CN106282417B - A kind of quick multi-fluorescence immunoassay primer, kit and method for distinguishing CAV, MDV, REV, IBDV - Google Patents
A kind of quick multi-fluorescence immunoassay primer, kit and method for distinguishing CAV, MDV, REV, IBDV Download PDFInfo
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Abstract
The invention discloses a kind of quick multi-fluorescence immunoassay primer, kit and method for distinguishing CAV, MDV, REV, IBDV.The present invention is easy to operate, obtains target amplification fragment by PCR, is then hybridized amplified production, fluorescence-encoded micro-beads and Streptavidin phycoerythrin, then reads MFI values by detector, offer an explanation different types of cause of disease.The method of the present invention, at the same time can accurately detect chicken infectious anemia virus, chicken Marek's disease virus, fowl Reticuloendotheliosis Virus and infections chicken cloacal bursa virus, and high specificity, high sensitivity are reproducible.Compared with traditional detection method, the method for the present invention is realized is carried out at the same time detection to a variety of different molecules of interest in same sample, and sample dosage is few, easy to operate, quick, can substantially reduce testing cost.The flexibility of the present invention is good, can add and subtract the species of detection cause of disease on this basis as needed.
Description
Technical field
The invention belongs to the Pathogen test field of aquaculture, and in particular to a kind of quick differentiation chicken infectious anemia virus
（CAV, Chicken infectious anemia virus）, chicken Marek's disease virus（MDV, Marek's disease
virus）, fowl Reticuloendotheliosis Virus（REV, Reticuloendotheliosis virus）And gumboro disease
Poison（IBDV, Infections Bursal Disease virus）Multi-fluorescence immunoassay primer, kit and method.
Background technology
The immune response of poultry immunity system or function are the main of the invasion of body resistance cause of disease, infection and breeding dispersal
Mechanism, is the important component of body defenses system.The immune organ of chicken body includes the bursa of farbricius, marrow, spleen, thymus gland, blind
Intestinal tonsil body.Immunosuppressive disease is by one or more single pathogenic factor collective effects, causes chicken immune system damage, immune function
The general name of disease caused by a variety of infectious agents and non-pathogenic factors reduced.Our most common clinical manifestations are chicken group
Chronic production performance is low, directly affects the economic performance index of chicken group, is primarily due in the case where immunosupress occurs
After vaccine immunity, antibody level is not high.Research shows that viral infection is to cause immunosuppressive principal element.Marek's disease
Virus, chicken infectious anemia virus, infectious bursa of Fabricius virus and reticuloendothelial cell hyperplasia disease virus, which are 4 kinds, to be caused
The virus of inhibitive ability of immunity disease, the infection in chicken group is very universal, but morbidity and mortality differ.The sense of these types of virus
Dye can decline Abwehrkraft des Koepers, cause the secondary sense of immuning failure and the Escherichia coli of the vaccines such as ewcastle disease, the bursa of farbricius etc.
Dye, very big economic loss is caused to aviculture.
These immunosuppressive virus are often in continuation subclinical infection, due to its atypical symptoms, it is difficult to make
Correctly diagnosis, its antidiastole usually need to mainly include pathogen separation by laboratory diagnostic technique, traditional detection method
Identification, serological test and enzyme-linked immunosorbent assay etc., but these methods often by clinical disease fresh material degree, pollution level or
The limitation of the factors such as the course of disease, operation is also very cumbersome, time-consuming, and sensitiveness is low, poor specificity, is not easy to detect mixing sense
Dye, is unfavorable for the timely diagnoses and treatment of disease, so as to cause great economic loss.In recent years, with the hair of molecular biology
Exhibition, PCR technologies have been widely used in the detection of these chicken infectious diseases, and establish multiplex PCR detection technique, examine at the same time
Several cause of diseases are surveyed, multiple RT-PCR is widely used in the mixing sense of poultry diease because of its sensitivity, specificity and the simplicity of operation,
But result judgement needs electrophoresis, time-consuming and laborious, and reaction product easily produces pollution and causes false positive, and multiplex PCR is to lean on
The size of fragment has generally been arrived more than triple come what is distinguished, and clip size difference is big, its every kind of viral amplification efficiency differs
Sample, causing result, there are deviation.Fluorescent quantitative PCR technique has merged a variety of advantages of PCR, is reacted by directly detecting PCR
The change of fluorescence signal, which is realized, in journey quantifies molecules of interest, it is not necessary to electrophoresis detection, and the complete stopped pipe type of whole process
Operation, pollution probability reduce, and avoid the false positive issue that Standard PCR is be easy to cause.Relatively conventional PCR, quantitative fluorescent PCR exist
Sensitiveness, specificity and speed etc. have advantage, but real-time fluorescence PCR technology is limited be subject to fluorescent species and instrument itself
System, can only at most be detected 5 targets, and the difficulty of Success in Experiment is very big.
The content of the invention
It is an object of the invention to provide a kind of quick multi-fluorescence immunoassay for distinguishing CAV, MDV, REV, IBDV to draw
Thing.
Another object of the present invention is to provide a kind of quick multi-fluorescence for distinguishing CAV, MDV, REV, IBDV to be immunized point
Analyse kit.
It is immunized point it is still another object of the present invention to provide a kind of quick multi-fluorescence for distinguishing CAV, MDV, REV, IBDV
Analysis method.
The technical solution used in the present invention is：
A kind of quick multi-fluorescence immunoassay primer for distinguishing CAV, MDV, REV, IBDV, the primer nucleotide sequences
It is as follows：
Primer C1：5’-CGACATCGGAGGAGACAG-3’ （SEQ ID NO：1）,
Primer C2：5’-GGAAGCGGATAGTCATAGTAGA-3’ （SEQ ID NO：2）；
Primer M1：5’-CCCATTCCCTCTTCTGCC-3’ （SEQ ID NO：3）,
Primer M2：5’-GCTGAGCGTAAACCGTC-3’ （SEQ ID NO：4）；
Primer R1：5’-GACTGCCTTGTGACTGCT-3’ （SEQ ID NO：5）,
Primer R2：5’-ACTCCCACTGTTGTCTAAATC-3’ （SEQ ID NO：6）；
Primer B1：5’-ATGCGGAGCCTTCTGA-3’ （SEQ ID NO：7）,
Primer B2：5’-ATTAGCCCTGACCCTGTG-3’ （SEQ ID NO：8）.
Further, the 5 ' ends of described wherein one primer of primer C1 and C2 are biotinylated；
5 ' the ends of described wherein one primer of primer M1 and M2 are biotinylated；
5 ' the ends of described wherein one primer of primer R1 and R2 are biotinylated；
5 ' the ends of described wherein one primer of primer B1 and B2 are biotinylated.
Further, the 5 ' of the primer not being biotinylated in the primer C1 and C2, M1 and M2, R1 and R2, B1 and B2
End is connected with tag sequences, and the tag sequences can be with the anti-tag sequence complementary pairings that are carried in fluorescence-encoded micro-beads.
Further, the tag sequences connected in this 4 groups of primer pairs of the primer C1 and C2, M1 and M2, R1 and R2, B1 and B2
Column selection is from SEQ ID NO:Tag sequences shown in 9 ~ 12, and the tag sequences connected in 4 groups of primer pairs are different.
A kind of quick multi-fluorescence immunoassay kits for distinguishing CAV, MDV, REV, IBDV, containing upper in the kit
State any primer.
Further, also containing streptavidin-phycoerythrin compound, the different fluorescence of 4 kinds of codings in mentioned reagent box
The fluorescence-encoded micro-beads of color.
Further, also containing the anti-tag sequences with tag sequences complementary pairing in primer in above-mentioned fluorescence-encoded micro-beads
Row, it is different to encode the anti-tag sequences contained in the fluorescence-encoded micro-beads of different iridescent.
A kind of quick multi-fluorescence immunoassay method for distinguishing CAV, MDV, REV, IBDV, includes the following steps：
1）Viral nucleic acid is extracted from sample；
2）Using the viral nucleic acid of extraction as template, RT-PCR amplifications are carried out with any of the above-described primer；
3）By upper step amplified production, the fluorescence-encoded micro-beads of the different iridescent of 4 kinds of codings, streptavidin-phycoerythrin
Hybridized；
4）After hybridization, analysis is detected hybrid product by Luminex systems, determines its viral type；
The above method is used for the diagnose and treat of non-disease.
Further, step 2）The reaction system of middle RT-PCR amplification is：
5×buffer 4µL
dNTP 0.8µL
2 μ L of primer mixed liquor
0.8 μ L of enzyme
1 μ L of template
ddH2O 11.4µL
Total 20µL；
Step 2）The response procedures of middle RT-PCR amplification are：50℃ 30min；94℃ 15min；94℃ 5min；94℃
30s, 60 DEG C of 25s, 72 DEG C of 20s；Circulation 35 times；72 DEG C of 10min, 4 DEG C of preservations.
Further, step 3）Described in the reaction system that hybridizes and program be：
4 kinds of 20 μ L of fluorescence-encoded micro-beads
75 μ L of streptavidin-phycoerythrin
5 μ L of amplified production
100 μ L of cumulative volume；37 DEG C of incubation 30min.
The beneficial effects of the invention are as follows：
1）The method of the present invention is at the same time to chicken infectious anemia virus, chicken Marek's disease virus, fowl reticular endothelium hyperplasia
When syndrome virus and infectious bursa of Fabricius virus are detected, by RT-PCR obtain target amplification fragment, then by amplified production,
Fluorescence-encoded micro-beads and streptavidin-phycoerythrin（SA-PE）Hybridized, when then reading MFI values by detector, point
Debate different types of cause of disease.
2）The method of the present invention can be at the same time to chicken infectious anemia virus, chicken Marek's disease virus, fowl reticular endothelium
Hyperplasia syndrome virus and infectious bursa of Fabricius virus are accurately detected, and high specificity, and high sensitivity is reproducible.With biography
System detection method is compared, and the method for the present invention is realized is carried out at the same time detection, sample to a variety of different molecules of interest in same sample
This dosage is few, easy to operate, quick, can substantially reduce testing cost.The present invention can guarantee that identical renaturation temperature and hybridization are imitated
Rate, and effectively avoid crisscrossing between the microballoon of different testing sample mark.
Brief description of the drawings
Fig. 1 is chicken infectious anemia virus, chicken Marek's disease virus, fowl Reticuloendotheliosis Virus and infectiousness
The electrophoretogram of the PCR of bursal disease virus and the multiple infection of manual simulation；
Fig. 2 is chicken infectious anemia virus, chicken Marek's disease virus, fowl Reticuloendotheliosis Virus and infectiousness
The multi-fluorescence immunoassay method test experience result figure of bursal disease virus；
Fig. 3 is chicken infectious anemia virus, chicken Marek's disease virus, fowl Reticuloendotheliosis Virus and infectiousness
The multi-fluorescence immunoassay method detection specificity experiments result figure of bursal disease virus；
Fig. 4 is chicken infectious anemia virus, chicken Marek's disease virus, fowl Reticuloendotheliosis Virus and infectiousness
The multi-fluorescence immunoassay method detection sensitivity experimental result picture of bursal disease virus；
Fig. 5 is chicken infectious anemia virus, chicken Marek's disease virus, fowl Reticuloendotheliosis Virus and infectiousness
The multi-fluorescence immunoassay method clinical detection experimental result picture of bursal disease virus.
Embodiment
A kind of quick multi-fluorescence immunoassay primer for distinguishing CAV, MDV, REV, IBDV, the primer nucleotide sequences
It is as follows：
Primer C1：5’-CGACATCGGAGGAGACAG-3’ （SEQ ID NO：1）,
Primer C2：5’-GGAAGCGGATAGTCATAGTAGA-3’ （SEQ ID NO：2）；
Primer M1：5’-CCCATTCCCTCTTCTGCC-3’ （SEQ ID NO：3）,
Primer M2：5’-GCTGAGCGTAAACCGTC-3’ （SEQ ID NO：4）；
Primer R1：5’-GACTGCCTTGTGACTGCT-3’ （SEQ ID NO：5）,
Primer R2：5’-ACTCCCACTGTTGTCTAAATC-3’ （SEQ ID NO：6）；
Primer B1：5’-ATGCGGAGCCTTCTGA-3’ （SEQ ID NO：7）,
Primer B2：5’-ATTAGCCCTGACCCTGTG-3’ （SEQ ID NO：8）.
Preferably, the 5 ' ends of described wherein one primer of primer C1 and C2 are biotinylated；
5 ' the ends of described wherein one primer of primer M1 and M2 are biotinylated；
5 ' the ends of described wherein one primer of primer R1 and R2 are biotinylated；
5 ' the ends of described wherein one primer of primer B1 and B2 are biotinylated.
Preferably, 5 ' ends of the primer not being biotinylated in the primer C1 and C2, M1 and M2, R1 and R2, B1 and B2
It is connected with tag sequences, the tag sequences can be with the anti-tag sequence complementary pairings that are carried in fluorescence-encoded micro-beads.
Preferably, the tag sequences connected in this 4 groups of primer pairs of the primer C1 and C2, M1 and M2, R1 and R2, B1 and B2
Selected from SEQ ID NO:Tag sequences shown in 9 ~ 12, and the tag sequences connected in 4 groups of primer pairs are different.
A kind of quick multi-fluorescence immunoassay kits for distinguishing CAV, MDV, REV, IBDV, containing upper in the kit
State any primer.
Preferably, also containing streptavidin-phycoerythrin compound, the different iridescent of 4 kinds of codings in mentioned reagent box
Fluorescence-encoded micro-beads.
Preferably, the anti-tag sequences with tag sequences complementary pairing in primer are also contained in above-mentioned fluorescence-encoded micro-beads,
It is different to encode the anti-tag sequences contained in the fluorescence-encoded micro-beads of different iridescent.
A kind of quick multi-fluorescence immunoassay method for distinguishing CAV, MDV, REV, IBDV, includes the following steps：
1）Viral nucleic acid is extracted from sample；
2）Using the viral nucleic acid of extraction as template, RT-PCR amplifications are carried out with any of the above-described primer；
3）By upper step amplified production, the fluorescence-encoded micro-beads of the different iridescent of 4 kinds of codings, streptavidin-phycoerythrin
Hybridized；
4）After hybridization, analysis is detected hybrid product by Luminex systems, determines its viral type；
The above method is used for the diagnose and treat of non-disease.
Preferably, step 2）The reaction system of middle RT-PCR amplification is：
5×buffer 4µL
dNTP 0.8µL
2 μ L of primer mixed liquor
0.8 μ L of enzyme
1 μ L of template
ddH2O 11.4µL
Total 20µL；
Step 2）The response procedures of middle PCR amplification are：50℃ 30min；94℃ 15min；94℃ 5min；94 DEG C of 30s,
60 DEG C of 25s, 72 DEG C of 20s；Circulation 35 times；72 DEG C of 10min, 4 DEG C of preservations.
Preferably, step 3）Described in the reaction system that hybridizes and program be：
4 kinds of 20 μ L of fluorescence-encoded micro-beads
75 μ L of streptavidin-phycoerythrin
5 μ L of amplified production
100 μ L of cumulative volume；37 DEG C of incubation 30min.
Preferably, step 3）In 4 kinds of different iridescent of coding fluorescence-encoded micro-beads in also contain and tag sequences in primer
The anti-tag sequences of complementary pairing, the anti-tag sequences contained in 4 kinds of fluorescence-encoded micro-beads are different.
With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.
1 design of primers of embodiment
After being screened to designed a large amount of primers, find primer pair C1 and C2, M1 and M2, R1 and R2, B1 and
The effect that B2 detects multi-fluorescence CAV, MDV, REV, IBDV at the same time is best, its base sequence is as follows.
Primer C1：5’-CGACATCGGAGGAGACAG-3’ （SEQ ID NO：1）,
Primer C2：5’-GGAAGCGGATAGTCATAGTAGA-3’ （SEQ ID NO：2）；
Primer M1：5’-CCCATTCCCTCTTCTGCC-3’ （SEQ ID NO：3）,
Primer M2：5’-GCTGAGCGTAAACCGTC-3’ （SEQ ID NO：4）；
Primer R1：5’-GACTGCCTTGTGACTGCT-3’ （SEQ ID NO：5）,
Primer R2：5’-ACTCCCACTGTTGTCTAAATC-3’ （SEQ ID NO：6）；
Primer B1：5’-ATGCGGAGCCTTCTGA-3’ （SEQ ID NO：7）,
Primer B2：5’-ATTAGCCCTGACCCTGTG-3’ （SEQ ID NO：8）.
The present invention distinguishes CAV, MDV, REV, IBDV using the method for multi-fluorescence analysis, therefore above-mentioned primer is made
Further modification, to meet that corresponding operation requires.5 ' the ends of wherein primer C1, M1, R1 and B1 are connected with tag sequences, institute
The tag sequences of connection are respectively：
The tag sequences of primer C1 are：5’-ATACTTTACAAACAAATAACACAC-3’ （SEQ ID NO：9）；
The tag sequences of primer M1 are：5’-TACTTAAACATACAAACTTACTCA-3’ （SEQ ID NO：10）；
The tag sequences of primer R1 are：5’-CTTAAACTCTACTTACTTCTAATT-3’ （SEQ ID NO：11）；
The tag sequences of primer B1 are：5’-TACTTCTTTACTACAATTTACAAC-3’ （SEQ ID NO：12）；
In addition, 5 ' the ends of primer C2, M2, R2 and B2 are also added with biotin labeling.
Embodiment 2 is used to quickly distinguish chicken infectious anemia virus, chicken Marek's disease virus, the increasing of fowl reticular endothelium
The multi-fluorescence immunoassay kits of raw syndrome virus and infectious bursa of Fabricius virus
Kit includes following components：
（1）Multi-fluorescence immunoassay primer sets designed by embodiment 1；
（2）The fluorescence-encoded micro-beads for including anti-tag sequences of the different iridescent of 4 kinds of codings, the anti-tag sequences
Row can correspondingly analyze the tag sequence complementary pairings in primer with multi-fluorescence；Four kinds of microballoons are purchased from luminex companies, its
The corresponding fluorescence-encoded micro-beads number of middle CAV, MDV, REV and IBDV for MTAG-A019, MTAG-A065, MTAG-A056 and
MTAG-015。
（3）Streptavidin-phycoerythrin compound.
Embodiment 3 quickly distinguishes chicken infectious anemia virus, chicken Marek's disease virus, fowl reticuloendotheliosis
The foundation of virus and the multi-fluorescence immunoassay method detection method of infectious bursa of Fabricius virus
（1）The structure of CAV, MDV, REV and IBDV plasmid
The nucleic acid that instrument extracts CAV, MDV, REV, IBDV cause of disease respectively is extracted automatically with the nucleic acid of Tiangeng（RNA/DNA）, point
Primer pair C1 and C2, M1 and M2, R1 and R2, B1 and B2 do not carry out RT-PCR amplifications designed by embodiment 1, by amplified production point
Do not detected into row agarose gel electrophoresis and cut glue purification.CDNA after purification is connected to the kit of TaKaRa companies
In pMD-19T carriers, connection product is converted to DH5a competent cells, selects monoclonal, bacterium colony PCR identifications is carried out, will reflect
The bacterium colony for being set to positive bacteria carries out plasmid extraction, send sequencing.
（2）Plasmid PCR expands
Designed by embodiment 1 primer pair C1 and C2, M1 and M2, R1 and R2, B1 and B2 respectively to CAV, MDV, REV,
IBDV primers carry out substance, Quadruple- PCR amplification.
The preparation of sense primer mixed liquor：By C1, M1, R1 and B1 with molar ratio 1:1:1:1 ratio is mixed；Draw in downstream
The preparation of thing mixed liquor：By C2, M2, R2 and B2 with molar ratio 1:1:1:1 ratio is mixed.Utilize four kinds of diseases of fowl immunosupress
Former specific template, and CAV, MDV, REV, IBDV quadruple template, the idiocrasy region viral to above-mentioned four kinds are expanded
Increase.The wherein preparation of quadruple template：By four kinds of plasmids with volume ratio 1:1:1:1 ratio is mixed.
PCR amplification answers system as follows：
Premix Ex Taq 10µL
1 μ L of sense primer mixed liquor
1 μ L of anti-sense primer mixed liquor
1 μ L of template
ddH2O 7µL
Total 20µL；
Wherein final concentration of 2 μM of upstream and downstream primer.
The response procedures of amplification are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 25s, 72 DEG C of extension 20s；
Circulation 35 times；72 DEG C extend 10min eventually.
Pcr amplification product is analyzed into row agarose gel electrophoresis, its electrophoretogram is as shown in Figure 1.In Fig. 1, M：DL2000
DNA marker, 1：CAV, 2：MDV, 3：REV, 4：IBDV, 5：The Quadruple- PCR carried out to CAV+MDV+REV+IBDV, 6：PCR
blank（PCR blank controls）.
From figure 1 it appears that the amplified production size of CAV is about 121bp, the amplified production size of MDV is about
The amplified production size of 114bp, REV are about 153bp, and the amplified production size of IBDV is about 128bp.Due to these four viruses
Amplified production size is close, so the electrophoretic band of Quadruple- PCR amplified production can not be differentiated.
（3）By gained PCR product and fluorescence-encoded micro-beads working solution, Streptavidin phycoerythrin（SA-PE）Working solution
Hybridization, comprises the following steps：
Be coated with 4 kinds of microballoons of special anti-tag sequences respectively, wherein anti-tag sequences can correspondingly with CAV,
Tag sequence complementary pairings on the viral primers of tetra- kinds of MDV, REV and IBDV.4 kinds of microballoons are purchased from luminex companies, specifically
The corresponding fluorescence-encoded micro-beads number of CAV, MDV, REV and IBDV for MTAG-A019, MTAG-A065, MTAG-A056 and
MTAG-015。
The preparation of fluorescence-encoded micro-beads working solution：By 2500/μ l fluorescence-encoded micro-beads with 1.1 × Tm
Hybrdization Buffer are diluted to 1 μ l and about contain 125/kind fluorescence-encoded micro-beads.
It is prepared by SA-PE working solutions：1mg/ml SA-PE are diluted to 10 μ g/ μ with 1 × Tm Hybrdization Buffer
l。
Fluorescence-encoded micro-beads working solution is fully resuspended, each sample well and background hole add 20 μ l of microballoon working solution, sample
5 μ l PCR products are added in hole, 5 μ l PCR blank products are added in background hole, the SA-PE working solutions of 75 μ l is added, fills
Divide and mix, 37 DEG C of incubation 30min in metal heater.
The 50 above-mentioned reaction solutions of μ l after hybridization are detected by the explanation according to 200 instruments of detector Luminex, as a result
As shown in Fig. 2, although the amplified production of quadruple can not be differentiated with electrophoresis, MFI values are read with 200 instruments of detector Luminex
When, hence it is evident that offer an explanation different types of virus.
As a result criterion（Note：The criterion is only for reference, and also result criterion can be adjusted）It is as follows：
Lowest detection threshold value（Cutoff values）Determine：Choose 10 healthy chicken tissue samples（The parallel repetition 3 of each sample
It is secondary）, MFI values are read respectively and calculate its average value and standard deviation.Using the MIF values of average value plus 3 times of standard deviations set its as
Cutoff values.It is 482.3 that the present invention, which obtains cutoff values, therefore the cutoff values of the present invention are set to 500.Only detect sample
When the MFI values of product are higher than 500, which could effectively be analyzed.
The analysis of sample to be tested judges：1）As the MFI value ＞ 500 of sample to be tested, it is judged as positive sample；2）Treat test sample
During this MFI values≤500, it is judged as negative, it is necessary to carry out repeatedly experiment or take other detection methods further to verify.
The multi-fluorescence immunoassay detection specificity experiments of embodiment 4 CAV, MDV, REV and IBDV
Infected respectively with chicken infectious anemia virus, chicken Marek's disease virus, fowl Reticuloendotheliosis Virus, chicken
Property bursal disease virus, Avianreovirus, infectious laryngotracheitis virus, infectious bronchitis virus, avian influenza virus and
Newcastle Disease Virus carries out multi-fluorescence immunoassay detection as template.
1）Viral nucleic acid is extracted from sample；
2）Using the viral nucleic acid of extraction as template, primer pair C1 and C2, M1 and M2, R1 and R2, B1 for being designed with embodiment 1
RT-PCR amplifications are carried out with B2 primers；
RT-PCR amplification reaction system be：
5×buffer 4µL
dNTP 0.8µL
2 μ L of sense primer mixed liquor
0.8 μ L of enzyme
1 μ L of template
ddH2O 11.4µL
Total 20µL；
Step 2）The response procedures of middle RT-PCR amplification are：50℃ 30min；94℃ 15min；94℃ 5min；94℃
30s, 60 DEG C of 25s, 72 DEG C of 20s；Circulation 35 times；72 DEG C of 10min, 4 DEG C of preservations.
3）By upper step amplified production, the fluorescence-encoded micro-beads of the different iridescent of 4 kinds of codings, streptavidin-phycoerythrin
Hybridized；
The reaction system and program of hybridization be：
4 kinds of 20 μ L of fluorescence-encoded micro-beads
75 μ L of streptavidin-phycoerythrin
5 μ L of amplified production
100 μ L of cumulative volume；37 DEG C of incubation 30min.
4）After hybridization, analysis is detected hybrid product by Luminex systems, determines its viral type.
Experimental result as shown in figure 3, only chicken infectious anemia virus, chicken Marek's disease virus, fowl reticular endothelium
Hyperplasia syndrome virus, infections chicken cloacal bursa virus are the positive, and others are feminine gender, illustrate that detection architecture specificity is good.
Embodiment 5：The multi-fluorescence immunoassay detection sensitivity experiment of CAV, MDV, REV, REO and IBDV
The plasmid of preparation is quantified, is diluted using 10 times of dilution methods, is diluted to 101Copies/ μ l, use are above-mentioned
The multi-fluorescence immunoassay method of foundation is detected.The multi-fluorescence immunoassay detection spirit of CAV, MDV, REV and IBDV
Quick property experimental result as shown in figure 4, test result indicates that, except IBDV sensitivity detection be limited to 104Copies/ul, other
Sensitivity detection be limited to 103copies/ul。
Embodiment 6：The detection of sample
The samples such as spleen, kidney, liver from chicken house collection, extract instrument extraction RNA/DNA with Tiangeng nucleic acid, use Qiagen automatically
RT-PCR kit expanded, using RNA/DNA as template, the multi-fluorescence using above-mentioned CAV, MDV, REV and IBDV is exempted from
Epidemic disease analyzing detecting method is detected, and amplified production hybridizes with fluorescence-encoded micro-beads and SA-PE, in 200 detectors of Luminex
Upper reading.For specific steps with reference to embodiment 3 or embodiment 4, experimental result is as shown in Figure 5.
The testing result of the method for the present invention shows that sample 4F, 4S, 4XW, 4W and 46 are negative sample；12 be infectiousness method
Family name's capsule viral sample；26 be chicken infectious anemia virus and the double mixed infection sample of fowl Reticuloendotheliosis Virus；11
For chicken infectious anemia virus, Marek's disease poison and the triple mixed infection samples of fowl Reticuloendotheliosis Virus；14、34、
22nd, 43,32,24,23 be chicken infectious anemia virus and the double mixed infection sample of Marek's disease poison；Others are chicken biography
Metachromia Anaemia Virus individually infects sample.By sequencing analysis, as a result unanimously.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention and from above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Experimental Animals Supervising Station, Guangdong Prov.
<120>A kind of quick multi-fluorescence immunoassay primer, kit and side for distinguishing CAV, MDV, REV, IBDV
Method
<130>
<160> 12
<170> PatentIn version 3.5
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cccattccct cttctgcc 18
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Claims (9)
1. a kind of quick multi-fluorescence immunoassay primer for distinguishing CAV, MDV, REV, IBDV, the primer nucleotide sequences are such as
Shown in lower：
Primer C1：5’-CGACATCGGAGGAGACAG-3’(SEQ ID NO：1),
Primer C2：5’-GGAAGCGGATAGTCATAGTAGA-3’(SEQ ID NO：2)；
Primer M1：5’-CCCATTCCCTCTTCTGCC-3’(SEQ ID NO：3),
Primer M2：5’-GCTGAGCGTAAACCGTC-3’(SEQ ID NO：4)；
Primer R1：5’-GACTGCCTTGTGACTGCT-3’(SEQ ID NO：5),
Primer R2：5’-ACTCCCACTGTTGTCTAAATC-3’(SEQ ID NO：6)；
Primer B1：5’-ATGCGGAGCCTTCTGA-3’(SEQ ID NO：7),
Primer B2：5’-ATTAGCCCTGACCCTGTG-3’(SEQ ID NO：8)；
The tag sequences connected in this 4 groups of primer pairs of the primer C1 and C2, M1 and M2, R1 and R2, B1 and B2 are selected from SEQ ID
NO:Tag sequences shown in 9~12, and the tag sequences connected in 4 groups of primer pairs are different.
2. primer according to claim 1, it is characterised in that：
5 ' the ends of described wherein one primer of primer C1 and C2 are biotinylated；
5 ' the ends of described wherein one primer of primer M1 and M2 are biotinylated；
5 ' the ends of described wherein one primer of primer R1 and R2 are biotinylated；
5 ' the ends of described wherein one primer of primer B1 and B2 are biotinylated.
3. the primer according to profit requires 1, it is characterised in that in the primer C1 and C2, M1 and M2, R1 and R2, B1 and B2
5 ' ends of the primer not being biotinylated are connected with tag sequences, and the tag sequences can be with carrying in fluorescence-encoded micro-beads
Anti-tag sequence complementary pairings.
A kind of 4. quick multi-fluorescence immunoassay kits for distinguishing CAV, MDV, REV, IBDV, it is characterised in that the reagent
Contain any primer of claims 1 to 3 in box.
5. kit according to claim 4, it is characterised in that also contain Streptavidin-algae red egg in the kit
The fluorescence-encoded micro-beads of white compound, the different iridescent of 4 kinds of codings.
6. kit according to claim 4, it is characterised in that in the fluorescence-encoded micro-beads also contain with primer
The anti-tag sequences of tag sequence complementary pairings, encode the anti-tag sequences contained in the fluorescence-encoded micro-beads of different iridescent
Arrange different.
7. a kind of quick multi-fluorescence immunoassay method for distinguishing CAV, MDV, REV, IBDV, it is characterised in that including as follows
Step：
1) viral nucleic acid is extracted from sample；
2) using the viral nucleic acid of extraction as template, RT-PCR amplifications are carried out with any primer of claims 1 to 3；
3) upper step amplified production, the fluorescence-encoded micro-beads of the different iridescent of 4 kinds of codings, streptavidin-phycoerythrin are carried out
Hybridization；
4) after hybridizing, analysis is detected hybrid product by Luminex systems, determines its viral type；
The above method is used for the diagnose and treat of non-disease.
8. according to the method described in claim 7, it is characterized in that：The reaction system of RT-PCR amplifications is in step 2)：
The response procedures of RT-PCR amplifications are in step 2)：50℃30min；94℃15min；94℃5min；94 DEG C of 30s, 60 DEG C
25s, 72 DEG C of 20s；Circulation 35 times；72 DEG C of 10min, 4 DEG C of preservations.
9. according to the method described in claim 7, it is characterized in that：The reaction system and program hybridized described in step 3) be：
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| CN110885899B (en) * | 2018-09-10 | 2023-04-14 | 中国动物疫病预防控制中心（农业部屠宰技术中心） | Freeze-drying microchip, kit and method for identifying 16 avian disease pathogens |
| CN110699485B (en) * | 2019-08-16 | 2023-03-28 | 广州千寻生物技术有限公司 | RPA primer pair, probe, kit and detection method for rapidly detecting Marek's disease virus |
| CN110564896A (en) * | 2019-09-26 | 2019-12-13 | 广西壮族自治区兽医研究所 | Primer group for identifying avian adenovirus type 4 and chicken infectious anemia viruses and application thereof |
| CN113897461B (en) * | 2021-11-18 | 2023-12-26 | 内蒙古自治区农牧业科学院 | ALV, REV, IBDV, CIAV, ARV and GHV six-fold PCR detection primer set and kit |
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