagt gtaatgcagc 300

acctccgtca tacgacgttt gatgtgcggc ggtttgagtt ttctccgacg gtgtttgaag 360

agtgtttgac tccatctttt accgctgtga ctgacactga ccctgtgagg tactttaata 420

ttgagcttcc gtcaactcac cgtctcctcc cttggcttcc cgttcttctg ttccaatcct 480

gtaaagtgca tgtttcttta gtacgtagat tctctttatg ctcgacttta tctgatattt 540

gtgagtacga ttgcaaattg cttccgtcta ttaacgctat tgtgtcgaat ccagtgtcga 600

gcgcggtttc atctatcgtc gttcactggg atggacggat taactcaaca gcagcgaaga 660

gaagtcgtgg ggttgatact gtcgttgact tcgagcgtga gtataagttc tggcgatttg 720

acgcaaattc gtgagcgtct ttccgctttg gaatctgcga ctgcgtcgct gaacgaatcc 780

gttaatacag ctttgtctag gttagtggat ttgtctgcat cgcttgataa cgtggcggcc 840

tcgttagcgg agacgaaagt ggaaatgaac tcactagttt ctgacgttca gggtttgcga 900

gcttcccttg actcttctgc ttcagagctg gcttctctat cttcattggt gcgtgatcac 960

ggctcttcga ttgctggcct acagagagaa gtaagtgcct tatcgagtga ggtaggcaac 1020

cttaaaacct cggtatcatc gcagggcctt actatcacta gccttgagaa acgagtgcaa 1080

gctttagaag gtggttctag tacgactctg tcatttgctg atcctcttaa gttagaggct 1140

gggaccgtgt cactcgaggt agatccgtat ttctgctctg tgaatcgtaa tctgacgtcg 1200

tattctgctg atgctcagtt gatgcaattt cagtggtctg tgaaagggga agatggcgcg 1260

gccaactcta ttgatatgga cgtgaacgct cactctcatg gttcacgcac tgattatctg 1320

atgtcaacca agcaatcatt gactgttaca acgtctcccg ctactcttgt ctttgaactg 1380

gataggatta ttgctcttcc ctccgacctt tctcgcctaa ttccatgtta tggttttcag 1440

caagccactt ttcccgttga tatctccttc cagcgagatg gcgtttcgca tacgtatcaa 1500

gtctatggga agtacacatc ttctcgcgtc ttcaagatta cgttctcgcc tggctcctca 1560

ggtcccgcag tgattaagtt tttgaccgtg cgtacgggca tcgataccta aggtgtggcg 1620

ccgtacgggg attggttatt catc 1644

<210> 11

<211> 1324

<212> DNA

<213>S2 genetic fragments

<400> 11

gctttttctc ccacgatggc gcgtgccgtg tacgacttct tttctacgcc tttcgggaat 60

cgtggtctag cgacgaatcg tactcaacta tcatcactac tatcaagctc gaattcccca 120

tggcaacgtt ttctatcatc aatgactcca ttgacagcgc cgggcatcgt ttcgacacct 180

gaagcaccct atccaggttc gttaatgtat caagagtcta tgctccacag tgctaccgtc 240

cctggagtac ttggcaatcg cgacgcttgg cgtacgttca atgtcttcgg actttcatgg 300

actgacgaag gactgtcagg actagtggct acccaagatc ctcctcccgc cgccccgtat 360

cagccagcct ctgctcagtg gtcggatctt ctcaactacc ccagatgggc aaacagacgt 420

cgtgagctgc aatctaagta cccgcttctg cttcgctcca ctctgctctc tgccatgcga 480

gctggtcctg ttctatatgt tgagacgtgg ccgaatatga tttctggacg attagctgat 540

tggtttatgt cccaatatgg caataatttc gttgacatgt gtgctaggtt gacccagtct 600

tgttcgaaca tgcctgttga acctgatggg aattatgatc aacagatgcg tgctttaatt 660

agtttgtggc ttctgtcata cattggggta atcaaccaaa ccaacaccat cagcggtttc 720

tacttctcct caaagactcg gggtcaagcg ttggacagtt ggactttgtt ctataccacg 780

aatactaatc gtgtccaaat tacgcagaga cattttgctt atgtgtgcgc ccgatctcct 840

gattggaacg tggacaaatc atggatcgct gctgcgaact taaccgccat tgttatggct 900

tgccgtcaac cgccgatgtt tgctaatcaa ggcgtcatta atcaggcgca gaaccgaccc 960

ggattctcca tgaatggggg gacgcccgtc cacgagctca acttacttac tactgcgcaa 1020

gagtgcatca ggcagtgggt ggtagcaggc ttggtgtcgg cagcaaaggg gcaagcacta 1080

acgcaggaag ctaatgactt ctcaaacctc atccaggcgg atctaggcca gatcaaggcg 1140

caggacgacg ctttgtacaa tcagcagccg ggatacgcga ggagaataaa acctttcgtt 1200

aatggtgact ggacaccagg tatgaccgct caagctctgg ccgttctagc cacttttacc 1260

gcctaggcgt agggtcgtac gctgcccgag tccagccctc cggcagcacg tggatgtact 1320

catc 1324

<210> 12

<211> 1202

<212> DNA

<213>S3 genetic fragments

<400> 12

gctttttgag tccttagcgt gcaagccgca atggaggtac gtgtgccaaa ctttcactcg 60

ttcgttgaag gaataacatc tagctacttg aagactcctg cttgctggaa tgcacaaaca 120

gcttgggata ctgtgacctt tcacgtccct gatgtaatta gagttggtaa cgcctactgt 180

tgttctcaat gttgtggtgt actctactac gggactctgc cctcggacgg taattatttc 240

cctcatcaca agtgtcatca gcaacagttt aggactgata ctccactgct tcgatacgtg 300

cgcattggta gaaccactga gcatctgatg gatcaatatg ctgtcgctct ggagtccatt 360

gctgaacact atgacgagat tagtcaacgt atggtcgatg agccagagaa tgacgaggtt 420

acacctcttg atatcgttac gcgtaccgaa tctatcagga gtgacaaggc agtcgaccca 480

gacttttgga catacccact tgagcggcgt tctgatgatt ctcgtagaga catcgcctca 540

gcatgctgga aaatgattga cgcgtcggcg cgtagtctca ctcttccaaa ttgcctcgtc 600

tccccctctt tgcactctcg ctccgtcttt ggtcagatgc aaacgaccac cactatatac 660

gatgttgcgg catcgggaaa ggccgttaag ttttcaccaa tggttgctac actagcgcaa 720

cgtgatgctg gccctgtaat gcttgcgaat gctgacccgg cggaaggcgt gtactctttc 780

tggacgtcgc acttcgcttt ctcaccgctc atcggcggag ttggaattac gggacagtac 840

gctcgtgagt cgtaccatca agtgggtcat ccagtgattg ggagtggtaa gaaggcatcg 900

cattacagga atctgttcat ggaagcgtgg cgcgggtggt cgaagtcagc tttcgcatgt 960

gctactggaa tggagccagc tgaatgtgaa tctcgtctga gaggacacgc tcgtactatg 1020

ctcggacgct ctctgccgcc cgtttgtgac gatgatgttg ctcagcagtc tggcgcggta 1080

ctgacttcgc tgcagaaaac gaacaagttc accgttgtgg agtgtggttg gtaagtacct 1140

ccgggtcaaa atgcacatag gctcccacct atgtgacggt tagcgggaca gatcggaaga 1200

gc 1202

<210> 13

<211> 1192

<212> DNA

<213>S4 genetic fragments

<400> 13

gctttttgag tccttgcgca gccatggaca acaccgtgcg tgttggagtt tcccgcaaca 60

catccggcgc agctggtcag actgttttta gaaactttta cttactacga tgcaatatct 120

cagctgacgg tcgtaatgca acaaaagctg tgcaatccca ttttccattc ctttctcgtg 180

ctgtccgttg cctatctcct ctagccgctc attgtgctga taggactctt cgtcgtgaca 240

atgtgaaaca aattcttact cgtgagctgc catttccatc ggatttaatc aattacgcac 300

atcatgtgaa ctcatcctcc cttactactt ctcagggtgt cgaggcggca cgtctagtgg 360

cccaagtcta tggagaacag ctatcgtttg atcacattta tcccactggt tccgcaactt 420

actgccctgg agcgattgct aatgcgattt cccgtatcat ggctggtttt gtgccccacg 480

aaggtgacaa ctttaccccg gacggttcta ttgactatct cgccgccgac ctggtcgcgt 540

ataagttcgt gctcccttac atgctagata ttgtggacgg acgtccgcag attgttcttc 600

catcacacac tgttgaggag atgctgtcca acacgagttt gcttaattcg attgacgctt 660

catttggtat tgaatcgaag agcgatcaac gcatgacccg tgacgcggct gaaatgagtt 720

ctcgctcact taatgagctt gaggatcatg agcagagggg tcgaatgcct tggaaaatca 780

tgacggcaat gttcgcggcg caattgaagg tggagttgga cgccctagct gatgagcgcg 840

ttgaatctca ggctaacgct catgtgacat cttttgggtc tcgtctgttc aaccaaatgt 900

ctgcttttgt cccaattgat cgtgagttga tggagctggc tctactcatc aaagagcagg 960

gtttcgcaat gaatccaggg caagtcgcat ctaaatggtc gctgatacga cgatctggcc 1020

ccactcgccc gctatcaggc gcacgccttg agatcaggaa tggcaactgg acaattcgtg 1080

aaggtgacca gacgcttctg tctgtctccc cagctaggat ggcgtaaacg ggacccatgg 1140

tgcgggtgag gggccgccac accctctgcc gcgacctgga ctcttattca tc 1192

<210> 14

<211> 93

<212> DNA

<213>Artificial sequence

<400> 14

atggcctatc tagccacacc tgtgctagga gtcggttctc gcattaccgc tttagatcgt 60

actattgatg ctatcacgtt gaaacctcga atc 93

Claims

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 avian reovirus or kit With；The deposit number of the novel avian reovirus is CCTCC NO：V201817.

3. a kind of fluorescence quantitative RT-PCR kit of the novel avian reovirus of detection, which is characterized in that the fluorescent quantitation PCR kit contains primer and probe described in claim 1.

4. fluorescence quantitative RT-PCR kit according to claim 3, which is characterized in that the fluorescence quantitative RT-RCR examination Further include in agent box：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 chicken of V201817 exhales The genome of the lonely virus of intestines, obtains amplified production, amplified production is connected on carrier, screening positive clone simultaneously extracts plasmid Standard items are prepared in DNA.

6. fluorescence quantitative RT-PCR kit according to claim 4, which is characterized in that the quantitative fluorescent PCR reaction Reagent includes：One Step RT-PCR Buffer、TaKaRa Ex Taq HS、PrimeScript RT Enzyme Mix Ⅱ。

7. a kind of detection reagent of the novel avian reovirus of detection, which is characterized in that the detection reagent contains claim 1 The primer and probe；The deposit number of the novel avian reovirus is CCTCC NO：V201817.

8. the detection reagent described in claim 3-6 any one of them fluorescence quantitative RT-PCR kit or claim 7 exists Application in any in (1)-(3) as follows：

9. a kind of detecting novel avian reovirus 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 Fluorescence PCR later, according to the concentration of standard items and Ct values, drawn glimmering Light quantitation curves；

(2) sample to be tested total serum IgE is extracted, Fluorescence PCR is carried out, collects sample to be tested fluorescence signal, fluorescence signal is carried 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 Fluorescence PCR is：

42 DEG C of 5min, 95 DEG C of 10sec, Reps：1；95 DEG C of 5sec, 60 DEG C of 34sec, Reps：40.

10. deposit number is CCTCC NO：The avian reovirus of V201817 causes the arthritic chicken of broiler chicken to exhale in preparation detection Application in the PCR kit for fluorescence quantitative of the lonely virus of intestines.