CN106191319B - A kind of multi-fluorescence immunoassay method of 6 kinds of fowl respiratory pathogens of quick differentiation - Google Patents
A kind of multi-fluorescence immunoassay method of 6 kinds of fowl respiratory pathogens of quick differentiation Download PDFInfo
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Abstract
The invention discloses a kind of multi-fluorescence immunoassay methods of quick 6 kinds of fowl respiratory pathogens of differentiation.The present invention is easy to operate, obtains target amplification segment by PCR, then hybridizes amplified production, fluorescence-encoded micro-beads and Streptavidin phycoerythrin, then reads MFI values by detector, offer an explanation different types of virus.The method of the present invention, avian influenza virus, avian infectious bronchitis virus, newcastle disease virus, avian infectious laryngotracheitis virus, chicken virus mycoplasma and chicken Mycoplasma synoviae can accurately be detected simultaneously, 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.
Description
Technical field
The invention belongs to the field of virus detection of aquaculture, and in particular to a kind of quick 6 kinds of fowl respiratory pathogens of differentiation
Multi-fluorescence immunoassay method.
Background technology
As demand of the market to birds is on the increase, aviculture obtains swift and violent development, and aviculture intensive degree is not
Disconnected to improve, the infection of fowl respiratory pathogens is in ascendant trend year by year.The breathing problem of fowl is always to lead to the great warp of aviculture
The principal element of Ji loss.The cause of disease of chicken respiratory tract disease has its complexity, it can have virus, mycoplasma and bacterium to cause hair
Disease clinically there is also various mixed infections.The generation of these diseases can not be ignored in poultry produces.The respiratory tract of chicken
Disease often occurs, and the chicken of various ages in days, which can infect, even results in death, and Adult Chicken, which is laid eggs, to be declined, while the respiratory tract of chicken
Disease incidence is high, easily causes the secondary infection of a variety of diseases, such as mycoplasma can induce the gentle capsulitis of pneumonia of chicken, so
Active prevention and timely treatment breathing problem have great meaning.
Avian influenza virus, avian infectious bronchitis virus, newcastle disease virus, avian infectious laryngotracheitis virus, chicken
Virus mycoplasma and chicken Mycoplasma synoviae are important fowl respiratory pathogens, and clinical symptoms and pathological change are very much like, are facing
It is difficult to distinguish on bed and histopathology.Chicken respiratory tract disease is mostly mixed infection, clinically be easy to cause mistaken diagnosis, is differentiated
Diagnosis usually need to by laboratory diagnostic technique, viral separation and identification, serological test, enzyme-linked immunosorbent assay and
Diagnosis of molecular biology.Traditional detection method sensibility is low, poor specificity, is not easy to detect mixed infection, and operation side
Method is cumbersome, is unfavorable for the timely diagnoses and treatment of disease, causes great economic loss.
With the fast development of molecular biology, multiplex PCR is widely used to the antidiastole of poultry diease mixed infection,
Principle is the multipair primer that design can amplify a variety of cause of disease specific fragment sizes to be measured simultaneously, is added in PCR reaction systems
Enter multipair primer, antidiastole can be carried out to a variety of cause of diseases.Multiple RT-PCR is simple because its sensitivity, specificity and operation
Property is widely used in the mixing sense of poultry diease, but result judgement needs electrophoresis, time-consuming and laborious, and reaction product easily generates pollution
And lead to false positive, and multiplex PCR is distinguished by the size of segment, has generally been arrived more than triple, clip size difference is big,
The amplification efficiency of its each virus is different, and causing result, there are deviations.Fluorescent quantitative PCR technique has merged a variety of excellent of PCR
Point is realized by the variation for directly detecting fluorescence signal in PCR reaction process and molecules of interest is quantified, and does not need to electrophoresis inspection
It surveys, and the complete stopped pipe type operation of whole process, pollution probability reduce, and avoid the false positive issue that Standard PCR is be easy to cause.
Relatively conventional PCR, quantitative fluorescent PCR have advantage, but real-time fluorescence PCR technology in sensibility, specificity and speed etc.
It is limited by fluorescent species and instrument itself, 5 targets can only be at most detected, and the difficulty of Success in Experiment is very big.
Invention content
In order to solve the deficiency in the presence of the prior art, it is an object of the present invention to provide one kind to be used for quick area
Divide the multi-fluorescence immunoassay primer sets of 6 kinds of fowl respiratory pathogens；
It is immunized point it is another object of the present invention to provide a kind of multi-fluorescence of quick 6 kinds of fowl respiratory pathogens of differentiation
Analyse kit；
It is immunized point it is another object of the present invention to provide a kind of multi-fluorescence of quick 6 kinds of fowl respiratory pathogens of differentiation
Analysis method.
The technical solution used in the present invention is：
A kind of multi-fluorescence immunoassay primer sets for quickly 6 kinds of fowl respiratory pathogens of differentiation, including：For examining
Survey avian influenza virus（AIV）Primer pair A1 and A2, for detecting avian infectious bronchitis virus（IBV）Primer pair B1 and
B2, for detecting newcastle disease virus（NDV）Primer pair N1 and N2, for detecting avian infectious laryngotracheitis virus（ILTV）
Primer pair L1 and L2, for detecting chicken virus mycoplasma（MG）Primer pair G1 and G2, for detecting chicken Mycoplasma synoviae
（MS）Primer pair S1 and S2, described for detecting the primer pair of avian influenza virus is the guarantor according to the M genes of avian influenza virus
Keep sequence design；The primer pair for being used to detect avian infectious bronchitis virus is according to infectious bronchitis
The N genes conserved sequence design of poison；The primer pair for being used to detect newcastle disease virus is according to newcastle disease virus
The conserved sequence design of F genes；The primer pair for being used to detect avian infectious laryngotracheitis virus is according to avian infectious
The conserved sequence design of the gD genes of laryngotracheitis virus；The primer pair for being used to detect chicken virus mycoplasma is according to chicken poison
The conserved sequence design of the PVPA genes of mycoplasma；The primer pair for being used to detect chicken Mycoplasma synoviae is slided according to chicken
The conserved sequence design of the vlhA genes of liquid capsule mycoplasma.
It is as preference, described as follows for the nucleotide sequence of primer pair that detects avian influenza virus：
A1：5 '-ATGAGTCTTCTACCCGAGG-3 ',
A2：5’-TCAGAGGTGACAGGATTG-3’；
It is as follows for detecting the nucleotide sequence of the primer pair of avian infectious bronchitis virus：
B1：5 '-AATCACGCTCAAGTTCAAGGC-3 ',
B2：5’-GCATTGTTCCTCTCCTCATC-3’；
It is as follows for detecting the nucleotide sequence of the primer pair of newcastle disease virus：
N1：5 '-CTCATCTCAGACAGGGTCAATC-3 ',
N2：5’-ATGGAGTCACCAAGGGG-3’；
It is as follows for detecting the nucleotide sequence of the primer pair of avian infectious laryngotracheitis virus：
L1：5 '-GCAGCGAAAAGAAGGC-3 ',
L2：5’-TCATCGTGCTCGGTGTCC-3’；
It is as follows for detecting the nucleotide sequence of the primer pair of chicken virus mycoplasma：
G1：5 '-CTTACAGATTACAGGAGCAG-3 ',
G2：5’-AAGCATTTCATTTGTTTTAC-3’；
It is as follows in the nucleotide sequence of the primer pair of detection chicken Mycoplasma synoviae：
S1：5 '-ATTAGCAGCTAGTGCAGTGG-3 ',
S2：5’-TTTGAGGATTATCAGTATTTG-3’.
5 ' the ends of primer A1, B1, N1, L1, G1 and S1 are also added with tag sequences by spacer sequence.
Primer A1, B1, N1, L1, G1 and S1 5 ' end tag sequences be respectively：
The tag sequences of primer A1 are：5’-TACTACTTCTATAACTCACTTAAA-3’；
The tag sequences of primer B1 are：5’-ATTAAACAACTCTTAACTACACAA-3’；
The tag sequences of primer N1 are：5’-ACTTATTTCTTCACTACTATATCA-3’；
The tag sequences of primer L1 are：5’-ATCTCAATTACAATAACACACAAA-3’；
The tag sequences of primer G1 are：5’-CACTACACATTTATCATAACAAAT-3’；
The tag sequences of primer S1 are：5’-CTTTCTTAATACATTACAACATAC-3’.
5 ' the ends of primer A2, B2, N2, L2, G2 and S2 are also added with biotin labeling.
A kind of multi-fluorescence immunoassay kits for quickly 6 kinds of fowl respiratory pathogens of differentiation, which is characterized in that institute
Kit is stated to include：（1）Above-described multi-fluorescence immunoassay primer sets；（2）6 kinds encode including for different iridescent
Have a fluorescence-encoded micro-beads of anti-tag sequences, the anti-tag sequences can correspondingly with multi-fluorescence immunoassay primer sets
Tag sequence complementary pairings on sense primer；（3）Streptavidin-phycoerythrin compound.
A kind of multi-fluorescence immunoassay method of 6 kinds of fowl respiratory pathogens of quick differentiation, includes the following steps：
1）Viral RNA/DNA is extracted from sample, using RNA/DNA as template, adds in the primer sets described in any of the above item
Carry out RT-PCR amplifications；
2）By amplified production, the fluorescence-encoded micro-beads and chain that include anti-tag sequences of the different iridescent of 6 kinds of codings
Mould Avidin-phycoerythrin hybridization；
3）It after hybridization, is analyzed using instrument, determines the type of its virus.
Step 2）In fluorescence-encoded micro-beads on be coated with special anti-tag sequences, the anti-tag sequences energy phase
It should ground and the tag sequence complementary pairings in sense primer.
The beneficial effects of the invention are as follows：
The tag sequences of specificity that the sense primer 5 ' of the present invention is held, can be with being coated with special anti-tag sequences
6 kinds of corresponding combinations of fluorescence-encoded micro-beads, and downstream primer 5 ' hold Biotin label, can be with Streptavidin-algae
Lactoferrin（SA-PE）Hybridized.The method of the present invention is simultaneously to avian influenza virus, avian infectious bronchitis virus, chicken new city
When epidemic disease poison, avian infectious laryngotracheitis virus, chicken virus mycoplasma and chicken Mycoplasma synoviae are detected, obtained by PCR
Target amplification segment, then by amplified production, fluorescence-encoded micro-beads and streptavidin-phycoerythrin（SA-PE）Hybridized,
When then reading MFI values by detector, different types of cause of disease is offered an explanation.
The method of the present invention is by multi-fluorescence immunoassay（MFIA）Combine with TAG technologies, TAG technologies use
By sequence label and the specificity complementary pairing of anti-sequence label, it is excellent to carry out nucleic acid experiment for universal tag proprietary Luminex
Change, product development and molecular diagnostic assay.TAG technologies can guarantee identical renaturation temperature and hybridization efficiency, and effectively avoid not
With crisscrossing between the microballoon of detection substance markers.
The method of the present invention, can be simultaneously to avian influenza virus, avian infectious bronchitis virus, newcastle disease virus, chicken
Infectious laryngotracheitis virus, chicken virus mycoplasma and chicken Mycoplasma synoviae are accurately detected, and high specificity, sensitivity
Height, it is reproducible.Compared with traditional detection method, the method for the present invention is realized to a variety of different molecules of interest in same sample
Detection is carried out at the same time, sample dosage is few, easy to operate, quick, can substantially reduce testing cost.
Description of the drawings
Fig. 1 is the electrophoretogram of 6 kinds of cause of disease RT-PCR of fowl respiratory tract；
Fig. 2 is the RT-PCR electrophoretograms of the multiple infection of 6 kinds of cause of disease manual simulations of fowl respiratory tract；
Fig. 3 is the multi-fluorescence immunoassay method test experience result figure of 6 kinds of cause of diseases of fowl respiratory tract；
Fig. 4 is the multi-fluorescence immunoassay method detection specificity experiments result figure of 6 kinds of cause of diseases of fowl respiratory tract；
Fig. 5 is the multi-fluorescence immunoassay method detection sensitivity experimental result picture of 6 kinds of cause of diseases of fowl respiratory tract；
Fig. 6 is the multi-fluorescence immunoassay method clinical detection experimental result picture of 6 kinds of cause of diseases of fowl respiratory tract.
Specific embodiment
With reference to embodiment, the invention will be further described, and however, it is not limited to this.
1 design of primers of embodiment
One group is designed for quickly distinguishing avian influenza virus, avian infectious bronchitis virus, newcastle disease virus, chicken biography
The multi-fluorescence immunoassay primer sets of metachromia laryngotracheitis virus, chicken virus mycoplasma and chicken Mycoplasma synoviae, wherein, it is described
Primer pair for detecting avian influenza virus is designed according to the conserved sequence of the M genes of avian influenza virus；It is described to be used to examine
The primer pair for surveying avian infectious bronchitis virus is designed according to the N genes conserved sequence of avian infectious bronchitis virus
's；The primer pair for being used to detect newcastle disease virus is designed according to the conserved sequence of the F genes of newcastle disease virus
's；Described for the primer pair that detects avian infectious laryngotracheitis virus is gD genes according to avian infectious laryngotracheitis virus
Conserved sequence design；Described for the primer pair that detects chicken virus mycoplasma is guarantor according to the PVPA genes of chicken virus mycoplasma
Keep sequence design；Described for the primer pair that detects chicken Mycoplasma synoviae is vlhA genes according to chicken Mycoplasma synoviae
Conserved sequence design.By repetition test with after screening, obtaining following primer sequence：
It is as follows for detecting the nucleotide sequence of the primer pair of avian influenza virus：
A1：5’-ATGAGTCTTCTACCCGAGG-3’（SEQ ID NO.1）,
A2：5’-TCAGAGGTGACAGGATTG-3’（SEQ ID NO.2）；
It is as follows for detecting the nucleotide sequence of the primer pair of avian infectious bronchitis virus：
B1：5’-AATCACGCTCAAGTTCAAGGC-3’（SEQ ID NO.3）,
B2：5’-GCATTGTTCCTCTCCTCATC-3’（SEQ ID NO.4）；
It is as follows for detecting the nucleotide sequence of the primer pair of newcastle disease virus：
N1：5’-CTCATCTCAGACAGGGTCAATC-3’（SEQ ID NO.5）,
N2：5’-ATGGAGTCACCAAGGGG-3’（SEQ ID NO.6）；
It is as follows for detecting the nucleotide sequence of the primer pair of avian infectious laryngotracheitis virus：
L1：5’-GCAGCGAAAAGAAGGC-3’（SEQ ID NO.7）,
L2：5’-TCATCGTGCTCGGTGTCC-3’（SEQ ID NO.8）；
It is as follows for detecting the nucleotide sequence of the primer pair of chicken virus mycoplasma：
G1：5’-CTTACAGATTACAGGAGCAG-3’（SEQ ID NO.9）,
G2：5’-AAGCATTTCATTTGTTTTAC-3’（SEQ ID NO.10）；
It is as follows for detecting the nucleotide sequence of the primer pair of chicken Mycoplasma synoviae：
S1：5’-ATTAGCAGCTAGTGCAGTGG-3’（SEQ ID NO.11）,
S2：5’-TTTGAGGATTATCAGTATTTG-3’（SEQ ID NO.12）.
Tag sequences, tag sequences difference are added by spacer sequence at the 5 ' ends of primer A1, B1, N1, L1, G1 and S1
For：
The tag sequences of primer A1 are：TACTACTTCTATAACTCACTTAAA（SEQ ID NO.13）；
The tag sequences of primer B1 are：ATTAAACAACTCTTAACTACACAA（SEQ ID NO.14）；
The tag sequences of primer N1 are：ACTTATTTCTTCACTACTATATCA（SEQ ID NO.15）；
The tag sequences of primer L1 are：ATCTCAATTACAATAACACACAAA（SEQ ID NO.16）；
The tag sequences of primer G1 are：CACTACACATTTATCATAACAAAT（SEQ ID NO.17）；
The tag sequences of primer S1 are：CTTTCTTAATACATTACAACATAC（SEQ ID NO.18）.
5 ' the ends of primer A2, B2, N2, L2, G2 and S2 are added with biotin labeling.
Embodiment 2 is used to quickly distinguish the foundation of the multi-fluorescence immunoassay kits of 6 kinds of fowl respiratory pathogens
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 6 kinds of codings, the anti-tag sequences
Row can correspondingly with the tag sequence complementary pairings on multi-fluorescence immunoassay primer sets sense primer；Six kinds of microballoons are purchased from
Luminex companies, the corresponding fluorescence-encoded micro-beads number of specific AIV, NDV, IBV, ILT, MS and MG are MTAG-A029,
MTAG-A034, MTAG-A036, MTAG-A067, MTAG-A025 and MTAG-042.
（3）Streptavidin-phycoerythrin compound；
（4）Pcr amplification reaction system.
The foundation of the multi-fluorescence immunoassay method detection method of 36 kinds of fowl respiratory pathogens of embodiment
1st, the structure of six kinds of fowl respiratory pathogens plasmids
The RNA/DNA that instrument extracts AIV, NDV, IBV, ILT, MG, MS cause of disease respectively is extracted automatically with the nucleic acid of Tiangeng, respectively
Corresponding primer designed by embodiment 1 carries out RT-PCR amplifications, and amplified production is detected respectively into row agarose gel electrophoresis
And cut glue purification.CDNA after purification is connected in pMD-19T carriers with the kit of TaKaRa companies, connection product is turned
Change to DH5a competent cells, select monoclonal, carry out bacterium colony PCR identifications, the bacterium colony for being accredited as positive bacteria is subjected to plasmid pumping
It carries, send sequencing.
2nd, plasmid PCR expands
Substance, double, quadruple, five weights, sixfold RT- are carried out with specific amplification AIV, NDV, IBV, ILT, MS, MG primer
PCR amplification.
The preparation of sense primer mixed liquor：By A1, B1, N1, L1, G1 and S1 with 1:1:1:1:1 ratio is mixed；Downstream
The preparation of primer mixed liquor：By A2, B2, N2, L2, G2 and S2 with 1:1:1:1:1 ratio is mixed.Utilize six kinds of fowl respiratory tract
The double template of specific template and AIV, NDV of cause of disease；AIV, NDV, MS, MG quadruple template, AIV, NDV, MS, MG, IBV
Five molality plates, AIV, NDV, IBV, ILT, MS, MG sixfold template, the idiocrasy region viral to above-mentioned three kinds expand.Its
In double template preparation：Will two-by-two plasmid with 1:1 ratio is mixed, the preparation of quadruple template：By four kinds of plasmids with 1:
1:1:1 ratio is mixed, the preparation of five molality plates：By five kinds of plasmids with 1:1:1:1:1 ratio is mixed, sixfold mould
The preparation of plate：By six kinds of plasmids with 1:1:1:1:1:1 ratio is mixed.
Pcr amplification reaction system is as follows：
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；Cycle 35 times；
72 DEG C extend 10min eventually.
PCR product is analyzed into row agarose gel electrophoresis, and electrophoretogram is as shown in Figure 1, 2.In Fig. 1,1：PCR blank,
2：ILT, 3：IBV, 4：AIV, 5：MG, 6：MS, 7：NDV, M：100bp DNA marker.In Fig. 2, M：DL2000 DNA
Marker, 1：AIV+NDV+IBV+ILT+MS+MG, 2：AIV+NDV+MS+MG+IBV, 3：AIV+NDV+MS+MG, 4：AIV+NDV
From figure 1 it appears that the amplified production size of ILT is about 141bp, the amplified production size of IBV is about
The amplified production size of 162bp, AIV are about 186bp, and the amplified production size of MG is about 133bp, and the amplified production size of MS is about
For 141bp, the amplified production size of NDV is about 155bp.From figure 2 it can be seen that since this six kinds of viral amplified productions are big
It is small close, so the electrophoretic band of double, quadruple, five weights and sixfold pcr amplification product can not be differentiated.
3rd, PCR product and fluorescence-encoded micro-beads working solution, Streptavidin phycoerythrin（SA-PE）Working solution hybridizes, packet
Include following steps：
Be coated with 6 kinds of microballoons of special anti-tag sequences respectively, wherein anti-tag sequences can correspondingly with AIV,
Tag sequence complementary pairings on the viral primers of six kinds of NDV, IBV, ILT, MS and MG.Six kinds of microballoons are purchased from luminex companies,
The specific corresponding fluorescence-encoded micro-beads number of AIV, NDV, IBV, ILT, MS and MG are MTAG-A029, MTAG-A034,
MTAG-A036, MTAG-A067, MTAG-A025 and MTAG-042.
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 in 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 mixing, 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 figure 3, although the amplified production of double, quadruple, five weights and sixfold template can not be differentiated with electrophoresis, detector is used
When 200 instruments of Luminex read MFI values, 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 868.2 that the present invention, which obtains cutoff values, therefore the cutoff values of the present invention are set to 900.Only detect sample
When the MFI values of product are higher than 900, which could effectively be analyzed.
The analytical judgment of sample to be tested：1）As the MFI value ＞ 900 of sample to be tested, it is judged as positive sample；2）Treat test sample
During this MFI values≤900, it is judged as feminine gender, carries out repeating experiment or other detection methods is taken further to verify.
The multi-fluorescence immunoassay detection specificity experiments of embodiment 4 AIV, NDV, IBV, ILT, MS and MG
Respectively with avian influenza virus, newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, cunning
Liquid capsule mycoplasma, chicken virus mycoplasma, infectious bursa of Fabricius virus, avian leukosis virus, avian infectious anemia virus, Marek's disease
Poison, Avianreovirus and Reticuloendotheliosis Virus carry out multi-fluorescence immunoassay detection as template.Experimental result
As shown in figure 4, only avian influenza virus, newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, cunning
Liquid capsule mycoplasma, is the positive at chicken virus mycoplasma, and others are feminine gender, illustrates that detection architecture specificity is good.
Embodiment 5：The multi-fluorescence immunoassay detection sensitivity experiment of AIV, NDV, IBV, ILT, MS and MG
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 inspection of AIV, NDV, IBV, ILT, MS and MG
Surveying sensitivity experiments, the results are shown in Figure 5, the experimental results showed that, in addition to the sensitivity detection of ILT is limited to 102Copies/ μ l,
His sensitivity detection is limited to 103copies/μl。
Embodiment 6：The detection of sample
Lung, kidney, the liver sample acquired from chicken house extracts instrument extraction RNA/DNA with Tiangeng nucleic acid, uses Qiagen's automatically
RT-PCR kit are expanded, using RNA/DNA as template, using the multi-fluorescence of above-mentioned AIV, NDV, IBV, ILT, MS, MG
Immunoassay detection method is detected, and amplified production hybridizes with fluorescence-encoded micro-beads and SA-PE, is detected in Luminex 200
Reading on instrument.For specific steps with reference to embodiment 3, experimental result is as shown in Figure 6.
Through regular-PCR test experience, the result shows that, sample 1,2,3,5,7,8,9,11,12,14,15,16,17,19,20 is
Negative sample；4 be infectious laryngotracheitis virus sample；6 be avian influenza virus sample；10 be Mycoplasma synoviae sample；13
For infectious bronchitis virus sample；18 be newcastle disease virus sample；21 be chicken virus mycoplasma sample.By sequencing analysis,
As a result it is consistent.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by 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.
<110>Experimental Animals Supervising Station, Guangdong Prov.
<120>A kind of multi-fluorescence immunoassay method of 6 kinds of fowl respiratory pathogens of quick differentiation
<130>
<160> 18
<170> PatentIn version 3.5
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Claims (5)
1. it is a kind of for quickly distinguishing the multi-fluorescence immunoassay primer sets of 6 kinds of fowl respiratory pathogens, including：For detecting
The primer pair A1 and A2 of avian influenza virus, for detect avian infectious bronchitis virus primer pair B1 and B2, for detecting
The primer pair N1 and N2 of newcastle disease virus, for detect avian infectious laryngotracheitis virus primer pair L1 and L2, for examining
Survey primer pair G1 and G2, the primer pair S1 and S2 for detecting chicken Mycoplasma synoviae of chicken virus mycoplasma, it is characterised in that：Institute
Stating for the primer pair for detecting avian influenza virus is designed according to the conserved sequence of the M genes of avian influenza virus；It is described to be used for
The primer pair of detection avian infectious bronchitis virus is designed according to the N genes conserved sequence of avian infectious bronchitis virus
's；The primer pair for being used to detect newcastle disease virus is designed according to the conserved sequence of the F genes of newcastle disease virus
's；Described for the primer pair that detects avian infectious laryngotracheitis virus is gD genes according to avian infectious laryngotracheitis virus
Conserved sequence design；Described for the primer pair that detects chicken virus mycoplasma is guarantor according to the PVPA genes of chicken virus mycoplasma
Keep sequence design；Described for the primer pair that detects chicken Mycoplasma synoviae is vlhA genes according to chicken Mycoplasma synoviae
Conserved sequence design；
It is it is characterized in that, as follows for detecting the nucleotide sequence of the primer pair of avian influenza virus：
A1：5 '-ATGAGTCTTCTACCCGAGG-3 ',
A2：5’-TCAGAGGTGACAGGATTG-3’；
It is as follows for detecting the nucleotide sequence of the primer pair of avian infectious bronchitis virus：
B1：5 '-AATCACGCTCAAGTTCAAGGC-3 ',
B2：5’-GCATTGTTCCTCTCCTCATC-3’；
It is as follows for detecting the nucleotide sequence of the primer pair of newcastle disease virus：
N1：5 '-CTCATCTCAGACAGGGTCAATC-3 ',
N2：5’-ATGGAGTCACCAAGGGG-3’；
It is as follows for detecting the nucleotide sequence of the primer pair of avian infectious laryngotracheitis virus：
L1：5 '-GCAGCGAAAAGAAGGC-3 ',
L2：5’-TCATCGTGCTCGGTGTCC-3’；
It is as follows for detecting the nucleotide sequence of the primer pair of chicken virus mycoplasma：
G1：5 '-CTTACAGATTACAGGAGCAG-3 ',
G2：5’-AAGCATTTCATTTGTTTTAC-3’；
It is as follows for detecting the nucleotide sequence of the primer pair of chicken Mycoplasma synoviae：
S1：5 '-ATTAGCAGCTAGTGCAGTGG-3 ',
S2：5’-TTTGAGGATTATCAGTATTTG-3’；
5 ' the ends of primer A1, B1, N1, L1, G1 and S1 are also added with tag sequences by spacer sequence；
Primer A1, B1, N1, L1, G1 and S1 5 ' end tag sequences be respectively：
The tag sequences of primer A1 are：5’-TACTACTTCTATAACTCACTTAAA-3’；
The tag sequences of primer B1 are：5’-ATTAAACAACTCTTAACTACACAA-3’；
The tag sequences of primer N1 are：5’-ACTTATTTCTTCACTACTATATCA-3’；
The tag sequences of primer L1 are：5’-ATCTCAATTACAATAACACACAAA-3’；
The tag sequences of primer G1 are：5’-CACTACACATTTATCATAACAAAT-3’；
The tag sequences of primer S1 are：5’-CTTTCTTAATACATTACAACATAC-3’.
2. multi-fluorescence immunoassay primer sets according to claim 1, which is characterized in that primer A2, B2, N2, L2, G2
Biotin labeling is also added with the 5 ' ends of S2.
3. a kind of multi-fluorescence immunoassay kits for quickly 6 kinds of fowl respiratory pathogens of differentiation, which is characterized in that described
Kit includes：（1）Primer sets described in claim 1；（2）The different iridescent of 6 kinds of codings include anti-tag sequences
The fluorescence-encoded micro-beads of row, the anti-tag sequences can correspondingly with the tag sequence complementary pairings in claim 1；（3）Chain
Mould Avidin-phycoerythrin compound.
4. a kind of quick multi-fluorescence immunoassay method for distinguishing 6 kinds of fowl respiratory pathogens of non-disease diagnosis, including as follows
Step：
1）Viral RNA/DNA is extracted from sample, using RNA/DNA as template, primer sets described in claim 1 is added in and carries out
RT-PCR is expanded；
2）By amplified production, the fluorescence-encoded micro-beads for including anti-tag sequences of the different iridescent of 6 kinds of codings and strepto- parent
With element-phycoerythrin hybridization；
3）It after hybridization, is analyzed using instrument, determines the type of its virus.
5. according to the method described in claim 4, it is characterized in that：Step 2）In fluorescence-encoded micro-beads on be coated with it is special
Anti-tag sequences, the anti-tag sequences can correspondingly with the tag sequence complementary pairings in claim 1.
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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 |
| CN111073999B (en) * | 2018-10-22 | 2022-08-09 | 四川农业大学 | Visual chip for synchronously and jointly detecting six avian viruses |
| CN110218808B (en) * | 2019-06-28 | 2021-07-13 | 华中农业大学 | Quadruple PCR (polymerase chain reaction) detection kit for mycoplasma synoviae and mycoplasma gallisepticum |
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