CN107475462A - A kind of kit for differentiating the infection of common serotype chicken inclusion body hepatitis and its application - Google Patents
A kind of kit for differentiating the infection of common serotype chicken inclusion body hepatitis and its application Download PDFInfo
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Abstract
The invention belongs to technical field of biological, and in particular to a kind of kit for differentiating the infection of common serotype chicken inclusion body hepatitis and its application.The detection kit of the present invention for differentiating the infection of common serotype chicken inclusion body hepatitis, utilize the characteristics of round pcr is quick, accurate, sensitiveness is high, divide in same system intermediate energy region the chicken inclusion body hepatitis of 8a types, 8b types and 11 types these three domestic Major Epidemics using three pairs of specific primers of design, chicken inclusion body hepatitis serum 8a types, the infection of 8b types and 11 types virus or above-mentioned viral mixed infection can quickly be distinguished, clinically still belong to the first time report, and there are time saving, laborsaving, low cost and other advantages, there is important application value.
Description
Technical field
The invention belongs to technical field of biological, and in particular to one kind differentiates the infection of common serotype chicken inclusion body hepatitis
Kit and its application.
Background technology
Chicken inclusion body hepatitis (Inclusion body hepatitis in chicken, IBH) is by I group I fowl adenovirus
Cause the acute infectious disease of chicken, be mainly in the chicken group of 2-5 week old, wherein again most multiple with 3-4 week old, cardinal symptom is that chicken group is dead
Die rate increases suddenly, and the incidence of disease can reach 10-20%, but the death rate is relatively low, be usually no more than 5%, generally morbidity after 5-7 days again
Recover normal.Infect ill chicken it is main show as the shapes such as depressed spirit, poor appetite, drowsiness, cockscomb turn white, feather is mixed and disorderly；
Cut open inspection is visible to be turned to be yellow with liver, enlargement, become fragile etc. be main characteristics of lesion inclusion body hepatitis, it is less hydropericardium, portion occur
Point kidney enlargement, in faint yellow, spleen is also with enlargement, and marrow is in pale red to faint yellow；And most significant pathological change is
There is inclusion body in liver cell nuclear.
The disease is popular in China's broiler chicken in recent years and has the trend of rising, at present, is led in China by I group I fowl adenovirus
The disease of cause mainly has two kinds：First, hydropericardium-hepatitis syndrome, is mainly caused by the type of serum 4；Second, inclusion body hepatitis,
It is mainly caused by serum 8a types (FAV-8a), 8b (FAV-8b) types and 11 types (FAV-11) virus, but this 3 kinds of serotypes are intersected
Protectiveness is very low, and cannot be distinguished by from clinical symptoms and lesion, and the preventing and treating sick to this brings great difficulty, also urgent
A kind of method for differentiating the infection of common serotype chicken inclusion body hepatitis is needed, to improve the treatment specific aim of inclusion body hepatitis.
The content of the invention
Therefore, the technical problems to be solved by the invention are to provide a kind of discriminating common serotype chicken inclusion body hepatitis sense
The kit of dye, to solve the problems, such as that chicken inclusion body hepatitis is difficult to differentiate between in production.
Second technical problem that the present invention solves is to provide a kind of discriminating common serotype chicken inclusion body hepatitis infection
Methods and applications
A kind of in order to solve the above technical problems, examination for differentiating the infection of common serotype chicken inclusion body hepatitis of the present invention
Agent box, the kit is with 100 times with gauge, including following component：
The μ L of PCR reaction solutions system 4800：
The μ L of positive plasmid 200；
The positive plasmid be final concentration be 0.1ng/ μ L recombinant plasmid T-8a, T-8b and T-11 mixture.
The PCR reaction solutions system includes following component：
The detection primer FAV8a-1/FAV8a-2 is used to detect the group I fowl adenovirus of serum 8a types I, and amplified fragments size is
292bp；
The detection primer FAV8a-1/FAV8a-2 has following sequential structure：
FAV8a-1：5'-AACCCCTATGAGAATACCACT-3'；
FAV8a-2：5'-CCAAATATTTCGTGCTCCC-3'.
The detection primer FAV8b-1/FAV8b-2 is used to detect the group I fowl adenovirus of serum 8b types I, and amplified fragments size is
208bp；
The detection primer FAV8b-1/FAV8b-2 has following sequential structure：
FAV8b-1：5'-GCCAGCGTTTCAGGCTCT-3'；
FAV8b-2：5'-CGTAGTAAGGCGTTGTTCCA-3'.
The detection primer FAV11-1/FAV11-2 is used to detect the group I fowl adenovirus of 11 type of serum I, and amplified fragments size is
474bp；
The detection primer FAV11-1/FAV11-2 has following sequential structure respectively：
FAV11-1：5'-GAAGACTTTAGCGCCTCG-3'；
FAV11-2：5'-CTCGGTTCCCTTATCACCC-3'.
Described recombinant plasmid T-8a, T-8b and T-11 are prepared as follows：Respectively extract FAV-8a, FAV-8b and
FAV11 viral nucleic acids, and be utilized respectively primers F AV8a-1/FAV8a-2, FAV8b-1/FAV8b-2, FAV11-1/FAV11-2 and enter
Row positive PCR amplification purpose band, purpose band is reclaimed, is connected with carrier pMD18-T, converts to DH5 α competent cells,
And plasmid is extracted, send sequencing after PCR identifications are positive；It is T-8a, T-8b and T-11 to take the correct plasmid markers of sequence, respectively as
The positive control of FAV-8a, FAV-8b and FAV11 virus.
The positive PCR amplification step reaction system (50 μ L) includes following component：
ddH2μ L of O 38.5, the μ L of 10 × PCR buffer solutions 5, the μ L of 2.5mM dNTP 3, the μ of 5U/ μ L Taq archaeal dna polymerases 0.5
L, each 0.5 μ L of 20pmol/ μ L upstream and downstream primers, the μ L of sample nucleic 2；
The pcr amplification reaction program is：95 DEG C of 5min pre-degenerations；94 DEG C denaturation 15sec, 55 DEG C annealing 15sec, 72 DEG C
Extend 30sec, totally 30 circulations；72 DEG C of extension 5min.
The application method of the kit infected the invention also discloses described discriminating common serotype chicken inclusion body hepatitis,
Comprise the following steps：
(1) sample nucleic extracts：200 μ L pathological material of disease homogenates to be measured are taken, sample is extracted using Viral nucleic acid extraction reagent box
Nucleic acid, it is standby；
(2) PCR reacts：PCR reaction solutions 48 μ L, the μ L of sample nucleic 2 are taken, is carried out after mixing by following program
PCR reacts：95 DEG C of 5min pre-degenerations；94 DEG C of denaturation 15sec, 55 DEG C of annealing 15sec, 72 DEG C of extension 30sec, totally 30 circulations；
72 DEG C of extension 5min；
(3) positive control：The μ L of PCR reaction solutions 48 are taken, the μ L of positive plasmid 2 is added, following journey is pressed after mixing
Sequence is reacted：95 DEG C of 5min pre-degenerations；94 DEG C of denaturation 15sec, 55 DEG C of annealing 15sec, 72 DEG C of extension 30sec, totally 30 are followed
Ring；72 DEG C of extension 5min；
(4) electrophoresis observation：The above-mentioned PCR primers of 5 μ L and positive products are taken to add and carry out electricity in 1.5 ℅ Ago-Gels respectively
Swimming observation, if there is 292bp band, can determine whether as FAV-8a virus-positives；If there is 208bp band, can determine whether for
FAV-8b virus-positives；If there is 474bp band, can determine whether as FAV-11 virus-positives；If above-mentioned each band mixedly appears,
It is then mixed infection.
The invention also discloses the described kit for differentiating the infection of common serotype chicken inclusion body hepatitis is common in discriminating
Application in the infection of serotype chicken inclusion body hepatitis.
The invention also discloses a kind of method for differentiating the infection of common serotype chicken inclusion body hepatitis, including the use of the examination
The step of agent box is differentiated.
The detection kit of the present invention for differentiating the infection of common serotype chicken inclusion body hepatitis, using round pcr it is quick,
Accurately, the characteristics of sensitiveness is high, 8a types, 8b types and 11 are divided in same system intermediate energy region using three pairs of specific primers of design
The chicken inclusion body hepatitis of these three domestic Major Epidemics of type, it can quickly distinguish chicken inclusion body hepatitis serum 8a types, 8b types and 11
The infection or above-mentioned viral mixed infection of type virus, clinically still belong to the first time report, and has time saving, laborsaving, cost
Low advantage, there is important application value.
The detection kit of the present invention for differentiating the infection of common serotype chicken inclusion body hepatitis, to three kinds of serotypes
Limit of identification be below 10pg, have the advantages that sensitiveness is high, specificity is good.
Brief description of the drawings
In order that present disclosure is more likely to be clearly understood, specific embodiment and combination below according to the present invention
Accompanying drawing, the present invention is further detailed explanation, wherein,
Fig. 1 is the specific detection result of kit of the present invention；Wherein, M is DNA Marker DL2000,1-10
Corresponding virus is respectively I group of group I fowl adenovirus of 8a types I, the group I fowl adenovirus of 8b types I, the group I fowl adenovirus of 11 type I, 8a types fowl gland
Virus and I group of the group I fowl adenovirus mixture of 8b types I, the group I fowl adenovirus of 8a types I and the group I fowl adenovirus mixture of 11 type I, 8b types fowl
Adenovirus and I group of the group I fowl adenovirus mixture of 11 type I, the group I fowl adenovirus of 8a types I, the group I fowl adenovirus of 8b types I and 11 types fowl adenopathy
Malicious mixture, the group I fowl adenovirus of 1 type I, the group I fowl adenovirus of 2 type I, the group I fowl adenovirus of 4 type I；
Fig. 2 is sensitivity Detection result of the kit of the present invention for the group I fowl adenovirus of 8a types I；Wherein, M DNA
The content of viral DNA corresponding to Marker DL2000,1-7 be respectively 17.2ng, 1.72ng, 172pg, 17.2pg,
1.72pg、0.172pg、0.0172pg；
Fig. 3 is sensitivity Detection result of the kit of the present invention for the group I fowl adenovirus of 8b types I；Wherein, M DNA
The content of viral DNA corresponding to Marker DL2000,1-7 be respectively 8.6ng, 0.86ng, 86pg, 8.6pg, 0.86pg,
0.086pg、0.0086pg；
Fig. 4 is sensitivity Detection result of the kit of the present invention for the group I fowl adenovirus of 11 type I；Wherein M is DNA
The content of viral DNA corresponding to Marker DL2000,1-7 be respectively 10.5ng, 1.05ng, 105pg, 10.5pg,
1.05pg、0.105pg、0.0105pg；
Fig. 5 is replica test of the kit of the present invention for the group I fowl adenovirus of 8a types I；Wherein, M DNA
Corresponding to Marker DL2000,1-3 is to repeat testing result three times；
Fig. 6 is replica test of the kit of the present invention for the group I fowl adenovirus of 8b types I；Wherein, M DNA
Corresponding to Marker DL2000,1-3 is to repeat testing result three times；
Fig. 7 is replica test of the kit of the present invention for the group I fowl adenovirus of 11 type I；Wherein, M DNA
Corresponding to Marker DL2000,1-3 is to repeat testing result three times.
Embodiment
The design of the detection primer of embodiment 1
According to the conserved sequence of listed 8a types, 8b types and the group I fowl adenovirus Hexon genes of 11 type I in Genbank, profit
Pair of primers is separately designed with Primer5.0 softwares, the detection for 8a types, 8b types and the group I fowl adenovirus of 11 type I.
The primer for being used to detect the group I fowl adenovirus of serum 8a types I is designated as FAV8a-1/FAV8a-2, amplified fragments size
For 292bp, its sequential structure includes：
FAV8a-1 (sense primer)：5'-AACCCCTATGAGAATACCACT-3'；
FAV8a-2 (anti-sense primer)：5'-CCAAATATTTCGTGCTCCC-3'.
The primer for being used to detect the group I fowl adenovirus of serum 8b types I is designated as FAV8b-1/FAV8b-2, amplified fragments size
For 208bp, its sequential structure includes：
FAV8b-1 (sense primer)：5'-GCCAGCGTTTCAGGCTCT-3'；
FAV8b-2 (anti-sense primer)：5'-CGTAGTAAGGCGTTGTTCCA-3'.
The primer for being used to detect the group I fowl adenovirus of 11 type of serum I is designated as FAV11-1/FAV11-2, amplified fragments size
For 474bp, its sequential structure includes：
FAV11-1 (sense primer)：5'-GAAGACTTTAGCGCCTCG-3'；
FAV11-2 (anti-sense primer)：5'-CTCGGTTCCCTTATCACCC-3'.
The specificity of the multiplex PCR of embodiment 2
Serum 1 type, 2 types, 4 types, 8a types, 8b types and I group, 11 types are extracted respectively according to Viral nucleic acid extraction reagent box explanation
The nucleic acid of aviadenovirus, and enter performing PCR reaction respectively as template.
The PCR reaction systems (50 μ L) of each viral nucleic acid include：ddH2μ L of O 38.5, μ L of 10 × PCR buffer solutions 5,2.5mM
The μ L of dNTP 3, the μ L of 5U/ μ L Taq archaeal dna polymerases 0.5, each 0.5 μ L of 20pmol/ μ L upstream and downstream primers, the μ L of sample nucleic 2.It is placed in
PCR amplification instrument is reacted, response procedures：95 DEG C of 5min pre-degenerations；94 DEG C denaturation 15sec, 55 DEG C annealing 15sec, 72 DEG C prolong
30sec is stretched, totally 30 circulations；72 DEG C of extension 5min.
After reaction terminates, take 5 μ L PCR primers to add in 1.5 ℅ Ago-Gels and carry out electrophoresis observation, as a result such as Fig. 1 institutes
Show.It can be seen that multiple PCR method of the present invention can amplify size 292bp band from 8a type viruses, from 8b type viruses
Size 208bp band is amplified, size 474bp band is amplified from 11 type viruses, from 8a types and 8b type hybrid viruses
In amplify the band of size 292bp and 208bp two, amplify size 292bp and 474bp from 8a types and 11 type hybrid viruses
Two band, the band of size 208bp and 474bp two is amplified from 8b types and 11 type hybrid viruses, from 8a types, 8b types and 11
The band of size 292bp, 208bp and 474bp tri- is amplified in type hybrid virus, and is expanded from 1 type, 2 types, 4 types and 5 type viruses
It can not increase any band.
The sensitiveness of the multiplex PCR of embodiment 3
Illustrate to extract serum 8a types, 8b types and 11 types respectively according to Viral nucleic acid extraction reagent box (AXYGEN Products)
The nucleic acid of I group I fowl adenovirus, 10 times of dilutions are carried out using spectrophotometric determination DNA content, and by these DNA, utilization is excellent
After the reaction condition progress pcr amplification reaction changed terminates, PCR primer described in 5 μ L is respectively taken to add in 1.5 ℅ Ago-Gels
Row electrophoresis observation, as a result respectively as in Figure 2-4.It can be seen that the limit of identification of 8a type viral DNAs is 17.2pg, 8b types virus
DNA limit of identification is 8.6pg, and the limit of identification of 11 type viral DNAs is 10.5pg.
The repeatability of the multiplex PCR of embodiment 4
The pathological material of disease of 8a types, 8b types and 11 type virus-positives is expanded respectively using the multiple PCR method, respectively repeatedly three
It is secondary, its repeatability is determined, as a result as shown in accompanying drawing 5-7.It can be seen that through repeating amplify purpose band three times, and band
Brightness is basically identical.
Embodiment 5 differentiates the kit of common serotype chicken inclusion body hepatitis infection
Differentiate the kit of common serotype chicken inclusion body hepatitis infection described in the present embodiment, in terms of 100 dosages,
Including following component：
(1) the μ L of PCR reaction solutions 4800, are specifically included：
(2) positive plasmid：The μ L of recombinant plasmid T-8a, T-8b and T-11 mixture 200, the final concentration of each recombinant plasmid are
0.1ng/μL；
Described recombinant plasmid T-8a, T-8b and T-11 detailed preparation method are as follows：
Illustrate to extract FAV-8a, FAV-8b and FAV11 disease according to Viral nucleic acid extraction reagent box (AXYGEN Products)
Malicious nucleic acid, it is utilized respectively above-mentioned primers F AV8a-1/FAV8a-2, FAV8b-1/FAV8b-2, FAV11-1/FAV11-2 and carries out PC
R expands purpose band, and reaction condition is as follows：
PCR reaction systems (50 μ L)：ddH2μ L of O 38.5, the μ L of 10 × PCR buffer solutions 5,2.5mMdNTP 3 μ L, 5U/ μ L
Each 0.5 μ L of the μ L of Taq archaeal dna polymerases 0.5,20pmol/ μ L upstream and downstream primers, the μ L of sample nucleic 2；PCR amplification instrument is placed in carry out instead
Should, response procedures：95 DEG C of 5min pre-degenerations；94 DEG C of denaturation 15sec, 55 DEG C of annealing 15sec, 72 DEG C of extension 30sec, totally 30 are followed
Ring；72 DEG C of extension 5min；
Purpose band is reclaimed, is connected with carrier pMD18-T, converts DH5 α competent cells, extracts plasmid, PCR identification sun
Property after serve Hai Ying fine horses Bioisystech Co., Ltd sequencing.The correct plasmid markers of sequence are T-8a, T-8b and T-11, are made respectively
For the positive control of FAV-8a, FAV-8b and FAV11 virus；
Recombinant plasmid T-8a, T-8b and T-11 concentration are determined respectively, is diluted to 0.3ng/ μ L, and mixed in equal amounts is dense eventually
Degree is 0.1ng/ μ L.
Embodiment 6 differentiates the concrete operations of common serotype chicken inclusion body hepatitis infection using kit
Differentiate the method for common serotype chicken inclusion body hepatitis infection described in the present embodiment, utilize the examination described in embodiment 5
Agent box is carried out, and concrete operation step is as follows：
(1) sample nucleic extracts：200 μ L pathological material of disease homogenates to be measured are taken, (AXYGEN is public according to Viral nucleic acid extraction reagent box
Department's product) illustrate to extract viral nucleic acid, it is standby；
(2) PCR reacts：The μ L of PCR reaction solutions 48 in kit are taken, the μ L of sample nucleic 2 is added, following journey is pressed after mixing
Sequence is reacted：95 DEG C of 5min pre-degenerations；94 DEG C of denaturation 15sec, 55 DEG C of annealing 15sec, 72 DEG C of extension 30sec, totally 30 are followed
Ring；72 DEG C of extension 5min；
(3) positive control：The μ L of PCR reaction solutions 48 in kit are taken, add the μ L of positive plasmid 2, by following after mixing
Program is reacted：95 DEG C of 5min pre-degenerations；94 DEG C denaturation 15sec, 55 DEG C annealing 15sec, 72 DEG C extension 30sec, totally 30
Circulation；72 DEG C of extension 5min；
(4) electrophoresis observation：Take the above-mentioned PCR primers of 5 μ L to add in 1.5 ℅ Ago-Gels and carry out electrophoresis observation, if occurring
Size 292bp band, it can determine whether as FAV-8a virus-positives；If there is size 208bp band, can determine whether as FAV-8b diseases
It is malicious positive；If there is size 474bp band, can determine whether as FAV-11 virus-positives；If size 292bp and 208bp band
Occur, can determine whether as FAV-8a and FAV-8b virus mixed infections；If size 292bp and 474bp band occur, can sentence
Break as FAV-8a and FAV-11 virus mixed infections；If size 208bp and 474bp band occur, can determine whether as FAV-8b
With FAV-11 virus mixed infections；If size 292bp, 208bp and 474bp band occur, can determine whether as FAV-8a, FAV-
8b and FAV-11 virus three's mixed infections.
The clinical practice of embodiment 7
The sample that the present embodiment clinical practice is examined is 50 parts of the sample of different times difference chicken house censorship.Using this reagent
Box and Virus Isolation method detect two methods of submitted sample, the coincidence rate situation of lateral comparison simultaneously.
As a result show, it is 4 parts to detect 8a type virus-positives pathological material of disease using this kit according to step in embodiment 6, positive
Recall rate is 8%, and utilizes isolation of virus identification to detect positive 4 parts of pathological material of disease, positive rate 8%, the symbol of two methods
Conjunction rate is 100% (shown in table 1 specific as follows).
The kit of table 1 (FAV-8a) compared with Virus Isolation
Note：Detect coincidence rate=(common number positive+common negative number)/gross sample number × 100%
As a result show, this kit detection 8b type virus-positives pathological material of disease is 7 parts, positive rate 14%, and virus point
Positive 6 parts of pathological material of disease is detected from identification, positive rate 12%, the coincidence rate of two methods is the 98% (institute of table 2 specific as follows
Show).
The kit of table 2 (FAV-8b) compared with Virus Isolation
Note：Detect coincidence rate=(common number positive+common negative number)/gross sample number × 100%
As a result show, it is 4 parts that this kit, which detects 11 type virus-positive pathological material of diseases, positive rate 8%, and virus purification
Identification detects positive 3 parts of pathological material of disease, and positive rate 6%, the coincidence rate of two methods is 98% (as shown in table 3 below).
The kit of table 3 (FAV-11) compared with Virus Isolation
Note：Detect coincidence rate=(common number positive+common negative number)/gross sample number × 100%
Obviously, above-described embodiment is only intended to clearly illustrate example, and is not the restriction to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or
Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Claims (10)
1. a kind of kit for differentiating the infection of common serotype chicken inclusion body hepatitis, it is characterised in that the kit is with 100 times
With gauge, including following component：
The μ L of PCR reaction solutions system 4800：
The μ L of positive plasmid 200；
The positive plasmid be final concentration be 0.1ng/ μ L recombinant plasmid T-8a, T-8b and T-11 mixture.
2. the kit according to claim 1 for differentiating the infection of common serotype chicken inclusion body hepatitis, it is characterised in that
PCR reaction solutions system includes following component：
3. the kit according to claim 2 for differentiating the infection of common serotype chicken inclusion body hepatitis, it is characterised in that institute
State detection primer FAV8a-1/FAV8a-2 to be used to detect the group I fowl adenovirus of serum 8a types I, amplified fragments size is 292bp；
The detection primer FAV8a-1/FAV8a-2 has following sequential structure：
FAV8a-1：5'-AACCCCTATGAGAATACCACT-3'；
FAV8a-2：5'-CCAAATATTTCGTGCTCCC-3'.
4. the kit for differentiating the infection of common serotype chicken inclusion body hepatitis according to Claims 2 or 3, its feature exist
In the detection primer FAV8b-1/FAV8b-2 is used to detect the group I fowl adenovirus of serum 8b types I, and amplified fragments size is
208bp；
The detection primer FAV8b-1/FAV8b-2 has following sequential structure：
FAV8b-1：5'-GCCAGCGTTTCAGGCTCT-3'；
FAV8b-2：5'-CGTAGTAAGGCGTTGTTCCA-3'.
5. the kit for differentiating the infection of common serotype chicken inclusion body hepatitis according to claim any one of 2-4, it is special
Sign is that the detection primer FAV11-1/FAV11-2 is used to detect the group I fowl adenovirus of 11 type of serum I, and amplified fragments size is
474bp；
The detection primer FAV11-1/FAV11-2 has following sequential structure respectively：
FAV11-1：5'-GAAGACTTTAGCGCCTCG-3'；
FAV11-2：5'-CTCGGTTCCCTTATCACCC-3'.
6. the kit for differentiating the infection of common serotype chicken inclusion body hepatitis according to claim any one of 2-5, it is special
Sign is that described recombinant plasmid T-8a, T-8b and T-11 are prepared as follows：Respectively extract FAV-8a, FAV-8b and
FAV11 viral nucleic acids, and be utilized respectively primers F AV8a-1/FAV8a-2, FAV8b-1/FAV8b-2, FAV11-1/FAV11-2 and enter
Row positive PCR amplification purpose band, purpose band is reclaimed, is connected with carrier pMD18-T, converts to DH5 α competent cells,
And plasmid is extracted, send sequencing after PCR identifications are positive；It is T-8a, T-8b and T-11 to take the correct plasmid markers of sequence, respectively as
The positive control of FAV-8a, FAV-8b and FAV11 virus.
7. the kit according to claim 6 for differentiating the infection of common serotype chicken inclusion body hepatitis, it is characterised in that institute
Stating positive PCR amplification step reaction system (50 μ L) includes following component：
ddH2μ L of O 38.5, the μ L of 10 × PCR buffer solutions 5, the μ L of 2.5mM dNTP 3, μ L of 5U/ μ L Taq archaeal dna polymerases 0.5,
Each 0.5 μ L of 20pmol/ μ L upstream and downstream primers, the μ L of sample nucleic 2；
The pcr amplification reaction program is：95 DEG C of 5min pre-degenerations；94 DEG C of denaturation 15sec, 55 DEG C of annealing 15sec, 72 DEG C of extensions
30sec, totally 30 circulations；72 DEG C of extension 5min.
8. the user of the kit for differentiating the infection of common serotype chicken inclusion body hepatitis as described in claim any one of 1-7
Method, it is characterised in that comprise the following steps：
(1) sample nucleic extracts：200 μ L pathological material of disease homogenates to be measured are taken, sample nucleic is extracted using Viral nucleic acid extraction reagent box,
It is standby；
(2) PCR reacts：The μ L of PCR reaction solutions 48 are taken, the μ L of sample nucleic 2, enter performing PCR by following program after mixing
Reaction：95 DEG C of 5min pre-degenerations；94 DEG C of denaturation 15sec, 55 DEG C of annealing 15sec, 72 DEG C of extension 30sec, totally 30 circulations；72℃
Extend 5min；
(3) positive control：The μ L of PCR reaction solutions 48 are taken, the μ L of positive plasmid 2 is added, enters after mixing by following program
Row reaction：95 DEG C of 5min pre-degenerations；94 DEG C of denaturation 15sec, 55 DEG C of annealing 15sec, 72 DEG C of extension 30sec, totally 30 circulations；72
DEG C extension 5min；
(4) electrophoresis observation：The above-mentioned PCR primers of 5 μ L and positive products are taken to add and carry out electrophoresis sight in 1.5 ℅ Ago-Gels respectively
Examine, if there is 292bp band, can determine whether as FAV-8a virus-positives；If there is 208bp band, can determine whether as FAV-8b
Virus-positive；If there is 474bp band, can determine whether as FAV-11 virus-positives；If the fragment band of above-mentioned variant size
Mixedly appear, be then mixed infection.
9. the kit for differentiating the infection of common serotype chicken inclusion body hepatitis described in claim any one of 1-7 is common in discriminating
Application in the infection of serotype chicken inclusion body hepatitis.
A kind of 10. method for differentiating the infection of common serotype chicken inclusion body hepatitis, it is characterised in that including the use of claim 1-
The step of any one of 7 kits are differentiated.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110499296A (en) * | 2019-08-28 | 2019-11-26 | 扬州大学 | A kind of heat-resisting serum 8b type aviadenovirus recombinant vaccine Candidate Strain and its construction method |
| CN111575412A (en) * | 2020-06-23 | 2020-08-25 | 青岛易邦生物工程有限公司 | Method for distinguishing four serotypes of avian adenovirus group I |
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| CN102230029A (en) * | 2011-06-17 | 2011-11-02 | 河南省动物疫病预防控制中心 | Ternary RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and method of type O, type A and type Asial foot and mouth disease viruses |
| US20130295126A1 (en) * | 2009-12-15 | 2013-11-07 | University Of Guelph | Vaccines for inclusion body hepatitis |
| CN106435031A (en) * | 2016-11-16 | 2017-02-22 | 湖北省农业科学院畜牧兽医研究所 | PCR detection kit for specific detection of fowl adenovirus group I and detection method thereof |
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| US20130295126A1 (en) * | 2009-12-15 | 2013-11-07 | University Of Guelph | Vaccines for inclusion body hepatitis |
| CN102230029A (en) * | 2011-06-17 | 2011-11-02 | 河南省动物疫病预防控制中心 | Ternary RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and method of type O, type A and type Asial foot and mouth disease viruses |
| CN106435031A (en) * | 2016-11-16 | 2017-02-22 | 湖北省农业科学院畜牧兽医研究所 | PCR detection kit for specific detection of fowl adenovirus group I and detection method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110499296A (en) * | 2019-08-28 | 2019-11-26 | 扬州大学 | A kind of heat-resisting serum 8b type aviadenovirus recombinant vaccine Candidate Strain and its construction method |
| CN111575412A (en) * | 2020-06-23 | 2020-08-25 | 青岛易邦生物工程有限公司 | Method for distinguishing four serotypes of avian adenovirus group I |
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