CN107475445B - Kit for identifying virulent and attenuated strains of chicken infectious bursal disease virus based on RT-PCR and RFLP technologies and application thereof - Google Patents

Abstract

The invention discloses a kit for identifying virulent and attenuated strains of chicken infectious bursal disease virus based on RT-PCR and RFLP technologies and application thereof. The kit contains a primer pair for amplifying the gene VP2 of the chicken infectious bursal disease virus and SpeI, SacI and StuI restriction endonucleases. The differential diagnosis method based on RT-PCR and RFLP technology can distinguish the ultra-virulent strains and the attenuated strains of IBDV, has simple steps and convenient operation, and provides a new effective technical means for the rapid differential diagnosis of the virulent strains and the attenuated strains of IBDV.

Description

Kit for identifying virulent and attenuated strains of chicken infectious bursal disease virus based on RT-PCR and RFLP technologies and application thereof

Technical Field

The invention relates to a kit for identifying virulent and attenuated strains of chicken infectious bursal disease virus and application thereof, in particular to a kit for identifying virulent and attenuated strains of chicken infectious bursal disease virus based on RT-PCR and RFLP technologies and application thereof. The invention belongs to the technical field of virus detection.

Background

Infectious Bursal Disease (IBD) in chickens is an acute, highly contagious, lymphoblastic Infectious disease in chickens caused by IBDV. The IBD mainly infects chickens of 3-6 weeks old, and mainly attacks the bursa of Fabricius of central immune organs, resulting in immunosuppression. There are two distinct types of IBDV, called serogroup I and serogroup II. The serotype I virus is pathogenic to chickens; the serum II type is separated from turkey, and has no pathogenicity to chicken. IBDV serotype I isolates have varying degrees of virulence and replication efficiency in bursa of Fabricius cells. The viruses have differences in proliferation rate and pathogenicity, and can be classified into different types such as classical strains or attenuated vaccine strains (cIBDV), variant strains (vvIBDV), and very virulent strains (vvIBDV) according to virulence.

Vaccination of chicks is the primary prevention strategy for controlling IBD. However, in recent years, the occurrence of variant strains and ultra-virulent strains causes new characteristics of the occurrence and prevalence of the disease, thereby aggravating the harm to the poultry industry. Scholars at home and abroad carry out a great deal of research work on prevention and treatment of IBD, develop and develop various vaccines, but the most common vaccine is the traditional attenuated vaccine, but after the ultra-virulent strain appears, a plurality of problems are exposed. Because the super-strong virus spread cannot be effectively controlled, the morbidity and mortality of the disease are greatly increased, and the economic loss is huge. Therefore, the establishment of a rapid, sensitive and accurate diagnosis technology for distinguishing virulent strains from attenuated strains is very critical to the prevention and control of the infectious bursal disease of chicken.

In recent years, various diagnostic techniques for IBDV have been applied to the differentiation and diagnosis of IBDV strains. ELISA assays, virus neutralization assays (VNT), real-time RT-PCR and agar gel precipitation Assays (AGPT) have been used to detect and differentiate various IBDV strains, but are lacking in clinical detection methods that are sensitive, specific and rapidly differentiate virulent strains from attenuated strains, in particular to differentiate between infection and immunity. The RT-PCR combined restriction endonuclease detection method has the advantages of simplicity, rapidness, accuracy and the like, and is an IBDV detection method which is worthy of popularization. The detection method can be completed in a short time from sampling, RNA extraction, RT-PCR, enzyme digestion and product gel electrophoresis analysis. The method can detect not only the artificially infected chick embryo allantoic fluid and cell culture, but also clinical tissue morbid substances directly, so the method is a specific, rapid, sensitive and practical IBD detection technology. Therefore, the establishment of diagnostic techniques for differentiating IBDV virulent strains and virulent strains provides a basis for obtaining IBDV epidemiological information and also provides a powerful help for establishing a vaccination strategy to control diseases.

Disclosure of Invention

The invention aims to provide a kit capable of quickly identifying and diagnosing IBDV (infectious bursal disease Virus) super virulent strains and attenuated strains and a detection method thereof.

In order to achieve the purpose, the invention adopts the following technical means:

the invention selects 8 IBDV super-virulent strains D6948, Gx, IM, KKI, KSH, OKYM, SH95 and UK661, 6 classical low-virulent strains ZJ2000, B87, CEF94, D78, HZ-2 and JD-1 with representative morbidity from GenBank to carry out sequence analysis. Comparing the nucleotide sequences of the 14 strains by DNAMAN software, determining that the difference of the sequences of the virulent and attenuated IBDV strains is mainly concentrated in the 860 bp-1185 bp region of the VP2 gene which is an important structural protein of IBDV, analyzing the specific enzyme digestion sites of the base mutation parts in the sequence difference concentration region of the virulent and attenuated strains by NEBcut software, and finally determining SpeI, SacI and StuI as restriction endonucleases for distinguishing the virulent strains and the attenuated strains of IBDV. The target fragment of the super-virulent strain can be cut into two fragments of 531bp and 302bp by SpeI, and can also be cut into two fragments of 242bp and 591bp by StuI, but can not be cut by SacI enzyme; the target fragment of the attenuated strain can only be cut by SacI, and two fragments of 218bp and 615bp are cut out. Therefore, the invention establishes a method for detecting IBDV virulent and attenuated strains based on RT-PCR and RFLP technology by using the VP2 gene in combination with restriction enzymes SpeI, SacI and StuI according to the characteristics of Restriction Fragment Length Polymorphism (RFLP) sequence of the VP2 target gene.

Firstly, universal IBDV identification primers L2 and U2 recommended by OIE are applied, an RT-PCR method is adopted to amplify and identify 15 IBDV strains stored in a laboratory, namely CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8, 05-6, 99-3, JC-7, SD-F3 and GC-7, the result shows that all target fragments of about 604bp can be amplified, and the sequencing result shows that all the IBDV strains are IBDV strains. Then designing a pair of primers PAU and PAD according to the common conserved region of VP2 gene of IBDV virulent and attenuated strain, amplifying target segment of 521 bp-1354 bp common 833bp of IBDV VP2 gene by RT-PCR, wherein the segment of gene comprises gene sequence of the main concentrated region of base mutation of virulent and attenuated strain; in order to further identify the strong and weak virulence characteristics of each strain, three restriction endonucleases of SpeI, SacI and StuI are used for carrying out enzyme digestion identification on the purified PCR product, wherein HR, CF, SD, DB11, BC and DN-04 can be cut into two segments of 531bp and 302bp by SpeI, and can also be cut into two segments of 242bp and 591bp by StuI, but can not be cut by SacI, and the 6 strains are proved to be IBDV super-virulent strains; CA. C-8, 05-6, 99-3, JC-7, BD, CEF94, SD-F3 and GC-7 were only excised by SacI for two fragments of 218bp and 615bp, confirming that this 9 strain is an IBDV attenuated strain.

In order to verify the pathogenicity of IBDV virulent and attenuated strains identified by the method established in the research, the purity of the 15 strains of viruses is detected by a hemagglutination experiment after the 15 strains of viruses are rejuvenated by SPF (specific pathogen free) chick embryos of 10 days old; the copy number of each virus strain was determined by the established real-time fluorescent quantitative RT-PCR method, and the quantified viruses were diluted with sterile PBS and adjusted to 10 for 15 virus strains^8.747PermL, 100uL each of 40-day-old SPF chickens were spotted with PBS in the control group, and 6 SPF chickens per group were spotted with the control group. 48 hours after the challenge, all the chickens infected with HR, CF, SD, DB11, BC and DN-04 strains of viruses show the clinical symptoms of depressed spirit, reduced feed intake, plump feathers, drooping head and wings, cachexia, staying one corner and closed eyelids, reluctant ambulation and the like; the PBS control group of chickens appeared normal, and the chickens in other attenuated groups had no obvious clinical symptoms. On the third day after infection, 3 chickens are randomly selected from each group for killing, and the bursa of each group of chickens infected with 15 strains of viruses can be seen to have different degrees of characteristic lesions through the autopsy, wherein the eye lesions of the chickens in the group with strong toxicity are more obvious than those of the chickens in the group with weak toxicity, for example, the bursa of Fabricius presents yellow jelly-like edema, and the mucous membrane is covered with cream-colored cellulosic exudates; splenomegaly with bleeding symptoms individually; streak bleeding appears in the inner thigh muscles of chickens infected with strong virus. No lesions were found after necropsy in the PBS control group. Taking the bursa of Fabricius and the spleen, and making paraffin sections, wherein the bursa of Fabricius and the spleen tissue structures of the PBS control group tend to be normal, and no obvious histopathological change is observed; phaffia bursa disease histology after virus challenge of 6 super virulent strains HR, CF, SD, DB11, BC and DN-04Observation shows that a great deal of lymphocyte necrosis is accompanied with apoptosis, a great deal of heterophilic cell infiltration exists in the lymph follicles, vacuole formation and edema of the lamina propria exist in part of the follicles, and inflammatory cell infiltration exists in the lamina propria. The pathological histology observation of 9 strains of low virulent strains CA, C-8, 05-6, 99-3, JC-7, BD, CEF94, SD-F3 and GC-7 shows that lymphoid follicle atrophy is observed, wherein lymphocytes are obviously reduced, and inflammatory cell infiltration exists in an inherent layer. The spleen pathological histology observation of the 15 strains after virus attack shows that the hyper-virulent strains and the low virulent strains have no obvious difference, the lymphocytes can be obviously reduced, and a large number of macrophages are proliferated to form white gaps. Therefore, animal regression experiments prove that the RT-PCR and RFLP methods established in the invention can rapidly distinguish HR, CF, SD, DB11, BC and DN-04 strains stored in a laboratory as IBDV super virulent strains, and the CA, C-8, 05-6, 99-3, JC-7, BD, CEF94, SD-F3 and GC-7 strains as IBDV attenuated strains, and can be used for identification and detection of the IBDV super virulent strains and the attenuated strains.

On the basis of the research, the invention provides a kit for identifying virulent strains and attenuated strains of the chicken infectious bursal disease virus based on RT-PCR and RFLP technologies, the kit contains a primer pair for amplifying the VP2 gene of the chicken infectious bursal disease virus and SpeI, SacI and StuI restriction enzymes, and the sequence of the primer pair for amplifying the VP2 gene of the chicken infectious bursal disease virus is as follows:

PAU：5’ATCTTGGGTATGTGAGGCTG 3’

PAD：5’TATGGCCCGGATTATGTCTT 3’。

in the kit, preferably, the kit further comprises rTaq enzyme, 10 XPCR buffer solution, dNTP, RNase inhibitor, reverse transcriptase, 5 XPRT buffer solution and enzyme digestion buffer solution.

The kit is used for identifying the virulent and attenuated strains of the chicken infectious bursal disease virus according to the following method:

(1) extracting RNA and performing reverse transcription on the infectious bursal disease virus to be detected to obtain virus cDNA;

(2) PCR amplification;

taking the cDNA obtained in the step (1) as a template, taking PAU and PAD as upstream and downstream primers, and carrying out PCR amplification on a target gene fragment in a VP2 sequence, wherein the reaction system is as follows: cDNA 4 mu L, rTaq enzyme 0.5 mu L, 10 XPCR buffer 2.5 mu L, dNTP 3 mu L, primer PAU 1 mu L, primer PAD 1 mu L, deionized water 13 mu L, total volume 25 mu L;

after the sample is mixed uniformly, the sample is separated instantly and put into a PCR instrument, and the amplification procedure is as follows: 5min at 95 ℃; 30 cycles at 94 ℃ for 30sec, 57.1 ℃ for 45sec, 72 ℃ for 50 sec; 10min at 72 ℃; storing at 4 deg.C;

(3) recovery and purification of virus target gene sequence PCR product

Purifying and recovering the PCR product obtained in the step (2), and storing the collected DNA at-20 ℃;

(4) RFLP identification of IBDV strains

Performing enzyme digestion identification on the PCR purified product obtained in the step (3) by using three restriction enzymes of SpeI, SacI and StuI; the enzyme digestion system is as follows: 1 muL of SpeI or StuI or SacI of 10U/uL, 2 muL of enzyme digestion buffer solution, 7 muL of PCR purified product of 20-40 ng/muL and 10 muL of deionized water, wherein the total volume is 20 muL;

mixing, placing in water bath at 37 deg.C for 2h, mixing with 2uL 10 × Loading Buffer, performing electrophoresis with 0.8% agarose gel at 110V for 20min, and observing enzyme digestion result;

(5) determination of results

If the two fragments of 531bp and 302bp are cut out by SpeI, the two fragments of 242bp and 591bp are cut out by StuI, but the two fragments can not be cut by SacI enzyme, the virus strain is judged to be a super-virulent strain;

if only two fragments of 218bp and 615bp can be cut out by SacI and cannot be cut by SpeI and StuI, the strain is judged to be an attenuated strain.

Furthermore, the invention also provides application of the kit in preparing a reagent for identifying virulent and attenuated strains of the infectious bursal disease virus.

The differential diagnosis method based on RT-PCR and RFLP technology can distinguish the super virulent strain and the attenuated strain of IBDV, has simple steps and convenient operation, and provides a new method for the rapid differential diagnosis of IBDV.

Drawings

FIG. 1 shows sequence analysis and restriction enzyme site selection;

wherein, the circled squares respectively represent SacI enzyme cutting sites at 862 th to 867 th sites, StuI enzyme cutting sites at 888 th to 893 th sites and SpeI enzyme cutting sites at 1179 th to 1184 th sites of the nucleotide sequence;

FIG. 2 shows the results of PCR amplification for virus identification;

m: DNA Marker DL 2000; 1-15: HR, CF, SD, DB11, BC, DN-04, CA, C-8, 05-6, 99-3, JC-7, BD, CEF94, SD-F3 and GC-7 strains of PCR amplification products; 16: water control;

FIG. 3 is a PCR amplification of a gene sequence of interest for use in the RFLP identification method;

m: DNA Marker DL 2000; 1-15: HR, CF, SD, DB11, BC, DN-04, CA, C-8, 05-6, 99-3, JC-7, BD, CEF94, SD-F3 and GC-7 strains of PCR amplification products; 16: water control;

FIG. 4 shows the results of enzyme digestion identification of HR, CF, SD, DB11, BC and DN-04 strains;

m: DNA Marker DL 2000; 1: cutting with SpeI; 2: performing single cutting by SacI; 3: performing single cutting through StuI;

FIG. 5 shows the results of enzyme digestion identification of strains CA, C-8, 05-6, 99-3, JC-7, BD, CEF94, SD-F3 and GC-7;

m: DNA Marker DL 2000; 1: cutting with SpeI; 2: performing single cutting by SacI; 3: performing single cutting through StuI;

FIG. 6 shows the results of hemagglutination assay;

FIG. 7 shows the ocular lesions of bursa of Fabricius and spleen after challenge in the infected group;

FIG. 8 is a graph of leg muscle bleeding;

FIG. 9 shows ocular pathological changes in bursa of Fabricius and spleen in the infected group versus the control group;

from left to right, C represents PBS control organ, followed by 6 organs with very strong toxicity and 9 organs with weak toxicity;

FIG. 10 is a histopathological observation (40X) of bursa of Fabricius and spleen in PBS control group;

PBS control group chicken bursa of Fabricius; PBS control group chicken spleen;

FIG. 11 is the histopathological change of bursa of Fabricius after infection (40X);

A-O respectively infects HR, CF, SD, DB11, BC, DN-04, CA, C-8, 05-6, 99-3, JC-7, BD, CEF94, SD-F3 and GC-7 strains;

FIG. 12 is spleen histopathological changes (40X) after infection.

A-O infects HR, CF, SD, DB11, BC, DN-04, CA, C-8, 05-6, 99-3, JC-7, BD, CEF94, SD-F3 and GC-7 strains, respectively.

Detailed Description

The invention is further illustrated and verified by the following examples, all of which are intended to be illustrative only and not limiting to the scope of the invention. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Materials and sources thereof involved in this example:

1 laboratory animal

SPF embryos 10 days old, SPF chickens 40 days old, were purchased at Harbin veterinary institute of Chinese academy of agricultural sciences.

2 virus

IBDV CEF94 strain and CA strain were provided by the laboratory preservation; IBDV strains HR, CF, SD, BC, DB11, DN-04, C-8, 05-6, 99-3, JC-7, BD, SD-F3 and GC-7 were isolated and stored in the laboratory.

3 kit

QIAquick Gel Extraction Kit, available from QINGEN company; GoTaq qPCR Master Mix, purchased from Promega.

4 tool enzymes and related reagents

rTaq enzyme, restriction enzymes SpeI, StuI and SacI, RNase inhibitor (RRI), reverse transcriptase RT Ace, DNA Marker, universal primer oligo (dT) purchased from Dalibao Bio; 96-well V-shaped blood coagulation plates, purchased from wuxinhokang.

5 preparation of common solvent

PBS buffer: NaCl 4.0g, KCl 0.1g, KH₂PO₄ 0.1g，Na₂HPO₄·12H₂O1.45 g and ultrapure water to a constant volume of 500mL, sterilized and stored at 4 ℃.

TAE buffer (50 ×): 121.14g of Tris-Base, 28.55mL of glacial acetic acid, 9.3g of EDTA and the constant volume of ultrapure water to 500mL, adjusting the pH value to 8.0 and storing at 4 ℃ for later use.

Liquid A: dextrose 1.025g, sodium citrate 0.4g, NaCl 0.21g and citric acid 0.028g are dissolved in 50mL of ultrapure water, and the mixture is placed in a refrigerator at 4 ℃ for storage after autoclaving.

1% chicken red blood cell suspension: sucking the Ashi solution by using a syringe, collecting chicken blood and mixing uniformly (the ratio is about 1: 4). Adding PBS about ten times of blood, slowly reversing to mix blood and PBS, centrifuging at 2000rpm for 10min, discarding supernatant, sucking surface layer leukocyte and discarding, and repeating several times until supernatant is clear and transparent. Diluting the erythrocyte sediment with 1 volume of PBS with 9 volumes, shaking up to prepare 10% chicken erythrocyte, then adding 10% erythrocyte with 1 volume into PBS with 9 volumes for dilution, mixing evenly and putting into a refrigerator with 4 ℃ for standby.

6 main instrument and experimental equipment

Gradient PCR instrument: eppendoff Mustercycler Grudient PE 2400;

low temperature bench high speed centrifuge: beckman Avanti TM 30;

superclean bench: froma Scientific 1829. SN.17069-15;

electrophoresis apparatus: bio Rad MA type 120 MINI VERITICAL GEL SYSTEM;

horizontal electrophoresis tank DYC-31A type: six instrument factories in Beijing;

gel imager: UVR-800 UK;

a micro electronic balance: METTER AE260 type, Deltalange corporation;

M9B88 microwave oven: three stars in Korea;

a water purifier: millipore Milli-type QII;

a micropipette: gilson Corp;

constant temperature gas bath shaking table: model HWY-100B, hao cheng kui testing instruments ltd;

a vortex oscillator: h-1 type, Shanghai Lucy Instrument Mill;

-20 ℃ freezer refrigerator, -4 ℃ refrigerator: haier corporation;

-80 ℃ ultra low temperature refrigerator: revco Corp;

moist heat autoclave: sanyo, japan;

a desk centrifuge: shanghai third analytical instrumentation plant;

ultraviolet spectrophotometer: shimadzu UV-120-02;

the biological safety cabinet: beijing Dong Lihaar instruments Inc.;

ABI PRISM 7500 fluorescent quantitative PCR: ABI, USA.

Example 1 establishment of method for detecting IBDV by RT-PCR in combination with RFLP

1. Method of producing a composite material

1.1 sequence analysis and restriction site selection

14 IBDV strains with known virulence are selected for sequence analysis, namely, the strains OKYM, D6948, IM, KKI, KSH, SH95, Gx and UK661 as well as the strains ZJ2000, B87, CEF94, D78, HZ-2 and JD-1 as super virulent strains respectively, the nucleotide sequences of the selected strains are subjected to a great deal of research and are collected in an NCBI database, and the basic information of the strains is detailed in Table 1. Comparing the nucleotide sequences of the 14 strains by DNAMAN software, determining that the difference between the sequences of the IBDV super-virulent strain and the attenuated strain is mainly concentrated in the gene region of the important structural protein VP2 of IBDV, analyzing the specific enzyme digestion sites of the base mutation parts in the sequence difference concentration region of the virulent and attenuated strains by NEBcut software, and finally selecting SpeI, SacI and StuI as restriction endonucleases for distinguishing the virulent and attenuated strains of IBDV.

Sources and information on IBDV strains of strains Table 114

Type (2): VV (ultra-strong toxicity) ATT (weak toxicity)

1.2 design and Synthesis of PCR primers

A pair of primers PAU and PAD are designed by referring to the VP2 common conserved region of the 14 IBDV virulent and attenuated strains in GenBank and utilizing primer analysis software Oligo 6.0, and the sequences of the primers are detailed in Table 2. The primers can amplify target gene segments of 833bp in VP2 genes of IBDV virulent and attenuated different strains, and the segment of genes comprise gene sequences of main concentrated regions of base mutations of the virulent and attenuated strains; meanwhile, the gene can be used for enzyme digestion identification of IBDV strains by three different restriction enzymes SpeI, SacI and StuI in an RFLP method.

Meanwhile, primers L2 and U2 of the IBDV strain RT-PCR detection method recommended by the world animal health Organization (OIE) are synthesized for identifying IBDV strains stored in the laboratory, and the sequences of the primers are detailed in Table 2. The pair of primers is designed according to the VP2 gene sequence, and the expected size of the PCR product is 604 bp.

The real-time fluorescent quantitative PCR primers S1 and S2 for virus quantification are designed according to a VP4 conserved gene sequence and comprise a segment of sequence within 302bp to 540bp in a VP4 gene sequence.

The above primers were synthesized by Saibashi Gene technology, Inc.

TABLE 2 primer sequences

1.3 extraction and reverse transcription of viral RNA

15 IBDV strains preserved in a refrigerator at-80 ℃ in the laboratory, namely CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8, 05-6, 99-3, JC-7, SD-F3 and GC-7 are taken out and subjected to extraction and reverse transcription experiments of virus RNA, and the detailed method is as follows:

(1) 250uL of each virus-extracted solution was put into a 1.5mL EP tube and extracted.

(2) Add 500uL Trizol to the EP tube and turn over quickly up and down, shake for 2min, place on ice for 10min, shake for 1min in the middle.

(3) A separation stage: adding 350uL chloroform, tightly covering the EP tube, violently shaking to uniformly mix liquid in the EP tube, standing on ice for 10min, centrifuging at 12000rpm for 7min, after centrifugation is finished, layering the liquid in the tube, wherein the lowest layer is a red phenol-chloroform layer, the middle layer is a white film layer, the uppermost layer is a transparent water sample layer, and RNA is completely left in the water sample layer.

(4) Precipitation of RNA: the supernatant was slowly transferred to a clean, rnase-free 1.5mL EP tube (about 400-500 uL), isopropanol was added in equal volume, mixed well, placed at-20 ℃ for more than 1h and then centrifuged at 12000rpm for 7 min.

(5) Washing of RNA: the supernatant was carefully decanted and the pellet was retained. The RNA pellet was washed once by adding 1mL of freshly prepared 75% absolute ethanol (in DEPC water, pre-chilled) and then centrifuged at 12000rpm for 7 min.

(6) Re-solubilization of RNA: carefully pour off the supernatant, flip the tube onto absorbent paper, air dry in the bench, not allow the RNA pellet to dry completely. Then 22uL of DEPC water was added to the tube to dissolve, and total RNA was obtained, immediately followed by reverse transcription.

(7) 2uL of universal primer oligo (dT) is added, and water bath is carried out for 10min at 75 ℃ and ice bath is carried out for 5 min.

(8) RRI 2uL, RT Ace 2uL, 5 XTT Buffer 8uL and dNTP 4uL are added into an EP tube, and the total amount is 40uL and mixed evenly.

(9) The cDNA is obtained after the cDNA is separated from an EP tube, water bath is carried out at 42 ℃ for 1h and 30min, ice bath is carried out for 5min at 72 ℃ to obtain the cDNA, and the cDNA is stored at minus 20 ℃ for standby.

1.4PCR amplification

(1) PCR amplification for virus identification

Using cDNA obtained in 1.3 experiment as template, L2 and U2 as upstream and downstream primers, carrying out PCR amplification to identify 15 strains CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8, 05-6, 99-3, JC-7, SD-F3 and GC-7 preserved in laboratory, the reaction system is as follows:

instantly separating after the sample is uniformly mixed, putting the sample into a PCR instrument, and performing an amplification procedure: 5min at 94 ℃; 30sec at 94 ℃, 45sec at 56 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃; storing at 4 ℃.

After the PCR reaction, the PCR product was mixed with 2.5uL of 10 × Loading Buffer, and subjected to electrophoresis with 0.8% agarose gel at 110V for 20min using DNA Marker DL2000 as a control to observe the PCR amplification result.

(2) PCR amplification of target gene sequences for RFLP identification methods

Using cDNA obtained in 1.3 experiment as template, PAU and PAD as upstream and downstream primers, PCR amplifying target gene fragment in VP2 sequence, the reaction system is as follows:

after the sample is mixed uniformly, the sample is separated instantly and put into a PCR instrument, and the amplification procedure is as follows: 5min at 95 ℃; 30 cycles at 94 ℃ for 30sec, 57.1 ℃ for 45sec, 72 ℃ for 50 sec; 10min at 72 ℃; storing at 4 ℃.

After the PCR reaction, the PCR product was mixed with 2.5uL of 10 × Loading Buffer, and subjected to electrophoresis with 0.8% agarose gel at 110V for 20min using DNA Marker DL2000 as a control to observe the PCR amplification result.

1.5 recovery and purification of the product of PCR of the target Gene sequence of the Virus

The PCR product 50uL and 5uL10 × Loading Buffer were mixed together uniformly, subjected to 1% agarose Gel electrophoresis, and after the electrophoresis was completed, the purified PCR product was recovered using the QIAquick Gel Extraction Kit, using DNA Marker DL2000 as a control. According to the kit instruction. The collected DNA was stored at-20 ℃ for subsequent experiments.

1.6 sequencing of purified PCR products

PCR products of 15 strains of viruses amplified with primers L2, U2, PAU, and PAD were purified and sent to Sebain Gene technology Co., Ltd for sequencing.

1.7RFLP identification of IBDV strains

And (3) carrying out enzyme digestion identification on PCR purified products of the target gene sequences amplified by the primers PAU and PAD by using three restriction enzymes SpeI, SacI and StuI respectively. The enzyme digestion system is as follows:

mixing, placing in water bath at 37 deg.C for 2h, mixing with 2uL 10 × Loading Buffer, performing electrophoresis with 0.8% agarose gel at 110V for 20min, and observing the enzyme digestion result.

2 results

2.1 analysis of VP2 Gene sequence and selection of restriction enzyme sites

After multiple sequence comparison, a region with the length of 833bp exists between 521 bp-1354 bp of a VP2 gene sequence of the selected 14 IBDV strains with different virulence, and the base mutations of the virulent strains and the attenuated strains are mainly concentrated in the region. The attenuated strains ZJ2000, B87, CEF94, D78, HZ-2 and JD-1 all have a common restriction enzyme cleavage site SacI at the 862-867 bp nucleotide position in the region, the sequence is GAGCTC, but the other 8 strains D6948, Gx, IM, KKI, KSH, OKYM, SH95 and UK661 all have no SacI restriction enzyme cleavage site at the same position because the base 'G' is replaced by 'A', i.e. GAACTC. 8 super virulent strains have restriction enzyme sites StuI at the 888-893 bp nucleotide positions in the region, and the sequence is AGGCCT; the nucleotide positions of 1179-1184 bp have restriction enzyme sites SpeI, and the sequence is ACTAGT. However, the 6 attenuated strains had no StuI or SpeI restriction sites at the same positions, since base ` A ` was substituted with ` T ` at 888bp and ` A ` was substituted with ` G ` at 1179bp (see FIG. 1). Therefore, in the 833bp sequence region, the ultra-virulent strain can be cut out into two fragments of 531bp and 302bp by a restriction enzyme SpeI, and can also be cut out into two fragments of 242bp and 591bp by StuI, but can not be cut by SacI; the attenuated strain can only be cut by SacI enzyme, and two segments of 218bp and 615bp are cut out. Therefore, restriction endonucleases SacI, StuI and SpeI are selected as gene fragments with the length of 833bp between 521bp to 1354bp in the enzyme digestion VP2 gene sequence to establish an RT-PCR combined RFLP differential diagnosis method for distinguishing IBDV virulent strains and attenuated strains.

2.2 PCR amplification results for Virus identification

15 IBDV strains CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8, 05-6, 99-3, JC-7, SD-F3 and GC-7 stored in a laboratory are all subjected to RNA extraction and reverse transcription, cDNA obtained by reverse transcription is used as a template, and primers L2 and U2 recommended by OIE are used for carrying out PCR amplification for virus identification, so that a target band of 604bp can be seen, the sequence determination result is consistent with expectation, and the 15 strains stored in the laboratory are all IBDV strains. As shown in fig. 2.

2.3 PCR amplification results of target Gene sequences for RFLP identification method

15 IBDV strains CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8, 05-6, 99-3, JC-7, SD-F3 and GC-7 stored in a laboratory are all subjected to RNA extraction and reverse transcription, the obtained cDNA is used as a template, primers PAU and PAD are used for carrying out PCR amplification on a target gene sequence for the RFLP identification method, and the result shows that a 833bp target band is consistent with the expectation and is shown in FIG. 3.

2.4 sequencing of purified PCR products

The sequencing results of PCR products of 15 strains of viruses amplified by PAU and PAD are shown in SEQ ID No.1-15, the sequencing results show that the ultra-virulent strains have StuI and SpeI enzyme cutting sites, the attenuated strains have SacI enzyme cutting sites, and the sequencing results are correct.

2.5RFLP identification of IBDV strains

After being purified, PCR products of target gene sequences used for the RFLP identification method are respectively cut by three restriction enzymes of SpeI, SacI and StuI, and the results show that: HR, CF, SD, DB11, BC and DN-04 strains were all cut out with two fragments of 531bp and 302bp by SpeI, and also cut out with two fragments of 242bp and 591bp by StuI, but not cut by SacI (see FIG. 4); CA. The C-8, 05-6, 99-3, JC-7, BD, CEF94, SD-F3 and GC-7 strains were only able to excise two fragments of 218bp and 615bp in size by SacI, but not by SpeI and StuI (see FIG. 5). HR, CF, SD, DB11, BC and DN-04 strains are all IBDV super virulent strains, while CA, C-8, 05-6, 99-3, JC-7, BD, CEF94, SD-F3 and GC-7 strains are all IBDV attenuated strains.

Example 2 identification of IBDV Strain virulence Using animal regression experiments

1. Method of producing a composite material

1.1 rejuvenation of viruses

15 IBDV strains CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8, 05-6, 99-3, JC-7, SD-F3 and GC-7 stored in the laboratory were taken out from a refrigerator at-80 ℃, centrifuged at 4 ℃ for 10min at 7000rpm, the supernatant was aspirated by a disposable syringe, the virus was diluted with sterile PBS 100, the diluted chorioallantoic membrane was inoculated into 10-day-old SPF chick embryos, and two chick embryos were inoculated with each virus dilution. Each chick embryo was inoculated with about 200uL of virus solution. The inoculated chick embryos are sealed by paraffin and then placed in an incubator at 37 ℃ for incubation. And discarding the dead chick embryos within 24h, detecting the chick embryos twice a day, taking out the dead chick embryos at any time until all the chick embryos are taken out together within 96h, and placing the chick embryos in a refrigerator at 4 ℃ for 4h so as to carry out subsequent experiments.

Taking out the inoculated chick embryo from a refrigerator at 4 ℃ and placing the chick embryo into a biological safety cabinet, opening an eggshell at an artificial air chamber part of the chick embryo by using sterile forceps, sucking allantoic fluid into a sterile container by using a disposable syringe without a needle head, collecting a diseased fetus and an allantoic membrane at the same time, placing the diseased fetus and the allantoic membrane into a mortar, pouring liquid nitrogen, grinding by using a pestle, mixing with the allantoic fluid, centrifuging at 5000rpm for 20min at 4 ℃, collecting supernatant, and storing in the refrigerator at-80 ℃ for later use.

1.2 hemagglutination assay to identify the purity of rejuvenating viruses

(1) In 96-well type V hemagglutination plates, 25uL PBS was added to each well.

(2) Adding 25uL of virus liquid into the first hole, uniformly mixing, sucking 25uL into the second hole, sequentially diluting to the 11 th hole in a multiple ratio according to the method, and finally discarding 25uL of liquid, wherein the 12 th hole is used as a red blood cell control.

(3) Add 25uL PBS to each well in turn.

(4) 25ul of 1% suspension of chicken red blood cells was added to each well in sequence.

(5) And (3) oscillating the hemagglutination plate on an oscillator for 2min, standing at room temperature for 30min, and judging the result when the red blood cells in the control hole are obviously in a button shape.

(6) And (4) judging a result: the hemagglutination plate was tilted at an angle and was considered hemagglutination-free if the red blood cells settled at the bottom of the well and slide down in a tear-drop shape. Hemagglutination is considered to occur if the erythrocytes are flattened at the bottom of the well and do not slide down like a tear drop after the hemagglutination plate is inclined.

If the virus to be detected has no hemagglutination, which indicates that the virus has no avian orthomyxovirus and paramyxovirus, the sample is judged to be pure IBDV, and no other avian common virus mixed infection exists.

1.3 real-time fluorescent quantitative PCR method for detecting copy number of each virus

RNA of 15 rejuvenated viruses is extracted and subjected to reverse transcription to obtain cDNA by the experimental method of section 1.3 of example 1, the cDNA is used as a template, S1 and S2 are respectively used as upstream and downstream primers (table 1 of example 1), real-time PCR is carried out according to the GoTaq qPCR Master Mix instruction, the copy number of the 15 viruses is calculated according to the standard curve equation y of established IBDV in the laboratory, which is-3.482 x +51.696, and the amplification system is as follows:

and (3) instantly separating after the sample is uniformly mixed, and putting the sample into an instrument for amplification: 2min at 95 ℃; at 95 ℃ for 15sec, at 60 ℃ for 1min, and for 40 cycles; 15sec at 95 ℃; 15sec at 60 ℃; 95 ℃ for 15 sec.

1.4 animal infection experiments

Adjusting the dose of 15 strains of virus for virus attack to be the same copy number according to the calculated virus copy number, diluting the quantified virus with sterilized PBS, performing eye-attacking and virus-attacking on 40-day-old SPF chickens, wherein the virus-attacking amount is 100uL per chicken, and performing eye-attacking on a control group by PBS with the same volume. There were 6 SPF chickens in each group. Observing and recording clinical symptoms of all groups of chickens at any time after the challenge, randomly selecting 3 chickens per group for killing on the third day after infection, and recording the eye lesions presented after the cesarean examination of all groups of chickens. The bursa of Fabricius and the spleen are taken for paraffin section preparation and pathological histological observation.

1.5 preparation of pathological section

(1) Repairing and flushing: repairing the fixed tissue to a size of about 1.0cm × 1.0cm × 0.5cm, washing with running water, and if the tissue block is fixed for a long time, washing overnight;

(2) and (3) dehydrating: taking out the washed tissue block, and dehydrating the tissue block, wherein the sequence of 70% ethanol over night to 80% ethanol for 1.5h to 95% ethanol for 15min to 100% ethanol I for 20min to 100% ethanol II for 20min to acetone for 10min, and attention is paid to no excessive dehydration;

(3) and (3) transparency: after dehydration, firstly, the tissue blocks are transparent, xylene I is 10 min-xylene II is 15min in sequence, and the tissue blocks are soaked in soft wax at the temperature of 60 ℃ for 1 h;

(4) wax dipping: pouring soft wax into a prepared small paper groove, quickly putting tissue blocks into the small paper groove, paying attention to reasonable distance between tissues, and standing for more than 2h to fully solidify the soft wax;

(5) embedding: taking out the embedded small wax block, trimming the wax block into a proper size by using a knife, and finally, carefully fixing the wax block on the small wood block;

(6) slicing: during slicing, the thickness of the slices is required to be proper, the slices are spread in a constant-temperature water bath box, and the temperature is 45 ℃; finally, placing the cut slices in a 37 ℃ incubator for baking;

(7) dewaxing: the dewaxing sequence is xylene I5 min-xylene II 5 min-95% ethanol I3 min-95% ethanol II 3 min-80% ethanol 3 min-70% ethanol 3 min-distilled water 10 min;

(8) dyeing: adjusting the hematoxylin staining time according to the freshness and the old of the staining solution during staining, wherein the sequence is hematoxylin 7 min-distilled water for a moment, 1% hydrochloric acid alcohol differentiation 20 s-flowing water washing 2 h-eosin staining 8 min-distilled water for a moment;

(9) and (3) dehydrating: the sequence is 70% ethanol 20 s-80% ethanol 20 s-95% ethanol 5 min-100% ethanol I10 min-100% ethanol II 5 min;

(10) transparency and mounting: the sequence is 10min xylene I, 5min xylene II, gum sealing, drying at 37 ℃ and microscopic examination.

2. Results

2.1 rejuvenation of viruses

Virus rejuvenation was performed on 15 IBDV strains, BD, CA, HR, CF, SD, BC, DB11, DN-04, CEF94, C-8, 05-6, 99-3, JC-7, SD-F3 and GC-7, stored in this laboratory by inoculation of the chorioallantoic membrane of a 10-day-old SPF chick embryo. Collecting allantoic fluid, allantoic membrane and affected fetus, grinding with liquid nitrogen, mixing with allantoic fluid, centrifuging to obtain supernatant as harvested virus, and storing in-80 deg.C refrigerator.

2.2 hemagglutination assay to identify the purity of rejuvenating Virus

After rejuvenation and virus collection, 15 IBDV strains preserved in a laboratory are subjected to hemagglutination experiments, and the results show that red blood cells are deposited at the bottom of a hole and slide downwards in a tear drop shape after a hemagglutination plate is inclined at a certain angle (as shown in figure 6), so that the red blood cells have no hemagglutination, and 15 strains of CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8, 05-6, 99-3, JC-7, SD-F3 and GC-7 preserved in the laboratory are judged to be pure IBDV, but no other common mixed virus infection of poultry.

2.3 real-time fluorescent quantitative RT-PCR method for detecting copy number of each virus

RNA extraction and reverse transcription are carried out on 15 strains of viruses after chick embryo rejuvenation, the obtained cDNA is used as a template, S1 and S2 are respectively used as upstream and downstream primers, and real-time PCR is carried out according to GoTaq qPCR Master Mix.

The average number of cycles required for strains CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8, 05-6, 99-3, JC-7, SD-F3 and GC-7 to reach the fluorescence absorption threshold can be obtained according to the amplification curve of 15 strains of IBDV preserved in the laboratory, and the cycles are 19.876, 24.722, 30.389, 30.914, 28.458, 29.279, 31.886, 31.425, 31.977, 29.688, 28.043, 26.953, 23.957, 21.240 and 23.139. According to the standard curve equation y-3.482 x +51.696 established in the laboratory for IBDV, copies-10 can be obtained

The copy number of each virus was calculated, and the results are shown in table 3:

TABLE 315 Strain copy number

2.4 clinical symptoms and pathological anatomical changes after animal infection

48h after challenge, all the chickens infected with HR, CF, SD, DB11, BC and DN-04 strains of viruses showed the clinical symptoms of depressed spirit, decreased feed intake, disordered feathers, drooping head and wings, cachexia, loss of one corner, closed eyelids, reluctant to walk and the like; the PBS control group of chickens appeared normal, and the chickens in other attenuated groups had no obvious clinical symptoms.

On the third day after infection, 3 chickens are randomly selected from each group for killing, and the bursa of each group of chickens infected with 15 strains of viruses can be seen to have different degrees of characteristic lesions through the autopsy, wherein the eye lesions of the chickens in the group with strong toxicity are more obvious than those in the group with weak toxicity, such as yellow jelly-like edema of the bursa of Fabricius, and cream-colored cellulosic exudates (as shown in figure 7A) cover the mucous membrane; enlarged spleen with bleeding symptoms individually (as shown in fig. 7B); streak bleeding occurred in the medial thigh muscle of chicken infected with virulent virus (see fig. 8). The chicken infected with the attenuated group also has the phenomenon of bursal and spleen enlargement, but the condition is relatively lighter compared with the ultra-virulent group; there was no significant pathological change in the tissues and organs after the autopsy of the PBS control group (see FIG. 9).

2.5 histopathological changes after infection of animals

Taking the bursa of Fabricius and the spleen, and making paraffin sections, wherein the bursa of Fabricius and the spleen tissue structures of the PBS control group tend to be normal, and no obvious histopathological change is observed (as shown in FIG. 10); histological observation of the 6 hypervariable strains HR, CF, SD, DB11, BC and DN-04 on bursal disease after virus challenge shows that a large amount of lymphocyte necrosis accompanied with apoptosis phenomenon exists, a large amount of heterophilic cell infiltration exists in the lymph follicles, vacuole formation can be seen in part of the follicles, edema exists in the lamina propria, and inflammatory cell infiltration exists in the lamina propria (as shown in figures 11A-F). The 9 attenuated strains CA, C-8, 05-6, 99-3, JC-7, BD, CEF94, SD-F3 and GC-7 can be observed in the pathological and histological observation of the bursal disease after virus challenge, and the lymphatic follicular atrophy, wherein the lymphocytes are obviously reduced, and inflammatory cell infiltration exists in the inherent layer (as shown in figure 11G-O).

The pathological histological observation of spleen after challenge shows that 15 strains HR, CF, SD, DB11, BC, DN-04, CA, C-8, 05-6, 99-3, JC-7, BD, CEF94, SD-F3 and GC-7 stored in a laboratory have no obvious difference between the hyper-virulent strain and the hypo-virulent strain, and all the hyper-virulent strain and the hypo-virulent strain have obvious reduction of lymphocytes and are accompanied with the proliferation of a large number of macrophages to form white gaps (as shown in a picture 12A-O).

Sequence listing

<110> northeast university of agriculture

<120> kit for identifying virulent and attenuated strains of chicken infectious bursal disease virus based on RT-PCR and RFLP technology and application thereof

<130> KLPI170687

<160> 17

<170> PatentIn 3.5

<210> 1

<211> 773

<212> DNA

<213> IBDV HR Strain

<400> 1

cccaaaatgg tagcaacatg tgacagcagt gacaggccca gagtctacac cataactgca 60

gccgatgatt accaattctc atcacagtac caaacaggtg gagtaacaat cacactgttc 120

tcagctaata tcgatgccat cacaagcctc agcatcgggg gagaactcgt gtttcaaaca 180

agcgtccaag gccttatact gggtgctacc atctacctta taggctttga tgggactgcg 240

gtaatcacca gagctgtggc tgcagacaat gggctaacgg ccggcactga caaccttatg 300

ccattcaata ttgtgattcc aaccagcgag ataacccagc caatcacatc catcaaacta 360

gagatagtga cctccaaaag tggtggtcag gcgggagatc agatgtcatg gtcagcaagt 420

gggagcctag cagtgacgat ccacggtggc aactatccag gggccctccg tcccgtcaca 480

ctagtagcct acgaaagagt ggcaacagga tctgtcgtta cggtcgccgg ggtgagcaac 540

ttcgagctga tcccaaatcc tgaactagca aagaacctgg tcacagaata cggccgattt 600

gacccaggag ccatgaacta cacaaaattg atactgagtg agagggaccg tcttggcatc 660

aagaccgtct ggccaacaag ggagtacact gactttcgcg agtacttcat ggaggtggcc 720

gacctcaact ctcccctgaa gattgcagga gcattcggct tcaaagctcc ccg 773

<210> 2

<211> 833

<212> DNA

<213> IBDV CF Strain

<400> 2

cggtcgccgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca aagaacctgg 60

tcacagaata cggccgattt gacccaggag ccatgaacta cacaaaattg atactgagtg 120

agagggaccg tcttggcatc aagaccgtat ggccaacaag ggagtacact gactttcgcg 180

agtacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcagga gcatttggct 240

tcaaagacat aatccgggcc ctaaggagga tagctgtgcc ggtggtctct acactgttcc 300

cacccgccgc tcccctagcc catgcaattg gggaaggtgt agactacctg ctgggcgatg 360

aggcacaggc tgcttcagga actgctcgag ccgcgtcagg aaaagcaaga gctgcctcag 420

gccgcataag gcagctaact ctcgccgccg acaaggggta cgaggtagtc gcgaatctgt 480

tccaggtgcc ccagaatcct gtagtcgacg ggattctcgc ttcacccggg atactccgcg 540

gtgcacacaa cctcgactgc gtgttgagag agggtgccac gctattccct gtggtcatca 600

cgacagtgga agatgccatg acacccaaag cactgaacag caaaatgttt gctgtcattg 660

aaggcgtgcg agaagatctc caacctccat ctcaaagagg atccttcata cgaactctct 720

ccggacatag agtctatgga tatgctccag atggggtact tccactggag actgggagag 780

tttacaccgt ggtcccaata gatggtgtct gggacgacag cattatgctg tcc 833

<210> 3

<211> 773

<212> DNA

<213> IBDV SD Strain

<400> 3

cccaaaatgg tagccacatg tgacagcagt gacaggccca gagtctacac cataactgca 60

gccgatgatt accaattctc atcacagtac caatcaggtg gggtaacaat cacactgttc 120

tcagccaaca ttgatgctat cacaagcctc agcattgggg gagaactcgt gttccataca 180

agcgtccaag gccttgcact gaacgccacc atctacctta taggctttga tgggactaca 240

gtaatcacca gagctgtggc ctcagacaat gggctgacta ccggcatcga caatcttatg 300

ccattcaatc ttgtgattcc aaccaacgag ataacccagc caatcacatc catcaaactg 360

gagatagtga cctccaaaag tggtggtcag gcaggggacc agatgtcatg gtcggcaagt 420

gggagcctag cagtgacaat ccatggtggc aactatccag gggccctccg tcccgtcaca 480

ctagtagcct acgaaagagt ggcaacagga tccgtcgtta cggtcgccgg ggtgagcaac 540

ttcgagctga tcccaaatcc tgaactagca aagaacctgg ttacagaata cggccgattt 600

gacccaggag ccatgaacta cacaaaattg atactgagtg agagggaccg tcttggcatc 660

aagaccgtct ggccaacaag ggagtacact gactttcgtg agtacttcat ggaggtggcc 720

gacctcaact ctcccctgaa gattgcagga gcatttggct tcaaagctcc cgg 773

<210> 4

<211> 773

<212> DNA

<213> IBDV DB11 Strain

<400> 4

cccaaaaatg gtagccacat gtgacagcag tgacaggccc agagtctaca ccataactgc 60

agccgatgat taccaattct catcacagta ccaatcaggt ggggtaacaa tcacactgtt 120

ctcagccaac attgatgcta tcacaagcct cagcattggg ggagaactcg tgttccatac 180

aagcgtccaa ggccttgcac tgaacgccac catctacctt ataggctttg atgggactac 240

agtaatcacc agagctgtgg cctcagacaa tgggctgact accggcatcg acaatcttat 300

gccattcaat cttgtgattc caaccaacga gataacccag ccaatcacat ccatcaaact 360

ggagatagtg acctccaaaa gtggcggtca ggcaggggac cagatgtcat ggtcggcaag 420

tgggagccta gcagtgacaa tccatggtgg caactatcca ggggccctcc gtcccgtcac 480

actagtagcc tacgaaagag tggcaacagg atccgtcgtt acggtcgccg gggtgagcaa 540

cttcgagctg atcccaaatc ctgaactagc aaagaacctg gttacagaat acggccgatt 600

tgacccagga gccatgaact acacaaaatt gatactgagt gagagggacc gtcttggcat 660

caagaccgtc tggccaacaa gggagtacac tgactttcgt gagtacttca tggaggtggc 720

cgacctcaac tctcccctga agattgcagg agcattcggc ttcaagctcc ggg 773

<210> 5

<211> 777

<212> DNA

<213> IBDV BC Strain

<400> 5

cccacaaatg gtagccacat gtgacagcag tgacaggccc agagtctaca ccataactgc 60

agccgatgat taccaattct tatcacagta ccaaccaggt ggggtaacaa tcacactgtt 120

ctcagccaac attgatgcta tcacaagcct cagcgttggg ggagaactcg tgtttcaaac 180

aagcgttcaa ggccttgtac tgggcgccac catctacctt ataggctttg atgggactac 240

ggtaatcacc agggctgtgg ccgcagacaa tgggctgacg gccggcaccg acaatcttat 300

gccattcaat attgtgattc caaccaacga gataacccag ccaattacat ccatcaaact 360

ggagatagtg acctccaaaa gtggtggcca ggcaggggac cagatgtcat ggtcggcaag 420

tgggagccta gcagtgacga tccatggtgg caactatcca ggggccctcc gtcccgtcac 480

actagtagcc tacgaaagag tggcaacagg atccgtcgtt acggtcgctg gggtgagcaa 540

cttcgagctg atcccaaatc ctgaactagc aaagaacctg gttacagaat acggccgatt 600

tgacccagga gccatgaact acacaaaatt gatactgagt gagagggacc gtcttggcat 660

caagaccgtc tggccaacaa gggagtacac cgactttcgt gagtacttca tggaggtggc 720

cgacctcaat tctcccctga agattgcagg agcatttggc ttcaaagaca tatcccg 777

<210> 6

<211> 768

<212> DNA

<213> IBDV strain DN-04

<400> 6

ccaaaaatgg tagccacatg tgacagcagt gacaggccca gagtctacac cataactgca 60

gccgatgatt accaattctc atcacagtac caatcaggtg gggtaacaat cacactgttc 120

tcagccaaca ttgatgctat cacaagcctc agcattgggg gagaacttgt gttccataca 180

agcgtccaag gccttgcact gaacgccacc atctacctta taggctttga tgggactaca 240

gtaatcacca gagctgtggc ctcagacaat gggctgacta ccggcatcga caatcttatg 300

ccattcaatc ttgtgattcc aaccaacgag ataacccagc caatcacatc catcaaactg 360

gagatagtga cctccaaaag tggcggtcag gcaggggacc agatgtcatg gtcggcaagt 420

gggagcctag cagtgacaat ccatggtggc aactatccag gggccctccg tcccgtcaca 480

ctagtagcct acgaaagagt ggcaacagga tccgtcgtta cggtcgccgg ggtgagcaac 540

ttcgagctga tcccaaatcc tgaactagca aagaacctgg ttacagaata cggccgattt 600

gacccaggag ccatgaacta cacaaaattg atactgagtg agagggaccg tcttggcatc 660

aagaccgtct ggccaacaag ggagtacact gactttcgtg agtacttcat ggaggtggcc 720

gacctcaact ctcccctgaa gattgcagga gcatttggct tcaagctc 768

<210> 7

<211> 773

<212> DNA

<213> IBDV CA Strain

<400> 7

cccaaaatgg tagccacatg tgacagcagt gacaggccca gagtctacac cataactgca 60

gccgatgatt accaattctc atcacagtac caaccaggtg gggtaacaat cacactgttc 120

tcagccaaca ttgatgccat cacaagcctc agcgttgggg gagagctcgt gtttcaaaca 180

agcgtccacg gccttgtact gggcgccacc atctacctca taggctttgg tgggacagcg 240

gtaatcacca gggctgtggc cgcaaacaat gggctgacga ccggcaccga caaccttttg 300

ccattcaatc ttgtgattcc aacaaacgag ataacccagc caatcacatc catcaaactg 360

gagatagtga cctccaaaag tggtggtcag gcaggggatc agatgtcatg gtccgcaaga 420

gggagcctag cagtgacgat ccatggtggc aactatccag gggccctccg tcccgtcacg 480

ctagtggcct acgaaagagt ggcaacagga tccgtcgtta cggtcgctgg ggtgagcaac 540

ttcgagctga tcccaaatcc tgaactagca aagaacctgg ttacagaata cggccgattt 600

gacccaggag ccatgaacta cacaaaattg atactgagtg agagggaccg tcttggcatc 660

aagaccgtct ggccaacaag ggagtacact gactttcgtg aatacttcat ggaggtggcc 720

gacctcaact ctcccctgaa gattgcagga gcattcggct tcaaagacta ccc 773

<210> 8

<211> 774

<212> DNA

<213> IBDV strain C-8

<400> 8

cccaaaaatg gtagccacat gtgacagcag tgacaggccc agagtctaca ccataactgc 60

agccgatgat taccaattct catcacagta ccaaccaggt ggggtaacaa tcacactgtt 120

ctcagccaac attgatgcca tcacaagcct cagcgttggg ggagagctcg tgtttcgaac 180

aagcgtccac ggccttgtac tgggcgccac catctacctc ataggctttg atgggacaac 240

ggtaatcacc agggctgtgg ccgcaaacaa tgggctgacg accggcaccg acaaccttat 300

gccattcaat cttgtgattc caacaaacga gataacccag ccaatcacat ccatcaaact 360

ggagatagtg acctccaaaa gtggtggtca ggcaggggat cagatgtcat ggtcggcaag 420

agggagccta gcagtgacga tccatggtgg caactatcca ggggccctcc gtcccgtcac 480

gctagtggcc tacgaaagag tggcaacagg atccgtcgtt acggtcgctg gggtgagcaa 540

cttcgagctg atcccaaatc ctgaactagc aaagaacctg gttacagaat acggccgatt 600

tgacccagga gccatgaact acacaaaatt gatactgagt gagagggacc gtcttggcat 660

caagaccgtc tggccaacaa gggagtacac tgactttcgt gaatacttca tggaggtggc 720

cgacctcaac tctcccctga agattgcagg agcattcggc ttcaaagact accc 774

<210> 9

<211> 768

<212> DNA

<213> IBDV strain 05-6

<400> 9

cccaaaatgg tagccacatg tgacagcagt gacaggccca gagtctacac cataactgca 60

gccgatgatt accaattctc atcacagtac caaccaggtg gggtaacaat cacactgttc 120

tcagccaaca ttgatgccat cacaagcctc agcgttgggg gagagctcgt gtttcaaaca 180

agcgtccacg gccttgtact gggcgccacc atctacctca taggctttga tgggacagcg 240

gtaatcacca gggctgtggc cgcaaacaat gggctgacga ccggcaccga caacctttag 300

ccattcaatc ttgtgattcc aacaaacgag ataacccagc caatcacatc catcaaactg 360

gagatagtga cctccaaaag tggtggtcag gcaggggatc agatgtcatg gtccgcaaga 420

gggagcctag cagtgacgat ccatggtggc aactatccag gggccctccg tcccgtcacg 480

ctagtggcct acgaaagagt ggcaacagga tccgtcgtta cggtcgctgg ggtgagcaac 540

ttcgagctga tcccaaatcc tgaactagca aagaacctgg ttacagaata cggccgattt 600

gacccaggag ccatgaacta cacaaaattg atactgagtg agagggaccg tcttggcatc 660

aagaccgtct ggccaacaag ggagtacact gactttcgtg aatacttcat ggaggtggcc 720

gacctcaact ctcccctgaa gattgcagga gcattcggct tcaagctc 768

<210> 10

<211> 768

<212> DNA

<213> IBDV strain 99-3

<400> 10

atgtgtagcc acatgtgaca gcagtgacag gcccagagtc tacaccataa ctgcagccga 60

tgattaccaa ttctcatcac agtaccaacc aggtggggta acaatcacac tgttctcagc 120

caacattgat gccatcacaa gcctcagcgt tgggggagag ctcgtgtttc aaacaagcgt 180

ccacggcctt gtactgggcg ccaccatcta cctcataggc tttgatggga caacggtaat 240

caccagggct gtggccgcaa acaatgggct gacgaccggc accgacaacc ttatgccatt 300

caatcttgtg attccaacaa acgagataac ccagccaatc acatccatca aactggagat 360

agtgacctcc aaaagtggtg gtcaggcagg ggatcagatg tcatggtcgg caagagggag 420

cctagcagtg acgatccatg gtggcaacta tccaggggcc ctccgtcccg tcacgctagt 480

ggcctacgaa agagtggcaa caggatccgt cgttacggtc gctggggtga gcaacttcga 540

gctgatccca aatcctgaac tagcaaagaa cctggttaca gaatacggcc gatttgaccc 600

aggagccatg aactacacaa aattgatact gagtgagagg gaccgtcttg gcatcaagac 660

cgtctggcca acaagggagt acactgactt tcgtgaatac ttcatggagg tggccgacct 720

caactctccc ctgaagattg caggagcatt cggcttcaaa gactcccg 768

<210> 11

<211> 774

<212> DNA

<213> IBDV JC-7 Strain

<400> 11

cgagaaatgg tagccacatg tgacagcagt gacaggccca gagtctacac cataactgca 60

gccgatgatt accaattctc atcacagtac caaccaggtg gggtaacaat cacactgttc 120

tcagccaaca ttgatgccat cacaagcctc agcgttgggg gagagctcgt gtttcaaaca 180

agcgtccacg gccttgtact gggcgccacc atctacctca taggctttga tgggacaacg 240

gtaatcacca gggctgtggc cgcaaacaat gggctgacga ccggcaccga caaccttttg 300

ccattcaatc ttgtgattcc aacaaacgag ataacccagc caatcacatc catcaaactg 360

gagatagtga cctccaaaag tggtggtcag gcaggggatc agatgtcatg gtccgcaaga 420

gggagcctag cagtgacgat ccatggtggc aactatccag gggccctccg tcccgtcacg 480

ctagtggcct acgaaagagt ggcaacagga tccgtcgtta cggtcgctgg ggtgagcaac 540

ttcgagctga tcccaaatcc tgaactagca aagaacctgg ttacagaata cggccgattt 600

gacccaggag ccatgaacta cacaaaattg atactgagtg agagggaccg tcttggcatc 660

aagaccgtct ggccaacaag ggagtacact gactttcgtg aatacttcat ggaggtggcc 720

gacctcaact ctcccctgaa gattgcagga gcattcggct tcaaagacta cccc 774

<210> 12

<211> 761

<212> DNA

<213> IBDV BD Strain

<400> 12

gttttgactg gcccttagtc tacaccataa ctgcagccga tgattaccaa ttctcatcac 60

agtaccaacc aggtggggta acaatcacac tgttctcagc caacattgat gccatcacaa 120

gcctcagcgt tgggggagag ctcgtgtttc aaacaagcgt ccacggcctt gtactgggcg 180

ccaccatcta cctcataggc tttgatggga cagcggtaat caccagggct gtggccgcaa 240

acaatgggct gacgaccggc accgacaacc ttttgccatt caatcttgtg attccaacaa 300

acgagataac ccagccaatc acatccatca aactggagat agtgacctcc aaaagtggtg 360

gtcaggcagg ggatcagatg tcatggtccg caagagggag cctagcagtg acgatccatg 420

gtggcaacta tccaggggcc ctccgtcccg tcacgctagt ggcctacgaa agagtggcaa 480

caggatccgt cgttacggtc gctggggtga gcaacttcga gctgatccca aatcctgaac 540

tagcaaagaa cctggttaca gaatacggcc gatttgaccc aggagccatg aactacacaa 600

aattgatact gagtgagagg gaccgtcttg gcatcaagac cgtctggcca acaagggagt 660

acactgactt tcgtgaatac ttcatggagg tggccgacct caactctccc ctgaagattg 720

caggagcatt cggcttcaaa gacatatccc ggggcccata a 761

<210> 13

<211> 833

<212> DNA

<213> IBDV strain CEF94

<400> 13

cggtcgctgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca aagaacctgg 60

ttacagaata cggccgattt gacccaggag ccatgaacta cacaaaattg atactgagtg 120

agagggaccg tcttggcatc aagaccgtct ggccaacaag ggagtacact gactttcgtg 180

aatacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcagga gcattcggct 240

tcaaagacat aatccgggcc ataaggagga tagctgtgcc ggtggtctcc acattgttcc 300

cacctgccgc tcccctagcc catgcaattg gggaaggtgt agactacctg ctgggcgatg 360

aggcacaggc tgcttcagga actgctcgag ccgcgtcagg aaaagcaaga gctgcctcag 420

gccgcataag gcagctgact ctcgccgccg acaaggggta cgaggtagtc gcgaatctat 480

tccaggtgcc ccagaatccc gtagtcgacg ggattcttgc ttcacctggg gtactccgcg 540

gtgcacacaa cctcgactgc gtgttaagag agggtgccac gctattccct gtggttatta 600

cgacagtgga agacgccatg acacccaaag cattgaacag caaaatgttt gctgtcattg 660

aaggcgtgcg agaagacctc caacctcctt ctcaaagagg atccttcata cgaactctct 720

ctggacacag agtctatgga tatgctccag atggggtact tccactggag actgggagag 780

actacaccgt tgtcccaata gatgatgtct gggacgacag cattatgctg tcc 833

<210> 14

<211> 833

<212> DNA

<213> IBDV strain SD-F3

<400> 14

cggtcgctgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca aagaacctgg 60

ttacagaata cggccgattt gacccaggag ccatgaacta cacaaaattg atactgagtg 120

agagggaccg tcttggcatc aagaccgtct ggccaacaag ggagtacact gactttcgtg 180

aatacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcggga gcattcggct 240

tcaaagacat aatccgggcc ataaggagga tagctgtgcc ggtggtctcc acattgttcc 300

cacctgccgc tcccctagcc catgcaattg gggaaggtgt agactacctg ctgggcgatg 360

aggcacaggc tgcttcagga actgctcgag ccgcgtcagg aaaagcaaga gctgcctcag 420

gccgcataag gcagctgact ctcgccgccg acaaggggta cgaggtagtc gcgaatctat 480

tccaggtgcc ccagaatccc gtagtcgacg ggattcttgc ttcacctggg gtactccgcg 540

gtgcacacaa cctcgactgc gtgttaagag agggtgccac gctattccct gtggttatta 600

cgacagtgga agacgccatg acacccaaag cattgaacag caaaatgttt gctgtcattg 660

aaggcgtgcg agaagacctc caacctccat ctcaaagagg atccttcata cgaactctct 720

ctggacgcag agtctatgga catgctccag atggggtact tccactggag actgggagag 780

actacaccgt tgtcccaata gatgatgtct gggacgacag cattatgctg tcc 833

<210> 15

<211> 833

<212> DNA

<213> IBDV strain GC-7

<400> 15

cggtcgctgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca aagaacctgg 60

ttacagaata cggccgattt gacccaggag ccatgaacta cacaaaattg atactgagtg 120

agagggaccg tcttggcatc aagaccgtct ggccaacaag ggagtacact gactttcgtg 180

aatacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcagga gcattcggct 240

tcaaagacat aatccgggcc ataaggagga tagctgtgcc ggtggtctcc acattgttcc 300

cacctgccgc tcccctagcc catgcaattg gggaaggtgt agactacctg ctgggcgatg 360

aggcacaggc tgcttcagga actgctcgag ccgcgtcagg aaaagcaaga gctgcctcag 420

gccgcataag gcagctgact ctcgccgccg acaaggggta cgaggtagtc gcgaatctat 480

tccaggtgcc ccagaatccc gtagtcgacg ggattcttgc ttcacctggg gtactccgcg 540

gtgcacacaa cctcgactgc gtgttaagag agggtgccac gctattccct gtggttatta 600

cgacagtgga agacgccatg acacccaaag cattgaacag caaaatgttt gctgtcattg 660

aaggcgtgcg agaagacctc caacctcctt ctcaaagagg atccttcata cgaactctct 720

ctggacacag agtctatgga tatgctccag gtggggtact tccactggag actgggagag 780

actacaccgt tgtcccaata gatgatgtct gggacgacag cattatgctg tcc 833

<210> 16

<211> 20

<212> DNA

<213> artificial sequence

<400> 16

atcttgggta tgtgaggctg 20

<210> 17

<211> 20

<212> DNA

<213> artificial sequence

<400> 17

tatggcccgg attatgtctt 20

Claims (4)

1. The kit for identifying the hyper-virulent strain and the low virulent strain of the chicken infectious bursal disease virus based on RT-PCR and RFLP technology is characterized by comprising a primer pair for amplifying the VP2 gene of the chicken infectious bursal disease virus and SpeI, SacI and StuI restriction endonucleases, wherein the sequence of the primer pair for amplifying the VP2 gene of the chicken infectious bursal disease virus is as follows:

PAU：5’ATCTTGGGTATGTGAGGCTG 3’；

PAD：5’TATGGCCCGGATTATGTCTT 3’。

2. the kit of claim 1, further comprising rTaq enzyme, 10 XPCR buffer, dNTP, RNase inhibitor, reverse transcriptase, 5 XTR buffer, and digestion buffer.

3. A method for identifying a super virulent strain and a low virulent strain of chicken infectious bursal disease virus based on RT-PCR and RFLP technologies, which is used for non-disease diagnosis purposes, is characterized by comprising the following steps:

(1) extracting RNA and performing reverse transcription on the infectious bursal disease virus to be detected to obtain virus cDNA;

(2) PCR amplification;

using the cDNA obtained in the step (1) as a template, using the PAU and PAD described in claim 1 as upstream and downstream primers, and carrying out PCR amplification on a target gene fragment in a VP2 sequence, wherein the reaction system is as follows: cDNA 4 mu L, rTaq enzyme 0.5 mu L, 10 XPCR buffer 2.5 mu L, dNTP 3 mu L, primer PAU 1 mu L, primer PAD 1 mu L, deionized water 13 mu L, total volume 25 mu L;

after the sample is mixed uniformly, the sample is separated instantly and put into a PCR instrument, and the amplification procedure is as follows: 5min at 95 ℃; 30 cycles at 94 ℃ for 30sec, 57.1 ℃ for 45sec, 72 ℃ for 50 sec; 10min at 72 ℃; storing at 4 deg.C;

(3) recovery and purification of virus target gene sequence PCR product

Purifying and recovering the PCR product obtained in the step (2), and storing the collected DNA at-20 ℃;

(4) RFLP identification of IBDV strains

Performing enzyme digestion identification on the PCR purified product obtained in the step (3) by using three restriction enzymes of SpeI, SacI and StuI; the enzyme digestion system is as follows: 1 muL of SpeI or StuI or SacI of 10U/uL, 2 muL of enzyme digestion buffer solution, 7 muL of PCR purified product of 20-40 ng/muL and 10 muL of deionized water, wherein the total volume is 20 muL;

mixing, placing in water bath at 37 deg.C for 2h, mixing with 2uL 10 × Loading Buffer, performing electrophoresis with 0.8% agarose gel at 110V for 20min, and observing enzyme digestion result;

(5) determination of results

If the two fragments of 531bp and 302bp are cut out by SpeI, the two fragments of 242bp and 591bp are cut out by StuI, but the two fragments can not be cut by SacI enzyme, the virus strain is judged to be a super-virulent strain;

if only two fragments of 218bp and 615bp can be cut out by SacI and cannot be cut by SpeI and StuI, the strain is judged to be an attenuated strain.

4. Use of the kit of claim 1 or 2 for preparing a reagent for identifying a hyper-virulent strain and a low virulent strain of the chicken infectious bursal disease virus.

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