CN106191312B - A kind of quick multi-fluorescence immunoassay method and reagent for distinguishing AIV, NDV, MG and MS - Google Patents
A kind of quick multi-fluorescence immunoassay method and reagent for distinguishing AIV, NDV, MG and MS Download PDFInfo
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Abstract
The invention discloses a kind of quick multi-fluorescence immunoassay method and reagent for distinguishing avian influenza virus, newcastle disease virus, chicken virus mycoplasma and chicken Mycoplasma synoviae.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 avian influenza virus, newcastle disease virus, chicken virus mycoplasma and chicken Mycoplasma synoviae, 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 bird flu（AIV）, chicken new city
Epidemic disease poison（NDV）, chicken virus mycoplasma（MG）With chicken Mycoplasma synoviae（MS）Multi-fluorescence immunoassay method.
Background technology
As the demand of birds is on the increase in market, 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 cause 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 scabies secondary infection of a variety of diseases, such as mycoplasma to induce the gentle capsulitis of pneumonia of chicken, so
Active prevention and timely treatment breathing problem have great meaning.
Avian influenza virus, newcastle disease virus, chicken virus mycoplasma and chicken Mycoplasma synoviae are important fowl respiratory diseases
Original, clinical symptoms and pathological change are very much like, are difficult to distinguish in clinical and histopathology.Chicken respiratory tract disease is mostly mixed
Infection is closed, clinically be easy to cause mistaken diagnosis, its antidiastole usually need to be by laboratory diagnostic technique, viral separation and mirror
Fixed, serological test, enzyme-linked immunosorbent assay and diagnosis of molecular biology.Traditional detection method sensitiveness is low, specifically
Property is poor, is not easy to detect mixed infection, and operating 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, its
Principle is the multipair primer that design can amplify a variety of cause of disease specific fragment sizes to be measured at the same time, 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 produces pollution
And cause false positive, and multiplex PCR is distinguished by the size of fragment, has generally been arrived more than triple, clip size difference is big,
The amplification efficiency of its every kind of cause of disease is different, and causing result, there are deviation.Fluorescent quantitative PCR technique has merged a variety of excellent of PCR
Point, the change by directly detecting fluorescence signal in PCR reaction process, which is realized, quantifies molecules of interest, it is not necessary to which electrophoresis is examined
Survey, 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 in sensitiveness, specificity and speed etc., but in practical applications, when
When sample size is very big, substance fluorescent PCR is in cost and there is certain inferior position in terms of the time, and establishes a multi-fluorescence
PCR method is more more complex than substance, its requirement higher to reagent and primer, while needs to ensure that different probe is marked
Fluorophor between without interfering with each other, the fluorescence quantitative PCR instrument used has corresponding multiple sense channels, adds experiment difficulty
Greatly.
The content of the invention
It is an object of the invention to provide it is a kind of quick distinguish avian influenza virus, newcastle disease virus, chicken virus mycoplasma and
The reference of the multi-fluorescence immunoassay of chicken Mycoplasma synoviae.
The another purpose of the present invention is to provide a kind of quick differentiation avian influenza virus, newcastle disease virus, chicken virus mycoplasma
With the reagent of the multi-fluorescence immunoassay of chicken Mycoplasma synoviae.
It is an object of the invention to provide it is a kind of quick distinguish avian influenza virus, newcastle disease virus, chicken virus mycoplasma and
The method of the multi-fluorescence immunoassay of chicken Mycoplasma synoviae.
The technical solution used in the present invention is：
A kind of primer of the quick multi-fluorescence immunoassay for distinguishing AIV, NDV, MG and MS, the primer nucleotide sequences
It is as follows：
Primer A1：5’-ATGAGTCTTCTACCCGAGG-3’（SEQ ID NO:1）,
Primer A2：5’-TCAGAGGTGACAGGATTG-3’（SEQ ID NO:2）；
Primer N1：5’-CTCATCTCAGACAGGGTCAATC-3’（SEQ ID NO:3）,
Primer N2：5’-ATGGAGTCACCAAGGGG-3’（SEQ ID NO:4）；
Primer G1：5’-CTTACAGATTACAGGAGCAG-3’（SEQ ID NO:5）,
Primer G2：5’-AAGCATTTCATTTGTTTTAC-3’（SEQ ID NO:6）；
Primer S1：5’-ATTAGCAGCTAGTGCAGTGG-3’（SEQ ID NO:7）,
Primer S2：5’-TTTGAGGATTATCAGTATTTG-3’（SEQ ID NO:8）.
Further, the 5 ' ends of described wherein one primer of primer A1 and A2 are biotinylated；
5 ' the ends of described wherein one primer of primer N1 and N2 are biotinylated；
5 ' the ends of described wherein one primer of primer G1 and G2 are biotinylated；
5 ' the ends of described wherein one primer of primer S1 and S2 are biotinylated.
Further, the 5 ' of the primer not being biotinylated in the primer A1 and A2, N1 and N2, G1 and G2, S1 and S2
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 A1 and A2, N1 and N2, G1 and G2, S1 and S2
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 reagent of the quick multi-fluorescence immunoassay for distinguishing AIV, NDV, MG and MS, contains above-mentioned in the reagent
One primer.
Further, also containing streptavidin-phycoerythrin compound, the different iridescent of 4 kinds of codings in mentioned reagent
Fluorescence-encoded micro-beads.
Further, also containing the anti-tag sequences with tag sequences complementary pairing in primer in the 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 method of the quick multi-fluorescence immunoassay for distinguishing AIV, NDV, MG and MS, includes the following steps：
1）Viral RNA or/and DNA are extracted from sample；
2）Using the viral RNA of extraction or/and DNA 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 the type of its cause of disease；
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 DEG C of reverse transcription 30min；94 DEG C of pre-degeneration 15min；94 DEG C of changes
Property 30s, 60 DEG C annealing 25s, 72 DEG C extension 20s；Circulation 35 times；72 DEG C extend 10min eventually.
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 reaction 30min.
The beneficial effects of the invention are as follows：
1）The method of the present invention is at the same time to avian influenza virus, newcastle disease virus, chicken virus mycoplasma and chicken Mycoplasma synoviae
When being detected, target amplification fragment is obtained by RT-PCR, it is then that amplified production, fluorescence-encoded micro-beads and strepto- is affine
Element-phycoerythrin（SA-PE）Hybridized, when then reading MFI values by detector, offer an explanation different types of cause of disease.
2）The method of the present invention can be at the same time to avian influenza virus, newcastle disease virus, chicken virus mycoplasma and chicken bursa synovialis branch
Substance is accurately detected, and high specificity, and high sensitivity is reproducible.Compared with traditional detection method, the method for the present invention
Realize and detection is carried out at the same time to a variety of different molecules of interest in same sample, sample dosage is few, easy to operate, quick, can
Substantially reduce testing cost.The present invention can guarantee that identical renaturation temperature and hybridization efficiency, and effectively avoid different testing sample mark
Crisscrossing between the microballoon of note.
Brief description of the drawings
Fig. 1 is the four of avian influenza virus, newcastle disease virus, chicken virus mycoplasma and chicken Mycoplasma synoviae and manual simulation
The electrophoretogram of the RT-PCR of double infection dye；
Fig. 2 is the four of avian influenza virus, newcastle disease virus, chicken virus mycoplasma and chicken Mycoplasma synoviae and manual simulation
The multi-fluorescence immunoassay method test experience result figure of double infection dye；
Fig. 3 is that the multi-fluorescence of avian influenza virus, newcastle disease virus, chicken virus mycoplasma and chicken Mycoplasma synoviae is immunized
Analysis method detects specificity experiments result figure；
Fig. 4 is that the multi-fluorescence of avian influenza virus, newcastle disease virus, chicken virus mycoplasma and chicken Mycoplasma synoviae is immunized
Analysis method detection sensitivity experimental result picture；
Fig. 5 is that the multi-fluorescence of avian influenza virus, newcastle disease virus, chicken virus mycoplasma and chicken Mycoplasma synoviae is immunized
Analysis method clinical detection experimental result picture.
Embodiment
A kind of primer of the quick multi-fluorescence immunoassay for distinguishing AIV, NDV, MG and MS, the primer nucleotide sequences
It is as follows：
Primer A1：5’-ATGAGTCTTCTACCCGAGG-3’（SEQ ID NO:1）,
Primer A2：5’-TCAGAGGTGACAGGATTG-3’（SEQ ID NO:2）；
Primer N1：5’-CTCATCTCAGACAGGGTCAATC-3’（SEQ ID NO:3）,
Primer N2：5’-ATGGAGTCACCAAGGGG-3’（SEQ ID NO:4）；
Primer G1：5’-CTTACAGATTACAGGAGCAG-3’（SEQ ID NO:5）,
Primer G2：5’-AAGCATTTCATTTGTTTTAC-3’（SEQ ID NO:6）；
Primer S1：5’-ATTAGCAGCTAGTGCAGTGG-3’（SEQ ID NO:7）,
Primer S2：5’-TTTGAGGATTATCAGTATTTG-3’（SEQ ID NO:8）.
Preferably, the 5 ' ends of described wherein one primer of primer A1 and A2 are biotinylated；
5 ' the ends of described wherein one primer of primer N1 and N2 are biotinylated；
5 ' the ends of described wherein one primer of primer G1 and G2 are biotinylated；
5 ' the ends of described wherein one primer of primer S1 and S2 are biotinylated.
Preferably, 5 ' ends of the primer not being biotinylated in the primer A1 and A2, N1 and N2, G1 and G2, S1 and S2
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 A1 and A2, N1 and N2, G1 and G2, S1 and S2
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 reagent of the quick multi-fluorescence immunoassay for distinguishing AIV, NDV, MG and MS, contains above-mentioned in the reagent
One primer.
Preferably, different iridescent also are encoded containing streptavidin-phycoerythrin compound, 4 kinds in mentioned reagent
Fluorescence-encoded micro-beads.
Preferably, the anti-tag sequences with tag sequences complementary pairing in primer are also contained in the 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 method of the quick multi-fluorescence immunoassay for distinguishing AIV, NDV, MG and MS, includes the following steps：
1）Viral RNA or/and DNA are extracted from sample；
2）Using the viral RNA of extraction or/and DNA 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 the type of its cause of disease；
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 RT-PCR amplification are：50 DEG C of reverse transcription 30min；94 DEG C of pre-degeneration 15min；94 DEG C of changes
Property 30s, 60 DEG C annealing 25s, 72 DEG C extension 20s；Circulation 35 times；72 DEG C extend 10min eventually.
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 reaction 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 primer of embodiment
After being screened to designed a large amount of primers, find primer pair A1 and A2, N1 and N2, G1 and G2, S1 and
S2 pairs of fast detection bird flu at the same time（AIV）, newcastle disease virus（NDV）, chicken virus mycoplasma（MG）With chicken Mycoplasma synoviae
（MS）Effect it is best, its base sequence is as follows.
Primer A1：5’-ATGAGTCTTCTACCCGAGG-3’（SEQ ID NO:1）,
Primer A2：5’-TCAGAGGTGACAGGATTG-3’（SEQ ID NO:2）；
Primer N1：5’-CTCATCTCAGACAGGGTCAATC-3’（SEQ ID NO:3）,
Primer N2：5’-ATGGAGTCACCAAGGGG-3’（SEQ ID NO:4）；
Primer G1：5’-CTTACAGATTACAGGAGCAG-3’（SEQ ID NO:5）,
Primer G2：5’-AAGCATTTCATTTGTTTTAC-3’（SEQ ID NO:6）；
Primer S1：5’-ATTAGCAGCTAGTGCAGTGG-3’（SEQ ID NO:7）,
Primer S2：5’-TTTGAGGATTATCAGTATTTG-3’（SEQ ID NO:8）.
The present invention distinguishes 4 kinds of AIV, NDV, MG and MS cause of diseases using the method for multi-fluorescence immunoassay, therefore will
Above-mentioned primer makees further modification, to meet that corresponding operation requires.5 ' the ends of wherein primer A1, N1, G1 and S1 are connected with
Tag sequences, the tag sequences connected are respectively：
The tag sequences of primer A1 are：5’-TACTACTTCTATAACTCACTTAAA-3’（SEQ ID NO：9）；
The tag sequences of primer N1 are：5’-ACTTATTTCTTCACTACTATATCA-3’（SEQ ID NO：10）；
The tag sequences of primer G1 are：5’-CACTACACATTTATCATAACAAAT-3’（SEQ ID NO：11）；
The tag sequences of primer S1 are：5’-CTTTCTTAATACATTACAACATAC-3’（SEQ ID NO：12）.
In addition, 5 ' the ends of primer A2, N2, G2 and S2 are also added with biotin labeling.
Embodiment 2 is used to quickly distinguish bird flu（AIV）, newcastle disease virus（NDV）, chicken virus mycoplasma（MG）And chicken
Mycoplasma synoviae（MS）Multi-fluorescence immunological assay reagents
The reagent includes following components：
（1）The primer for multi-fluorescence immunoassay 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 with the tag sequence complementary pairings in multi-fluorescence immunoassay primer；4 kinds of microballoons are purchased from luminex companies,
Wherein the corresponding fluorescence-encoded micro-beads number of AIV, NDV, MS and MG for MTAG-A029, MTAG-A034, MTAG-A025 and
MTAG-042。
（3）Streptavidin-phycoerythrin compound.
3 bird flu of embodiment（AIV）, newcastle disease virus（NDV）, chicken virus mycoplasma（MG）With chicken Mycoplasma synoviae
（MS）Multi-fluorescence immunoassay method detection method foundation
（1）The structure of AIV, NDV, MG and MS plasmid
The RNA/DNA that instrument extracts AIV, NDV, MG, MS cause of disease respectively is extracted automatically with the nucleic acid of Tiangeng, respectively embodiment 1
Designed corresponding primer, carries out RT-PCR amplifications, amplified production is detected into row agarose gel electrophoresis respectively and cuts glue
Purifying.It will be purified with the kit of TaKaRa companies, cDNA after purification is respectively connected in pMD-19T carriers, will be connected
Product is converted to DH5a competent cells, selects monoclonal, carries out bacterium colony PCR identifications, and the bacterium colony for being accredited as positive bacteria is carried out
Plasmid extraction, send sequencing.
（2）Plasmid PCR expands
Carry out substance, quadruple RT-PCR amplifications to AIV, NDV, MS, MG primer respectively with the primer described in embodiment 1.
The preparation of sense primer mixed liquor：By A1, N1, G1 and S1 with molar ratio 1:1:1:1 ratio is mixed；Draw in downstream
The preparation of thing mixed liquor：By A2, N2, G2 and S2 with molar ratio 1:1:1:1 ratio is mixed.Utilize AIV, NDV, MS, MG 4
The specific template of kind cause of disease, and AIV, NDV, MS, MG 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 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；
Circulation 35 times；72 DEG C extend 10min eventually.
Electrophoresis detection result：Above-mentioned PCR product is analyzed into row agarose gel electrophoresis, its electrophoretogram is as shown in Figure 1.1：
PCR blank controls, M：DL2000bp DNA marker, 2：AIV, 3：MG, 4：MS, 5：NDV, 6：To AIV+NDV+MS+MG into
Capable Quadruple- PCR.From figure 1 it appears that the amplified production size of AIV is about 186bp, the amplified production size of NDV is about
The amplified production size of 155bp, MG are about 133bp, and the amplified production size of MS is about 141bp, due to these four viral expansions
It is close to increase primer size, so the electrophoretic band of Quadruple- PCR amplified production can not be differentiated.
（3）By the PCR product of gained and fluorescence-encoded micro-beads working solution, Streptavidin phycoerythrin（SA-PE）Work
Liquid hybridizes, and comprises the following steps：
Be coated with 4 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 tetra- kinds of NDV, MS and MG.Four kinds of microballoons are purchased from luminex companies, specifically
The corresponding fluorescence-encoded micro-beads number of AIV, NDV, MS and MG are MTAG-A029, MTAG-A034, MTAG-A025 and MTAG-
A042。
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, " AIV+NDV+MS+MG " represents Quadruple- PCR result in figure；" AIV ", " NDV ", " MS ", " MG " respectively to AIV,
The substance PCR that NDV, MS and MG are carried out；From figure 2 it can be seen that although the amplified production of quadruple template can not be differentiated with electrophoresis,
But when reading MFI values with 200 instruments of detector Luminex, hence it is evident that offer an explanation different types of cause of disease.
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 MFI values of average value plus 3 times of standard deviations set its as
Cutoff values.It is 450.8 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 AIV, NDV, MS and MG
Respectively with avian influenza virus, newcastle disease virus, chicken Mycoplasma synoviae, chicken virus mycoplasma, chicken infectivity throat gas
Pipe inflammation virus, avian infectious bronchitis virus, avian infectious anemia virus, Marek's disease poison carry out multi-fluorescence as template
Immunoassay detects.Experimental result is as shown in figure 3, only avian influenza virus, newcastle disease virus, Mycoplasma synoviae, chicken are malicious
Mycoplasma is the positive, and others are feminine gender, illustrates that detection architecture specificity is good.
Embodiment 5：The multi-fluorescence immunoassay detection sensitivity experiment of AIV, NDV, 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 detection sensitivity of AIV, NDV, MS and MG
Experimental result as shown in figure 4, test result indicates that, the sensitivity of all cause of diseases detection is limited to 102copies/µl。
Embodiment 6：The detection of sample
From the larynx tracheae of chicken house collection, lung, kidney, liver sample, instrument extraction RNA/DNA is extracted automatically with Tiangeng nucleic acid, is used
The RT-PCR kit of Qiagen are expanded, using RNA/DNA as template, using the multi-fluorescence of above-mentioned AIV, NDV, 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 5.
Through regular-PCR test experience, the result shows that, sample 1,4,6,7,10,11,12,13,17,18,19,20,21 is the moon
Property sample；5th, 9,14 be avian influenza virus sample；10 be newcastle disease virus sample；2nd, 8 be Mycoplasma synoviae sample；3、
15th, 16 be chicken virus mycoplasma 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.
<110>Experimental Animals Supervising Station, Guangdong Prov.
<120>A kind of quick multi-fluorescence immunoassay method and reagent for distinguishing AIV, NDV, MG and MS
<130>
<160> 12
<170> PatentIn version 3.5
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Claims (9)
1. a kind of primer of the quick multi-fluorescence immunoassay for distinguishing AIV, NDV, MG and MS, the primer nucleotide sequences are such as
Shown in lower：
Primer A1：5’-ATGAGTCTTCTACCCGAGG-3’(SEQ ID NO:1),
Primer A2：5’-TCAGAGGTGACAGGATTG-3’(SEQ ID NO:2)；
Primer N1：5’-CTCATCTCAGACAGGGTCAATC-3’(SEQ ID NO:3),
Primer N2：5’-ATGGAGTCACCAAGGGG-3’(SEQ ID NO:4)；
Primer G1：5’-CTTACAGATTACAGGAGCAG-3’(SEQ ID NO:5),
Primer G2：5’-AAGCATTTCATTTGTTTTAC-3’(SEQ ID NO:6)；
Primer S1：5’-ATTAGCAGCTAGTGCAGTGG-3’(SEQ ID NO:7),
Primer S2：5’-TTTGAGGATTATCAGTATTTG-3’(SEQ ID NO:8)；
The tag sequences connected in this 4 groups of primer pairs of the primer A1 and A2, N1 and N2, G1 and G2, S1 and S2 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 A1 and A2 are biotinylated；
5 ' the ends of described wherein one primer of primer N1 and N2 are biotinylated；
5 ' the ends of described wherein one primer of primer G1 and G2 are biotinylated；
5 ' the ends of described wherein one primer of primer S1 and S2 are biotinylated.
3. the primer according to profit requires 1, it is characterised in that in the primer A1 and A2, N1 and N2, G1 and G2, S1 and S2
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.
4. a kind of reagent of the quick multi-fluorescence immunoassay for distinguishing AIV, NDV, MG and MS, it is characterised in that in the reagent
Contain any primer of claims 1 to 3.
5. reagent according to claim 4, it is characterised in that also answered in the reagent containing streptavidin-phycoerythrin
The fluorescence-encoded micro-beads of compound, the different iridescent of 4 kinds of codings.
6. reagent according to claim 4, it is characterised in that also contain and tag in primer in the fluorescence-encoded micro-beads
The anti-tag sequences of sequence complementary pairing, it is mutual to encode the anti-tag sequences contained in the fluorescence-encoded micro-beads of different iridescent
Differ.
A kind of 7. method of the quick multi-fluorescence immunoassay for distinguishing AIV, NDV, MG and MS, it is characterised in that including as follows
Step：
1) viral RNA or/and DNA are extracted from sample；
2) using the viral RNA of extraction or/and DNA as template, RT-PCR expansions are carried out with any primer of claims 1 to 3
Increase；
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 the type of its cause of disease；It is above-mentioned
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 DEG C of reverse transcription 30min；94 DEG C of pre-degeneration 15min；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.
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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| CN104531901A (en) * | 2014-12-31 | 2015-04-22 | 四川圣迪乐村生态食品股份有限公司 | PCR detection method for detecting four kinds of poultry respiratory disease pathogens simultaneously |
| CN105671204A (en) * | 2016-03-15 | 2016-06-15 | 山东省动物疫病预防与控制中心 | Newcastle disease virus/avian influenza virus H9 subtype/infectious bronchitis virus triplex fluorescence quantification detection reagent and detection method |
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| CN104531901A (en) * | 2014-12-31 | 2015-04-22 | 四川圣迪乐村生态食品股份有限公司 | PCR detection method for detecting four kinds of poultry respiratory disease pathogens simultaneously |
| CN105671204A (en) * | 2016-03-15 | 2016-06-15 | 山东省动物疫病预防与控制中心 | Newcastle disease virus/avian influenza virus H9 subtype/infectious bronchitis virus triplex fluorescence quantification detection reagent and detection method |
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| Title |
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| 应用复合RT-PCR 诊断禽支原体与其他呼吸道病原的混合感染;张秀美等;《中国预防兽医学报》;20030531;第25卷(第3期);第232-234页，尤其是第232页摘要，第232-233页第1.4节 * |
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