CN106480173B - The poultry immunities such as bird flu inhibit pathogen Multiple detection reagent and its application - Google Patents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
This application discloses the poultry immunities such as bird flu to inhibit pathogen Multiple detection reagent and its application.The reagent of the application, including six specific probes, respectively sequence shown in Seq ID No.1 to Seq ID No.6, be sequentially avian influenza virus, newcastle disease virus, 1 type of marek virus, infectious bursa of Fabricius virus, Chicken Infectious Anemia Virus and J hypotype avian leukosis virus specific probe.The reagent of the application, contain the specific probe that six kinds of poultry immunities such as bird flu inhibit pathogen, the target sequence of pathogen can be inhibited to carry out specificity capture to six kinds of poultry immunities, inhibit the high throughput of pathogen quickly to detect for six kinds of poultry immunities and lay a good foundation.The present processes based on liquid-phase chip, inhibit pathogen to provide a kind of fast and accurately detection method using the reagent of the application for six kinds of poultry immunities.
Description
Technical field
This application involves the detection fields that the poultry immunities such as bird flu inhibit pathogen, are used for fowl more particularly to one kind
Reagent, detection method and the application of the poultry immunities such as influenza inhibition pathogen Multiple detection.
Background technique
There are many kinds of the inhibitive ability of immunity pathogen of poultry, in addition to 1 type of marek virus (MDV), infectious bursa of Fabricius
Other than virus, Chicken Infectious Anemia Virus, J hypotype avian leukosis virus, newcastle disease virus and avian influenza virus can also be generated very
Strong immunosuppressive action.
Bursal Disease, also known as gompaauw sieve disease, are by infectious bursa of Fabricius virus (Infectious Bursal
Disease Virus, abridge IBDV) caused by one kind is acute, social disease.With bursa of farbricius inflammation, necrosis, atrophy and
Bursa of farbricius endolymph cell, which is badly damaged, to be characterized.So as to cause the immunodeficiency disease of chicken, the immune effect of various vaccines is interfered
Fruit.Disease incidence is high, and almost up to 100%, the death rate is generally 5%~15%, but it is currently popular pass through variation generate it is superpower
The strain death rate reaches 60%-100%, is one of most important disease of current aviculture.
Chicken infectious anemia is by Chicken Infectious Anemia Virus (Avian Infectious Anaemia Virus, contracting
Write CIAV) caused by, it is a kind of immunosuppressive disease of chicken.The disease is with alpastic anemia, lymphoid organ Telatrophy, subcutaneous
It is characterized with hemorrhage of muscle, the reduction of general haemocyte；This disease is present in all main aviculture countries.1979 for the first time in day this newspaper
Road, China were separated to disease virus in 1992 with Later Zhou Dynasty, one of the Five Dynasties's text equality on Henan, Shandong, Jiangsu, Liaoning, Jilin and other places for the first time
It is also separated to CIAV successively in chicken group.This disease causes clinical and subclinical infection by vertical and horizontal propagation；Make chicken to horse
Garrick virus (MDV), reticuloendothelial cell hyperplasia virus (REV), bursal disease virus (IBDV), newcastle disease virus (NDV), adenopathy
The neurological susceptibility enhancing of poison, respiratory tract and alimentary canal virus infection etc., even results in immune failure.It chicken infectious anemia and its lures
The disease of hair is that aviculture causes huge economic loss every year.
J hypotype fowl myelomatosis is by J hypotype avian leukosis virus (Aivan Leukosis Virus
Subgroup J, abridge ALV-J) caused by a kind of infectious disease for taking place mostly in meat type chicken.It mainly causes the marrow of chicken
Cytoma.The disease is found in Britain in 1988 for the first time, and there are the report of the disease in subsequent many countries and regions.China is also in 1999
Year is isolated to ALV-J for the first time.The pathogenicity of the virus show its not only pass through myelocytome cause in broiler chicken it is higher dead
It dies outside rate, can also cause chicken weight degradation, its growth performance reduces after Chickens Infected.It can by horizontal transmission and hang down
It direct transfers to broadcast and promptly infects entire chicken group, make chicken group in a short time by extinction.
Marek's disease is by 2 type of herpetoviridae chicken herpetoviruses or 1 type of marek virus (Serotype I
Marek's Disease Virus), a kind of caused infectiousness neoplastic disease.The disease is in 1907 by Hungary's veterinary pathology
Family's horse Garrick has found that most valued chicken disease since being the external fifties is in worldwide distribution for the first time, with lymphadenia and
Tumour formation is characterized, and is generated immunosupress, is interfered the immune effect of various vaccines.Viral air-borne transmission, contagiousness is strong, quilt
Infected chicken generates tumour and death, causes huge economic losses to poultry husbandry.
Newcastle disease (newcastle disease, abridge ND) is a kind of urgency as caused by newcastle disease virus (abbreviation NDV)
Property, hot, septic and highly contagious disease.With high fever, expiratory dyspnea, diarrhea, neurological disorders, mucous membrane and serous coat bleeding
It is characterized.It is a kind of Infectious Diseases for endangering aviculture with very high disease incidence and case fatality rate.OIE is classified as A class epidemic disease
Disease.
Bird flu is commonly called as " checken pest ", is the poultry disease as caused by avian influenza virus (AIV), morbidity and mortality are all
It is very high.Influenza virus belongs to the orthomyxovirus section of RNA virus, divides first, second, the third 3 types；Avian influenza virus belongs to Flu-A disease
Poison, is mainly in birds, and part influenza A virus can also infect the various mammals such as pig, horse, sea dog and whale and the mankind.A type
Influenza virus is in pleomorphism, and wherein 80~120nm of spherical diameter, there is cyst membrane.Genome is segmented sub-thread strand RNA.Foundation
The difference of its outer membrane hemagglutinin (H)/and neuraminidase (N) protein antigenicity, can be divided into 16 H hypotypes (H1~H16) and 9
N hypotype (N1~N9).The avian influenza virus subtype of infection people is mainly H5N1, H9N2, H7N7, wherein the disease of infection H5N1
Feelings weight, case fatality rate are high.
The detection method of the inhibitive abilities of immunity pathogen such as existing poultry bird flu is mainly separately cultured, Electronic Speculum inspection,
The methods of agar gel diffusion test, virus neutralization tests, enzyme-linked immunosorbent assay；But these detection methods are not the presence of time-consuming
It is long, cumbersome, the disadvantages of being exactly poor specificity.Therefore, it needs to establish a kind of fast and accurately for fowl immunity inhibition phase
The detection method of virus is closed, to reduce the loss caused by aquaculture of the inhibitive abilities of immunity pathogen such as poultry bird flu, improves house
The safety of fowl breeding production.
Summary of the invention
The purpose of the application is to provide a kind of reagent for the inhibitive abilities of immunity pathogen Multiple detection such as poultry bird flu,
Application based on the multiple detection method of the detection reagent and the detection reagent and method.
The application uses following technical scheme:
The one side of the application discloses a kind of examination for the inhibitive abilities of immunity pathogen Multiple detection such as poultry bird flu
Agent, the reagent include six specific probes, and six specific probes are respectively the avian flu of sequence shown in Seq ID No.1
Sequence shown in the new castle disease virus specific probe of sequence shown in malicious specific probe, Seq ID No.2, Seq ID No.3
Infectious bursa of Fabricius virus specific probe, the Seq of sequence shown in 1 type specificity probe of marek virus, Seq ID No.4
The J hypotype fowl of sequence shown in the Chicken Infectious Anemia Virus specific probe and Seq ID No.6 of sequence shown in ID No.5
Leukemia virus specific probe；
Seq ID No.1:5 '-TCAGGCCCCCTCAAAGCCGA-3 '
Seq ID No.2:5 '-TTCTCTAGCAGTGGGACAGCCTGC-3 '
Seq ID No.3:5 '-ACCGCCCCAAATTCGCACTTGA-3 '
Seq ID No.4:5 '-CAACCGGACCGGCGTCCATTC-3 '
Seq ID No.5:5 '-CACCTCAAGCGACTTCGACGAA-3 '
Seq ID No.6:5 '-ATTTCCGTTGTCCCAGGGGTGG-3 '.
The poultry immunities such as the bird flu of the application inhibit pathogen Multiple detection reagent, i.e., for including bird flu
Six kinds of fowl inhibitive ability of immunity pathogen Multiple detections reagent.It should be noted that the application is by a large amount of research and in fact
It tramples, finally develops for the multiple of six kinds of important six including poultry bird flu kind inhibitive ability of immunity pathogen
The specific probe that detection reagent, i.e. six kinds of poultry immunities inhibit pathogen；It is appreciated that this six specific probe conducts
The entirety of one combination, can be used together, to inhibit pathogen to carry out Multiple detection six kinds of poultry immunities, when
So, under some special use environments, it also can be used alone wherein one or several specific probe.
It should also be noted that, the six kinds of poultry immunities that are integrated to that six specific probes of the application are capable of specificity press down
On the nucleic acid of pathogen processed, therefore, in a kind of application mode of the application, this six specific probes are used for liquid phase core
Piece is coupled with the microballoon of liquid-phase chip, to realize that six kinds of poultry immunities inhibit the multiple liquid-phase chip detection of pathogen.It can be with
Understand, since six specific probes of the application have very strong specificity, accordingly it is also possible to design primer appropriate, uses
In real-time fluorescence PCR；Alternatively, being not specifically limited herein for genetic chip as capture probe.
Preferably, the reagent for inhibitive abilities of immunity pathogen Multiple detections such as poultry bird flus of the application, Liu Tiaote
In specific probes, 5 ' ends of every specific probe all have amido modification.
In a kind of implementation of the application, 5 ' end amido modifications, specifically, being the 12nd of 5 ' first base in end
The carbon-based upper connection amido-NH in position2。
Preferably, the reagent for the inhibitive abilities of immunity pathogen Multiple detection such as poultry bird flu of the application further includes
Six groups of primer pairs, six groups of primer pairs are respectively to expand the first primer of avian influenza virus to draw to the second of, amplification newcastle disease virus
Object to, amplification 1 type of marek virus third primer pair, expand infectious bursa of Fabricius virus the 4th primer pair, amplification chicken
5th primer pair of infectious anemia virus, and the 6th primer pair of amplification J hypotype avian leukosis virus；The first primer pair
Upstream and downstream primer is respectively sequence shown in Seq ID No.7 and Seq ID No.8, and the upstream and downstream of the second primer pair draws
Object is respectively sequence shown in Seq ID No.9 and Seq ID No.10, and the upstream and downstream primer of third primer pair is respectively Seq
Sequence shown in ID No.11 and Seq ID No.12, the upstream and downstream primer of the 4th primer pair be respectively Seq ID No.13 and
Sequence shown in Seq ID No.14, the upstream and downstream primer of the 5th primer pair are respectively Seq ID No.15 and Seq ID
Sequence shown in No.16, the upstream and downstream primer of the 6th primer pair are respectively shown in Seq ID No.17 and Seq ID No.18
Sequence.
It should be noted that the application devises the specific probe that six kinds of poultry immunities inhibit pathogen, for side
Just it uses, while devising six groups of primer pairs, inhibit pathogen to carry out nucleic acid amplification six kinds of poultry immunities to facilitate.It can be with
Understand, on the basis of six specific probes of the application, other primers can also be used, as long as can be by six specificity
The specific binding region of probe covers into, have to not necessarily use six groups of primer pairs of the application；Certainly, the application
As a preferred implementation, and by what is be specifically designed and study, six groups of primer pairs can be used as six groups of primer pairs
The entirety of one combination, the six kinds of poultry immunities that amplify that can be specific inhibit pathogens, also, six groups of primer pairs
Between interfere with each other and influence minimum.In fact, six groups of primer pairs of the application, can also be individually used for six kinds of poultry immunities
Inhibit the sixfold PCR amplification detection of pathogen；The introducing of six specific probes is intended merely to further ensure that detection is special
It is anisotropic.
It should also be noted that, six groups of primer pairs of the application introduce a Duan Xiangtong at 5 ' ends of six upstream primers
20bp sequence, meanwhile, the sequence of another section of identical 20bp has also been introduced at 5 ' ends of six downstream primers；This is this Shen
Special construction design in a kind of preferred embodiment please, by the design, as soon as group super primer can be used, to six
Target sequence is carried out while being expanded, and be can reduce in sixfold PCR amplification and is interfered between multipair primer to PCR amplification efficiency in this way
It influences.It is appreciated that the identical sequence of the 20bp introduced respectively in the upstream and downstream primer of six groups of primer pairs, is a Duan Yuliu
Sequence of the genome of kind inhibitive ability of immunity pathogen all without any relationship, which will not be to six kinds of poultry immunity inhibitions
The genome of pathogen generates non-specific amplification；Also, the sequence of the 20bp introduced respectively in upstream and downstream primer, alkali
Radix amount and particular sequence are all that can be changed according to different test platforms, this will not influence the spy of six groups of primer pairs
It is anisotropic；Even, the sequence of this 20bp can be deleted completely, certainly, in the preferred embodiment of the application, and it is not recommended that in this way.
Preferably, the reagent for the inhibitive abilities of immunity pathogen Multiple detection such as poultry bird flu of the application further includes
One group of super primer, the upstream and downstream primer of super primer are respectively sequence shown in Seq ID No.19 and Seq ID No.20
Column.
It should be noted that the super primer of the application, actually draws for the upstream and downstream of six groups of primer pairs
The sequence of the 20bp introduced respectively in object and design, by the super primer, six target sequences can be expanded simultaneously
Increase.For example, carrying out nucleic acid amplification to sample to be tested in advance using six groups of primer pairs, super primer pair nucleic acid amplification is then used
Product carries out a PCR amplification again, and amplification while one group of super six target of primer pair thus may be implemented.
Preferably, the reagent for inhibitive abilities of immunity pathogen Multiple detections such as poultry bird flus of the application, it is super
In primer, 5 ' ends of the downstream primer of sequence shown in Seq ID No.20 have biotin labeling.
In a kind of implementation of the application, 5 ' end biotin labelings, specifically, being the of 5 ' first base in end
Biotin is marked on 6 or the 10th hydroxyl.
It should be noted that 5 ' ends of the downstream primer of sequence shown in the Seq ID No.20 of super primer have biotin
Label, this is also to detect and design for liquid-phase chip, can also be without biotin mark if not liquid-phase chip is used for
Note.
It is super therefore it should also be noted that, using the design scheme of super primer in the preferred embodiment of the application
5 ' ends of grade primer carry out biotin labeling；If not using the design scheme of super primer, i.e., in the upstream of six groups of primer pairs
With the identical sequence that 20bp is not introduced in downstream primer, at this time, it is necessary to directly adopt the core of the mix primer of six groups of primer pairs
Sour amplified production carries out liquid-phase chip detection, and therefore, it is necessary to 5 ' ends of the downstream primer in six groups of primer pairs to carry out biotin mark
Note.
The another side of the application disclose the application for the inhibitive abilities of immunity pathogen Multiple detection such as poultry bird flu
Reagent poultry immunosuppressive substance multiple gene chip detection, multiple liquid-phase chip detection, multiple Standard PCR inspection
Application in survey or multiple real time fluorescence PCR detection.
It is appreciated that the specificity that the reagent of the application includes six kinds of inhibitive ability of immunity pathogen such as poultry bird flu is visited
Needle or specific probe add six group-specific primers pair, these probes and primer pair all have very strong specificity, not only may be used
To be used for liquid-phase chip, can be used in the detection of nucleic acids such as the genetic chip, Standard PCR, real-time fluorescence PCR of solid phase.
The application's discloses a kind of examination for the inhibitive abilities of immunity pathogen Multiple detection such as poultry bird flu on one side again
Agent box, the reagent for inhibitive abilities of immunity pathogen Multiple detections such as poultry bird flus in the kit containing the application.
It is appreciated that kit can be made in the reagent of the application completely, to facilitate six kinds of poultry bird flu etc. immune suppressions
The specific detection of pathogen processed.
The application's discloses a kind of Multiple detection side for the inhibitive abilities of immunity pathogen such as poultry bird flu on one side again
Method includes the following steps,
(1) the microballoon coupling of numbers different from six of liquid-phase chip respectively, six specificity by six specific probes
Probe distinguishes the newcastle disease of sequence shown in the avian influenza virus specific probe of sequence shown in Seq ID No.1, Seq ID No.2
Shown in the 1 type specificity probe of marek virus of sequence shown in virus-specific probe, Seq ID No.3, Seq ID No.4
The Chicken Infectious Anemia Virus specificity of sequence shown in the infectious bursa of Fabricius virus specific probe of sequence, Seq ID No.5
The J hypotype avian leukosis virus specific probe of sequence shown in probe and Seq ID No.6；
(2) mix primer for using six groups of primer pairs carries out nucleic acid amplification to sample to be tested, and six groups of primer pairs are respectively to expand
Increase avian influenza virus the first primer to, amplification newcastle disease virus the second primer pair, expand 1 type of marek virus third
Primer pair, the 4th primer pair for expanding infectious bursa of Fabricius virus, the 5th primer pair for expanding Chicken Infectious Anemia Virus, and
Expand the 6th primer pair of J hypotype avian leukosis virus；The upstream and downstream primer of the first primer pair is respectively Seq ID No.7
With sequence shown in Seq ID No.8, the upstream and downstream primer of the second primer pair is respectively Seq ID No.9 and Seq ID
Sequence shown in No.10, the upstream and downstream primer of third primer pair are respectively shown in Seq ID No.11 and Seq ID No.12
Sequence, the upstream and downstream primer of the 4th primer pair are respectively sequence shown in Seq ID No.13 and Seq ID No.14, and the 5th
The upstream and downstream primer of primer pair is respectively sequence shown in Seq ID No.15 and Seq ID No.16, the 6th primer pair it is upper
Trip and downstream primer are respectively sequence shown in Seq ID No.17 and Seq ID No.18；
(3) there is the microballoon of six specific probes using the coupling of step (1), liquid is carried out to the amplified production of step (2)
The detection of phase chip.
Preferably, the multiple detection method of the application further includes, before step (3), using one group of super primer, to step
Suddenly the amplified production of (2) carries out a PCR amplification again, and pcr amplification product is then used for liquid-phase chip detection；It is described super to draw
The upstream and downstream primer of object is respectively sequence shown in Seq ID No.19 and Seq ID No.20.
Wherein, in super primer, 5 ' ends of the downstream primer of sequence shown in Seq ID No.20 have biotin labeling.
The beneficial effects of the present application are as follows:
The reagent for inhibitive abilities of immunity pathogen Multiple detections such as poultry bird flus of the application contains six kinds and is immunized
Inhibit the specific probe of pathogen, the target sequence of pathogen can be inhibited to carry out specificity to six kinds of poultry immunities and caught
It obtains, inhibits the high throughput of pathogen quickly to detect for six kinds of poultry immunities and lay a good foundation.The multiple detection method of the application,
Using the reagent of the application, based on liquid-phase chip, one is provided for six kinds of inhibitive ability of immunity pathogen such as poultry bird flu
Fast and accurately detection method is planted, is used particularly suitable for departments such as inspection and quarantine, to ensure production safety, controls poultry fowl
The immunosuppressives pathogen such as influenza provides strong science tools.
Detailed description of the invention
Fig. 1 is the agarose gel electrophoresis figure that multiple RT-PCR expands single strain in the embodiment of the present application.
Specific embodiment
The application is described in further detail below by specific embodiment.Following embodiment only to the application carry out into
One step explanation, should not be construed as the limitation to the application.
Embodiment
One, reagent consumptive material
1. strain and chicken embryo
Avian influenza virus H 5 N 1 (Re-6) standard antigen hemagglutinating antigen, the Newcastle Disease standard antigen hemagglutinating antigen of this example use, which are purchased from, to be breathed out
That shore institute of viruses, 1 plant of 1 type of Marek's disease poison, 1 plant of infectious bursa of Fabricius virus, 1 plant of Chicken Infectious Anemia Virus and J are sub-
It 1 plant of type avian leukosis virus, is saved and is provided by the dynamic inspection laboratory of Shenzhen Entry-Exit Inspection and Quarantine Bureau.
In addition, infectious bronchitis virus, infectious laryngotracheitis virus, the egg-decreasing syndrome disease as negative reference
Poison etc. infects virus and Escherichia coli O 157, salmonella, the single bacterium for increasing the infection poultry such as Listeria of poultry,
Also it is saved and is provided by the dynamic inspection laboratory of Shenzhen Entry-Exit Inspection and Quarantine Bureau.
9 days~11 days no-special pathogen (Specific pathogen free, SPF) chicken embryos are purchased from Beijing Cimmeria
Wei Tong experimental animal Technology Co., Ltd., breeding and identification for poultry immunity inhibition virus.
2. material and facility
The reagent of this example refers both to analytical reagents in addition to particular provisions.
The concentration that carboxyl is contained on surface is 1.25 × 107Fluorescence-encoded micro-beads, 1- ethyl-(the 3- dimethyl-the third of a/mL
Alkane) hydrogen chloride imidodicarbonic diamide (abbreviation EDC), pH 4.5 0.5M 2- (N- morphine base) ethanesulfonic acid (abbreviation MES), 0.02% (w/
V) Tween-20,0.1% (w/v) lauryl sodium sulfate SDS, 5MTetramethylammonium chloride solution
(abbreviation TMAC), Qiagen One-Step RT-PCR Kit, EZ1Virus mini Kit2.0.
Microballoon dilution is 1.5 × TMAC hybridization solution, and preparation is shown in Table 1；Detection buffer is 1 × TMAC hybridization solution, is prepared
It is shown in Table 2.
1 microballoon diluent preparing table (250mL total amount) of table
| Reagent name | Article No. | Ultimate density | Quantity/250mL |
| 5M TMAC | Sigma T-3411-1L | 4.5M | 225mL |
| 20% musculamine acyl | Sigma L7414 | 0.15% | 1.88mL |
| 1M Tris-HCl,pH 8.0 | Sigma T-3038-1L | 75Mm | 18.75mL |
| 0.5M EDTA,pH 8.0 | Sigma 03690-100ml | 6mM | 3.0mL |
| Deionized water | 1.37mL |
Table 2 detects buffer table (250mL total amount)
| Reagent name | Article No. | Ultimate density | Quantity/250mL |
| 5M TMAC | Sigma T-3411-1L | 3M | 150mL |
| 20% musculamine acyl | Sigma L7414 | 0.1% | 1.25mL |
| 1M Tris-HCl,pH 8.0 | Sigma T-3038-1L | 50Mm | 12.5mL |
| 0.5M EDTA,pH 8.0 | Sigma 03690-100ml | 4mM | 2.0mL |
| Bovine serum albumin(BSA) | Sigma A9418 | 0.2% | 0.5g |
| Deionized water | 84.25mL |
The detection device that this example uses includes that liquid-phase chip detects work station, full-automatic nucleic acid extraction work station, high speed platform
Formula refrigerated centrifuge, gradient nucleic acid augmentative instrument, oscillator, ultrasonoscope, desk centrifuge, timer, assay balance etc..
3. primer, probe sequence and base modification
This example is infected according to avian influenza virus, newcastle disease virus, 1 type of marek virus, infectious bursa of Fabricius virus, chicken
Property anemia virus and J hypotype avian leukosis virus, the nucleic acid design of the inhibitive abilities of immunity pathogen such as six kinds of poultry bird flus is special
Property probe and respective primer.Also, the random sequence of 20bp is introduced at 5 ' ends of various primers；Specifically, on all
5 ' the ends for swimming primer introduce the random sequence of one section of identical 20bp, introduce an other Duan Xiangtong at 5 ' ends of all downstream primers
20bp random sequence；And according to introduced random sequence, one group of super primer is devised.
In order to cooperate liquid-phase chip to detect, this example holds the 6th of first base in the reverse primer (RSP) 5 ' of super primer
Biotin (biotin) is marked on position or the 10th hydroxyl, each disease specific probe 5 ' holds 12 carbon of first base
Amido (- NH is connected on base2), it is required to carry out chromatographic grade (HPLC) purifying after all primed probe synthesis.Probe and primer
Specifically it is shown in Table 3.
The primer and probe of six kinds of inhibitive ability of immunity pathogen such as 3 poultry bird flu of table
Two, strain breeding and nucleic acid extraction
1. strain is bred
Using SPF chicken embryo, respectively in addition to bird flu and newcastle disease other for examination virus or bacterium cultivate, chicken embryo
Dead or after culture seven days, acquisition allantoic fluid is detected using corresponding standard, to determine whether chicken embryo is specific dead
It dies or infects.Chicken embryo is being determined for specificity after dead or infection, take chicken embryo tissue for virus or bacterial nucleic acid extraction.Together
When, the tissue of the SPF chicken embryo without virus or bacterium infection is taken, extracts nucleic acid as negative control.
Acquisition, preservation and the transport of the sample of this example all meet the related request of GB/T 18088.
2. nucleic acid extraction
For tissue sample, it is fully ground in mortar with sterile scissors and tweezers clip measuring samples 2.0g, then plus
PBS of the 10mL containing 10 000IU/mL of penicillin, 10 000 μ g/mL of streptomysin is mixed, or is placed in tissue homogenizer, is added
Tissue suspension, is then transferred to sterile by PBS homogenate of the 10mL containing 10 000IU/mL of penicillin, 10 000 μ g/mL of streptomysin
4 DEG C of centrifugation 15min of 3000r/min, take supernatant to be transferred in another sterile 1.5mL centrifuge tube in 1.5mL centrifuge tube, number standby
With.
The nucleic acid extraction method that can extract viral DNA and viral RNA simultaneously, such as tradition can be used in the nucleic acid extraction of this example
Phenol-chloroform method, can also be used automatic nucleic acid extracting instrument and mating nucleic acid extraction reagent carries out nucleic acid extraction.Specifically, this
Example extracts nucleic acid using phenol-chloroform method.It is specific as follows:
(1) the 1.5mL centrifuge tube for taking sterilizing, carries out mark.
(2) 600 μ L lysates are added in every pipe, are separately added into each 200 μ L of tested sample, negative control, positive control, then plus
Enter 200 μ L chloroforms, oscillation mixes 5s on vortex mixer, is centrifuged 15min in 4 DEG C of 12000g.
(3) the 1.5mL centrifuge tube of sterilizing is taken, -20 DEG C of 400 μ L isopropanols of pre-cooling are added, is drawn in each pipe of this standard 6.2
Supernatant is transferred in corresponding pipe, is avoided that middle layer is sucked out, is mixed by inversion.
(4) it is centrifuged 15min in 4 DEG C of 12000g, abandons supernatant, is inverted on blotting paper, is stained with dry liquids；600 μ L75% are added
Pre-cooled ethanol overturns washing.
(5) it is centrifuged 10min in 4 DEG C of 12000g, abandons supernatant, is inverted on blotting paper, is stained with dry liquids.
(6) 10000g is centrifuged 10s, is blotted with micro sample adding appliance, drying at room temperature (5~10) min.
(7) 15 μ L DEPC water are added, dissolve the RNA of tube bottom, 5000g is centrifuged 5s, saves backup on ice, if need to protect for a long time
- 70 DEG C of refrigerators should be placed by depositing.
Three, liquid-phase chip detects
The laboratory testing of this example meets the related request of GB 19489 and SN/T 1193.
1. oligonucleotide probe and the fluorescence-encoded micro-beads of carboxylated are coupled
The specific probe of different fowl inhibitive ability of immunity diseases is carried out even with the microballoon of corresponding encoded according to table 4
Connection.
4 probe of table and coupling microballoon coding
At least 30min, all operations should all be protected from light all reagents of this example at room temperature, most handy aluminium platinum film Bao Yan
Real centrifuge tube operation.The specific method that probe is coupled with microballoon includes:
(1) six specific probes are dissolved respectively to 0.2mM with the 0.1M MES of pH 4.5.
(2) storage tube that microballoon is housed is taken out, first disperses 3min with ultrasonoscope, is shaken at full speed on vortex oscillator
At least 5min.
(3) taken out from storage tube 20 μ L microballoons storage liquid, about 5 × 104A microballoon, until the polypropylene of 1.5mL it is micro from
In heart pipe, 5min is centrifuged with 10000g.
(4) it removes supernatant, the 0.1M MES of the pH 4.5 of 50 μ L is added, in shaking 5min on vortex oscillator at full speed, then use
Ultrasonoscope disperses about 5min.
(5) specific probe of 3 μ L 0.2mM is added, in instantaneously shaking on vortex oscillator.
(6) before use, plus 1.0mL deionized water into the EDC of 10mg, add the fresh EDC solution of 2.5 μ l to microballoon with
Instantaneous to shake in the mixed liquor of probe, room temperature, which is protected from light, is incubated for 30min.
(7) step (6) are repeated once with fresh EDC.
(8) Tween-20 for adding 1.0mL 0.02% (w/v), after shaking 1min at full speed, 12000g is centrifuged 10min.
(9) supernatant is removed, the SDS solution of 1.0mL 0.1% (w/v) is added into microballoon, after shaking 5min at full speed, 12000g
It is centrifuged 10min.
(10) supernatant is abandoned, the 0.1M MES of the pH 4.5 of 100 μ L is added, shakes 10imn, ultrasonic wave settling flux microballoon at full speed
About 10imn.
The microballoon of preparation counts: using dH2The microspheres solution of coupling is resuspended in O 1:100 dilution, and mixing fullys shake, takes 10 μ L
Haemocytometer is added to, microballoon counting is carried out to 4 big angles of haemocytometer grid.Microballoon number/the big angle μ l=4 microballoon is total
With × 2.5 × 100.Microballoon maximum number is 50000 microballoons/μ L.
Microballoon is diluted to 1.25 × 10 with 1.5 × TMAC hybridization solution5, storable half a year is protected from light at 2~8 DEG C.
Liquid-phase chip detection is carried out with PCR product after coupling, and sets blank control, to determine coupling efficiency.When every kind it is glimmering
Pumped FIR laser microballoon number is no less than 100, and blank control fluorescence intensity is not higher than 300, shows that microballoon is coupled successfully.
2.RT-PCR nucleic acid amplification
The nucleic acid amplification of this example due to six kinds of existing DNA virus of poultry immunity inhibition disease, and has RNA virus.In order to
Guarantee to amplify all virus simultaneously, it is unified to carry out nucleic acid amplification using RT-PCR method.
The preparation of reaction solution:
In PCR solution preparation area, prepared referring to each kit corresponding instructions, specifically, every part of test sample
RT-PCR system is 50 μ L, and system is shown in Table 5.Each PCR reaction reagent of preparation is mixed well, sample is transferred to after brief centrifugation
Present treatment area.
5 RT-PCR reaction system of table
Super primer and mix primer, the upstream and downstream primer of super primer have been separately added into the reaction system of table 5
That is FSP and RSP.Mix primer is the mixture of six groups of primer pairs, and the mixing match of mix primer is shown in Table 6.
The preparation of 6 mix primer of table
Sample-adding: positive control, negative control are set up.It is separately added into the PCR pipe for having dispensed PCR reaction mixture
PCR pipe is put into PCR detector by the 5 μ L of nucleic acid extracted, lid upper tube cap, records sample placement order.This example specifically uses
Six positive controls, two negative controls, a water blank control.
The setting of RT-PCR amplification condition is shown in Table 7.
7 RT-PCR amplification condition of table
3. microsphere probe hybridizes with nucleic acid amplification product
At least 30min, all operations are protected from light all reagents at room temperature, and most handy aluminium platinum film packet is tightly centrifuged
Pipe operation.The storage tube that conjugated probes microballoon is housed is taken out, first disperses 1min with ultrasonoscope, on vortex oscillator at full speed
Shake at least 20s.The 10 μ L of various fluorescence-encoded micro-beads being coupled is taken respectively, the fluorescence-encoded micro-beads that six kinds are encoded mix,
Every kind of microballoon is finally diluted to 200 fluorescence-encoded micro-beads/μ L with 1.5 × TMAC hybridization solution.It is prepared according to the proportion of table 8 miscellaneous
Hand over reaction system.
8 microsphere probe of table and nucleic acid amplification product hybridization reaction system
| Component | Amount/reaction (μ L) |
| Six kinds of inhibitive ability of immunity disease microballoon bulk crossing liquid | 33 |
| RT-PCR product | 5 |
| 1×TE(PH8.0) | 12 |
| Total | 50 |
Hybridization reaction designs blank control pipe and positive control pipe, directly adds 5 μ L of deionized water in blank control pipe and replaces
RT-PCR product is changed, the complementary series of six specific probes of 5 μ L synthesis is added in positive control pipe.
After reaction system prepares, gently piping and druming is mixed repeatedly, is reacted as follows in PCR instrument: 94 DEG C of 15min, and 52
℃30min。
After reaction, 10000r/min is centrifuged 3min.After centrifugation, each pipe supernatant is abandoned, the report that 75 μ L are added is mixed
Liquid is closed, slight piping and druming mixes several times, in 52 DEG C of incubation 5min in PCR instrument.Then, in being detected in liquid-phase chip detector.
Report that mixed liquor is that SA-PE is diluted to 4 μ g/mL with 1 × TMAC hybridization solution, that is, report mixed liquor is made.
Liquid-phase chip detector detection specifically includes:
(1) it when PCR amplification carries out, opens liquid-phase chip and detects all pertinent instruments, and the loading of instrument is heated and is made pottery
Porcelain bottom plate is previously heated to 52 DEG C.
(2) according to liquid-phase chip detector operational manual, the setting of parameters is carried out.
This example is provided that using 100 liquid-phase chip instrument of Luminex
(a) when each sample detection, simultaneous selection is answered to detect 6 kinds of fluorescence-encoded micro-beads (33,34,35,36,37,38), and
It fills in every kind of fluorescent microsphere and corresponds to disease name.
(b) detection volume is 45 μ L, flow velocity be it is quick, every kind of fluorescent microsphere, which is arranged, is counted as 100, and gate time is
120s, every kind of fluorescence-encoded micro-beads are at least >=20 when detection every time.
(3) sample is sequentially placed into blank control pipe, each negative control pipe, positive control pipe and measuring samples pipe, and
It is marked accordingly in software interface.
4. specific detection
(1) single plant strain specific detects
According to the method and system in " 2.RT-PCR nucleic acid amplification ", respectively to the avian influenza virus of extraction, Newcastle Disease
Malicious 1 type of poison, Marek's disease, infectious bursa of Fabricius virus, Chicken Infectious Anemia Virus and J hypotype avian leukosis virus be nucleic acid into
Row RT-PCR nucleic acid amplification.And water blank control 1 and infectious bronchitis virus, infectious laryngotracheitis are set
Poison, egg-decreasing syndrome virus, Escherichia coli O 157, salmonella, single increasing 6 negative controls of Listeria.Part amplification produces
Object is observed with agarose gel electrophoresis.
In addition, by avian influenza virus, newcastle disease virus, malicious 1 type of Marek's disease, infectious bursa of Fabricius virus, avian infectious
Anemia virus and the amplified production of J hypotype avian leukosis virus according to " 3. microsphere probes hybridize with nucleic acid amplification product " method
And condition, carry out liquid-phase chip detection.
(2) more strains mix specific detection
The mixing that this example has prepared the mixing nucleic acid samples of six strain combination of two respectively and six strains combine together
Nucleic acid samples are tested.And water blank control 1 and infectious bronchitis virus, infectious laryngotracheitis are set
Poison, egg-decreasing syndrome virus, Escherichia coli O 157, salmonella, single increasing 6 negative controls of Listeria.Part amplification produces
Object is observed with agarose gel electrophoresis.
In addition, by avian influenza virus, newcastle disease virus, malicious 1 type of Marek's disease, infectious bursa of Fabricius virus, avian infectious
Anemia virus and the positive amplification product of J hypotype avian leukosis virus combination are according to " 3. microsphere probes and nucleic acid amplification product are miscellaneous
The method and condition of friendship " carries out liquid-phase chip detection.
Four, testing result
1. agarose gel electrophoresis
The agarose gel electrophoresis results of single plant strain specific detection show that water blank control and 6 negative controls are all
There is no an amplified band, and it is malicious 1 type of avian influenza virus, newcastle disease virus, Marek's disease, infectious bursa of Fabricius virus, avian infectious
Six strains of anemia virus and J hypotype avian leukosis virus have amplified band, as shown in Figure 1, in figure, first swims partial results
The amplified band that road is 100bp DNA Ladder Marker, the second swimming lane is avian influenza virus about 174bp, third swimming lane are
The amplified band of newcastle disease virus about 145bp, the 4th swimming lane are the amplified band about 198bp of infectious bursa of Fabricius virus, the 5th
The amplified band that swimming lane is the amplified band about 245bp of malicious 1 type of Marek's disease, the 6th swimming lane is J hypotype avian leukosis virus is about
185bp, the amplified band about 122bp that the 7th swimming lane is Chicken Infectious Anemia Virus, are consistent with expected results.
The agarose gel electrophoresis results of more strains mixing specific detections show, water blank control and 6 negative controls
All without amplified band, and malicious 1 type of avian influenza virus, newcastle disease virus, Marek's disease, infectious bursa of Fabricius virus, chicken are infected
Property anemia virus and J hypotype avian leukosis virus combination of two or the mixing samples of six combinations have corresponding amplification item
Band, statistical result are as shown in table 9.
More than the 9 strain mixing RT-PCR amplifications of table
"+" indicates detection in table 9, and blank then indicates not detect
Table 9 the results show that six viral nucleic acid combination of two can be it is anticipated that amplify corresponding target piece
Section, six viral nucleic acid combine together can also amplify six target fragments, and size is consistent with expection.
2. liquid-phase chip detects
The qualitative ratio result of liquid-phase chip (Luminex qualitative ratio result, LQRR) is equal to sample school
The average value of fluorescence intensity median (Median florescence intensity, MFI) and blank control MFI after just
(MFIB) ratio.
LQRR=MFIS/mMFIB.
According to the analysis software operation of instrument instrument data, the output of testing result is obtained.
LQRR >=3 are determined as positive sample.
LQRR < 2 are then determined as feminine gender.
2≤LQRR < 3, then be determined as suspicious, and suspicious specimen should re-apply other standards method and carry out detection judgement.
After purification by single plant strain RT-PCR amplified production, directly hybridized with mixed probe, as a result each participation meter
Several fluorescence-encoded micro-beads are equal >=20 between 30~243, show that the fluorescence-encoded micro-beads quantity for counting has
Effect, generated MFI value is credible；The blank control MFI of each fluorescence-encoded micro-beads is between 143~2839, < 3000,
Show that result is effective, test can be determined；The MFI value of each strain RT-PCR reaction amplified production is between 12776~15840
Between.Have hybridizing for specificity between mixed probe and the RT-PCR amplified production of each strain, LQRR value between 7~
Between 109.7, show to be the positive, detailed results are as shown in table 10.
10 single plant strain specific testing result of table
After purification by the RT-PCR amplified production of double strain mixing samples, directly and on fluorescence-encoded micro-beads in mixed liquor
Probe is hybridized, and as a result each fluorescence-encoded micro-beads for participating in counting are equal >=20 between 27~246, shows to use
Effective in the fluorescence-encoded micro-beads quantity of counting, generated MFI value is credible；The blank control MFI of each fluorescence-encoded micro-beads
Also < 3000, show that result is effective, test can be determined；RT-PCR react the MFI of amplified production between 7747~
Between 17824.Have between mixed probe and the RT-PCR amplified production of each double strains specificity hybridize, LQRR value between
Between 3.7~93.1, show to be the positive, detailed results are as shown in table 11.
Strain combines specific detection result to table 11 two-by-two
The RT-PCR amplified production of six strains combination after purification, is directly hybridized with mixed probe, as a result each ginseng
Fluorescence-encoded micro-beads with counting are equal >=20 between 28~215, show the fluorescence-encoded micro-beads foot for counting
Enough, generated MFI value is credible；The blank control MFI of each fluorescence-encoded micro-beads < 3000, shows each fluorescence-encoded micro-
The background noise value of ball is less high, and test can be determined；RT-PCR reacts the MFI of amplified production between 6848~16400.
Mixed probe and six strain RT-PCR amplified productions have hybridizing for specificity, LQRR value between 3~99.9, show be
It is positive.
The foregoing is a further detailed description of the present application in conjunction with specific implementation manners, and it cannot be said that this Shen
Specific implementation please is only limited to these instructions.For those of ordinary skill in the art to which this application belongs, it is not taking off
Under the premise of from the application design, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to the protection of the application
Range.
Claims (3)
1. a kind of kit for inhibiting pathogen Multiple detection for poultry immunities such as bird flus, it is characterised in that: the examination
Contain six specific probes, six groups of primer pairs and one group of super primer in agent box；
Six specific probes are respectively the avian influenza virus specific probe of sequence shown in Seq ID No.1, Seq ID No.2
1 type specificity of marek virus of sequence shown in the new castle disease virus specific probe of shown sequence, Seq ID No.3 is visited
The chicken of sequence shown in the infectious bursa of Fabricius virus specific probe of sequence shown in needle, Seq ID No.4, Seq ID No.5 passes
The J hypotype avian leukosis virus specific probe of sequence shown in metachromia anemia virus specific probe and Seq ID No.6；
In six specific probes, 5 ' ends of every specific probe all have amido modification；
Six groups of primer pairs be respectively expand the first primer of avian influenza virus to the second primer pair of, amplification newcastle disease virus, expand
The third primer pair for increasing 1 type of marek virus, the 4th primer pair for expanding infectious bursa of Fabricius virus, amplification are avian infectious poor
5th primer pair of blood virus, and the 6th primer pair of amplification J hypotype avian leukosis virus；The upstream of the first primer pair is under
Swimming primer is respectively sequence shown in Seq ID No.7 and Seq ID No.8, and the upstream and downstream primer of the second primer pair is respectively
Sequence shown in Seq ID No.9 and Seq ID No.10, the upstream and downstream primer of third primer pair is respectively Seq ID
Sequence shown in No.11 and Seq ID No.12, the upstream and downstream primer of the 4th primer pair are respectively Seq ID No.13 and Seq
Sequence shown in ID No.14, the upstream and downstream primer of the 5th primer pair are respectively Seq ID No.15 and Seq ID No.16 institute
Show sequence, the upstream and downstream primer of the 6th primer pair is respectively sequence shown in Seq ID No.17 and Seq ID No.18；
The upstream and downstream primer of the super primer is respectively sequence shown in Seq ID No.19 and Seq ID No.20, and
And 5 ' ends of the downstream primer of sequence shown in Seq ID No.20 have biotin labeling；
Seq ID No.1:5 '-TCAGGCCCCCTCAAAGCCGA-3 '
Seq ID No.2:5 '-TTCTCTAGCAGTGGGACAGCCTGC-3 '
Seq ID No.3:5 '-ACCGCCCCAAATTCGCACTTGA-3 '
Seq ID No.4:5 '-CAACCGGACCGGCGTCCATTC-3 '
Seq ID No.5:5 '-CACCTCAAGCGACTTCGACGAA-3 '
Seq ID No.6:5 '-ATTTCCGTTGTCCCAGGGGTGG-3 '
Seq ID No.7:
5’-CAGGCCACGTATTGTCATGCAGATGAGTCYTCTAACCGAGGTCG-3’
Seq ID No.8:
5’-TTCTTGGCGTTATGTCGCTGTGCAAARACAYYTTCMAGTCTCTG-3’
Seq ID No.9:
5’-CAGGCCACGTATTGTCATGCAGTGATGTGCTCGGACCTTC-3’
Seq ID No.10:
5’-TTCTTGGCGTTATGTCGCTGCCTGAGGAGAGGCATTTGCTA-3’
Seq ID No.11:
5’-CAGGCCACGTATTGTCATGCAGGTGAGCCAATCGGATA-3’
Seq ID No.12:
5’-TTCTTGGCGTTATGTCGCTGAAGAGAGAAGGAAC TCG-3’
Seq ID No.13:
5’-CAGGCCACGTATTGTCATGCTTCATACGGAGCCTTCTGATT-3’
Seq ID No.14:
5’-TTCTTGGCGTTATGTCGCTGACAATTAGCCCTGACCCT-3’
Seq ID No.15:
5’-CAGGCCACGTATTGTCATGC GCCGATTTTACGCCTTCA-3’
Seq ID No.16:
5’-TTCTTGGCGTTATGTCGCTG TACCGCTGTCTCCTCCG-3’
Seq ID No.17:
5’-CAGGCCACGTATTGTCATGCAGAAAGACCCGGAGAAGAC-3’
Seq ID No.18:
5’-TTCTTGGCGTTATGTCGCTGACACGTTTCCTGGTTGTT-3’
Seq ID No.19:5 '-CAGGCCACGTATTGTCATGC-3 '
Seq ID No.20:5 '-TTCTTGGCGTTATGTCGCTG-3 '.
2. according to claim 1 inhibit the kit of pathogen Multiple detection to be in for poultry immunities such as bird flus
It is non-in the detection of multiple gene chip, the detection of multiple liquid-phase chip or the detection of multiple Standard PCR of fowl inhibitive ability of immunity pathogen
The application of diagnoses and treatment purpose.
3. a kind of multiple detection method for the non-diagnostic therapeutic purposes for inhibiting pathogen for poultry immunities such as bird flus, special
Sign is: includes the following steps,
(1) the microballoon coupling of numbers different from six of liquid-phase chip respectively, six specificity by six specific probes
Probe distinguishes the newcastle disease of sequence shown in the avian influenza virus specific probe of sequence shown in Seq ID No.1, Seq ID No.2
Shown in the 1 type specificity probe of marek virus of sequence shown in virus-specific probe, Seq ID No.3, Seq ID No.4
The Chicken Infectious Anemia Virus specificity of sequence shown in the infectious bursa of Fabricius virus specific probe of sequence, Seq ID No.5
The J hypotype avian leukosis virus specific probe of sequence shown in probe and Seq ID No.6；
(2) mix primer for using six groups of primer pairs carries out nucleic acid amplification to sample to be tested, and six groups of primer pairs are respectively to expand
Increase avian influenza virus the first primer to, amplification newcastle disease virus the second primer pair, expand 1 type of marek virus third
Primer pair, the 4th primer pair for expanding infectious bursa of Fabricius virus, the 5th primer pair for expanding Chicken Infectious Anemia Virus, and
Expand the 6th primer pair of J hypotype avian leukosis virus；The upstream and downstream primer of the first primer pair is respectively Seq ID No.7
With sequence shown in Seq ID No.8, the upstream and downstream primer of the second primer pair is respectively Seq ID No.9 and Seq ID
Sequence shown in No.10, the upstream and downstream primer of third primer pair are respectively shown in Seq ID No.11 and Seq ID No.12
Sequence, the upstream and downstream primer of the 4th primer pair are respectively sequence shown in Seq ID No.13 and Seq ID No.14, and the 5th
The upstream and downstream primer of primer pair is respectively sequence shown in Seq ID No.15 and Seq ID No.16, the 6th primer pair it is upper
Trip and downstream primer are respectively sequence shown in Seq ID No.17 and Seq ID No.18；
(3) there is the microballoon of six specific probes using the coupling of step (1), liquid phase core is carried out to the amplified production of step (2)
Piece detection；
It further include before step (3), using one group of super primer, a PCR being carried out to the amplified production of step (2) again and is expanded
Increase, pcr amplification product is then used for liquid-phase chip detection；The upstream and downstream primer of the super primer is respectively Seq ID
Sequence shown in No.19 and Seq ID No.20；In the super primer, the 5 ' of the downstream primer of sequence shown in Seq ID No.20
End has biotin labeling.
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| CN1858249A (en) * | 2006-03-21 | 2006-11-08 | 中国科学院武汉病毒研究所 | Method for detecting bird flue virus H5N1 subtype based on liquid phase chip |
| CN101824488A (en) * | 2010-04-09 | 2010-09-08 | 山东农业大学 | Kit for detecting chicken multipartite virus by nucleic acid probe and dot hybridization |
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| CN1858249A (en) * | 2006-03-21 | 2006-11-08 | 中国科学院武汉病毒研究所 | Method for detecting bird flue virus H5N1 subtype based on liquid phase chip |
| CN101824488A (en) * | 2010-04-09 | 2010-09-08 | 山东农业大学 | Kit for detecting chicken multipartite virus by nucleic acid probe and dot hybridization |
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| Title |
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| Simultaneous detection of eight immunosuppressive chicken viruses using a GeXP analyser-based multiplex PCR assay;Tingting Zeng等;《Virology Journal》;20151230;第12卷;1-12 |
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