CN105886502A - Primer pair for preparing kit for detecting type-4 avian adenovirus and application thereof - Google Patents
Primer pair for preparing kit for detecting type-4 avian adenovirus and application thereof Download PDFInfo
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Abstract
The invention discloses a primer pair for preparing a kit for detecting type-4 avian adenovirus and an application thereof. The primer pair comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown by SEQ ID No.1; and the nucleotide sequence of the downstream primer is shown by SEQ ID No.2. The invention also discloses a kit comprising the primer pair and a method for detecting the type-4 avian adenovirus. The primer pair can specifically amplify the Hexon gene segment of the type-4 avian adenovirus in a sample, the detection sensitivity is high, the lowest detection limit reaches 7.2*10<4>ng/mu L, the result is accurate, and the primer pair can be applied to the detection and identification of the type-4 avian adenovirus; the primer pair is universal in detecting different strains of the type-4 avian adenovirus; and the rapid, specific and accurate detection method of the type-4 avian adenovirus provides a detection tool for detecting and controlling the prevalence of the disease.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of for prepare detection aviadenovirus 4 type test kit primer to and
Its application.
Background technology
Aviadenovirus (Fowl adenovirus, FAdV) is a kind of double-stranded DNA virus without cyst membrane, can be divided into I, II, III 3
Group, wherein I crowd of FAdV has 12 serotypes (FAV1～12).Pathogenic to birds of the different serotypes of I group I fowl adenovirus
Having nothing in common with each other, quick and precisely clinical diagnosis and treatment are of great significance by the different serotypes of differentiating I group I fowl adenovirus.
The major structural protein of aviadenovirus coding has six adjacent bodies (Hexon), penton (matrix, IIIa), fine prominent, pV I, pV
Ⅱ、pVⅢ.Have proven to hexon with main genus and subgenus specific epitope and secondary species-specific antigen
Determinant, can cause extremely strong neutralization to react, and is neutralized immune deficiency when six adjacent bodies substitute or suddenly change by causing.Therefore, Hexon
The albumen infection important in inhibiting to diagnosing I group I fowl adenovirus.
Li Haiying etc. (2011) have carried out Hexon complete genome sequence and have measured and polymerase chain reaction-restriction 12 serotype strains
Property fragment length polymorphism (PCR-RFLP) analyze.Three pairs of primers (A, B, C) are utilized to carry out 12 of I group I fowl adenovirus respectively
The Hexon complete genome sequence PCR amplification of serotype strain, and carry out sequencing and PCR-RFLP analysis, research shows,
Hexon full length gene is about 2800 nucleotide, encodes about 940 aminoacid, and the homology between different serotypes exists
75.2%-99.5%, range of variation is 0.5-34.6%.The amino acid identity derived exists in 20.7%-98.8%, diversity
1.1%-213.0%.Between 12 serotypes, Hexon complete genome sequence information is sent out for the germline evaluating I group I fowl adenovirus family
Educating relation, the cladogram of structure demonstrates five main branches.Select restricted enzyme Hae II, utilize fragment D special
Property can effectively distinguish major part serotype.
12 serotypes of I crowd of FAdV all may result in inclusion body hepatitis, but only 4 type adenoviruss (FAdV4) can cause pericardium
Hydrops, it is sick that this disease is referred to as hepatitis-pericardial effusion syndrome, also referred to as Ankara.
This disease has been shown in there is Sporadic cases the most as far back as 1985, and in March, 1987, the Ankara near Pakistan Karachi was regional
One broiler field occurs fulminant popular.Ankara disease is gained the name the most therefrom.This disease main harm 3～6 week old broiler, and standby
Hen and laying hen only occur once in a while.This disease is short for incubation period, and morbidity is anxious, involves rapidly full group after morbidity.Well-developed chicken group
Morbidity is serious.Sickness rate is high, population risk 100%, individual average attack rate 30%, and serious chicken group's sickness rate is higher than 80%.
Mortality rate is high, and average mortality 10%, chicken group's mortality rate that only a few is serious is up to 60%.
Ankara is sick is generally only fragmentary outburst in China.But since 2012, this disease is in China's nationwide report case
Rise, since in June, 2015 especially in Jiangsu, Anhui, Shandong, Liaoning, Henan, the ground such as Inner Mongol the most popular and
Break out this disease, bring the biggest economic loss to the poultry husbandry of China.Therefore, one is set up special, sensitive and quickly sick
Former detection method is significant for the qualification of aviadenovirus 4 type.
Summary of the invention
The invention provides a primer pair, the gene sheet of this primer Hexon to can specifically amplify aviadenovirus 4 type
Section, it is possible to for Rapid identification aviadenovirus 4 type.
Primer pair, including forward primer and downstream primer, the nucleotide sequence of forward primer as shown in SEQ ID NO.1, downstream
The nucleotide sequence of primer is as shown in SEQ ID NO.2.
Described primer is to being the gene design synthesis of structural protein Hexon according to aviadenovirus high conservative, to aviadenovirus
The detection of FAdV4 difference strain has versatility.
The invention provides the PCR detection kit of a kind of described primer pair, this test kit includes:
(1) DNA extraction reagent, including Digestion Buffer, BD Buffer, Proteinase K, PW Solution, Wash
Solution and Elution Buffer, carrys out spontaneous work UNIQ-10 pillar viral genome extraction agent box.
(2) PCR reaction reagent, including ExTaq enzyme, the upstream and downstream primer of 10mM of 20U/ μ L, the dNTP of 2.5mM,
10×PCR buffer；
Described forward primer is 5'-ATACCAACACGAGCACCTC-3'；Downstream primer is
5'-TTATCCCTGAACCCGATG-3'；
(3) positive control, can be selected for the nucleic acid samples of the FAdV4ZJ2015-1 strain that this laboratory separates, with this nucleic acid samples is
Template, utilize above-mentioned primer to carry out PCR amplification obtain 403bp product, nucleotide sequence as shown in SEQ ID NO.3,
Sequence analysis shows that the DNA fragmentation of amplification is Hexon genetic fragment；
(4) negative control, the DNA extracted in normal chick embryo allantoic liquid.
The invention provides a kind of described primer to the application in preparation detection aviadenovirus 4 type test kit.
The invention provides a kind of method detecting aviadenovirus 4 type, including:
(1) STb gene of testing sample is extracted；
(2) set up PCR reaction system, with described STb gene as template, use the primer described in claim 1 to carrying out PCR
Amplification；
(3) pcr amplification product is carried out detected through gel electrophoresis；
As occurred the band of 403bp in amplified production, then containing aviadenovirus 4 type in explanation testing sample, otherwise, explanation is treated
Without aviadenovirus 4 type in test sample product.
Described testing sample is birds embryo allantoic liquid or in vitro pathological tissue.Described in vitro pathological tissue be die of illness birds liver,
The segregative tissue such as spleen.From the birds that dies of illness, dissect the testing sample obtained carry out pretreatment, be used further to PCR amplification.
As preferably, described birds is chicken.
Described PCR reaction system includes: PCR reaction buffer is final concentration of 1 ×, each 0.2mM of ExTaq enzyme 1.0U, dNTP,
The each 0.4mM of upstream and downstream primer, the nucleotide sequence of described upstream and downstream primer is respectively such as SEQ ID NO.1 and SEQ ID NO.2
Shown in.
The condition of PCR reaction is: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 35
Individual circulation, 72 DEG C extend 10min.
With above-mentioned PCR reaction system and reaction condition, the lowest detection of aviadenovirus 4 type nucleic acid is limited to 0.0072ng/ μ L.
The present invention compared with prior art, has the advantage that
(1) the Hexon genetic fragment of the primer of present invention aviadenovirus 4 type to amplifying specifically in sample, detects sensitive
Degree height, lowest detectable limit reaches 7.2 × 10-4Ng/ μ L, result is accurate, can apply it to the detection to aviadenovirus 4 type and qualification
In the middle of.
(2) primer of the present invention has versatility to the detection of the different strains to aviadenovirus 4 type.
(3) present invention establishes a kind of detection method quick, special, aviadenovirus 4 type accurately, for stream to this disease from now on
Row detection and prevention and control provide detection instrument.
Accompanying drawing explanation
Fig. 1 is aviadenovirus 4 type PCR amplification figure of the present invention, and wherein M is DNA marker；1 divides for ZJ2015-1
From strain positive findings；2 and 3 is negative control.
Fig. 2 is the aviadenovirus 4 type PCR specific amplification result figure of embodiment 3, and wherein M is DNA marker；1-3
Separate strain (ZJ2015-1, ZJ2015-2, ZJ2015-3) for 3, aviadenovirus 4 type and infect allantoic fluid nucleic acid extraction thing PCR result；
4-9 respectively subtracts egg drop syndrome virus (adenovirus 1 type), pAeasy-1 adenovirus vector (Adenovirus Type 5), avian influenza
Poison, infectious bronchitis virus, newcastle disease virus and infections chicken cloacal bursa virus nucleic acid extraction thing PCR result.
Fig. 3 is the aviadenovirus PCR detection sensitivity result figure of embodiment 4, and wherein M is DNA marker；1,7.2 × 103
ng/μL；2,7.2 × 102ng/μL；3,7.2 × 101ng/μL；4,7.2 × 100ng/μL；5,7.2 × 10-1ng/μL；6,7.2 × 10-2
ng/μL；7,7.2 × 10-3ng/μL；8,7.2 × 10-4ng/μL；9,7.2 × 10-5ng/μL；10,7.2 × 10-6ng/μL；11,
7.2×10-7ng/μL。
Fig. 4 is the clinical sample testing result of embodiment 5 test kit, and wherein M is DNA marker；1-7 is 7 parts of duck clinics
The PCR testing result of sample.
Detailed description of the invention
Below in conjunction with specific embodiment, technical scheme is described in detail, but following embodiment is for illustration purposes only,
Rather than restriction the scope of the present invention.
The test kit composition that the present invention utilizes includes:
(1) DNA extraction reagent, including Digestion Buffer, BD Buffer, Proteinase K, PW Solution, Wash
Solution and Elution Buffer, carrys out spontaneous work UNIQ-10 pillar viral genome extraction agent box.
(2) PCR reaction reagent, draws including the ExTaq enzyme of 20u/ μ L, the I group I fowl adenovirus 4 type specificity upstream of 10mM
FAdV4 specific Down Stream primers F AdV-R of thing FAdV-F, 10mM, the dNTP of 2.5mM, 10 × buffer；
Described aviadenovirus general forward primer FAdV-F is 5'-ATACCAACACGAGCACCTC-3'；
Described aviadenovirus general downstream primer FAdV-R is 5'-TTATCCCTGAACCCGATG-3'；
(3) positive control: the nucleic acid samples that the chick embryo allantoic liquid of the new FAdV4ZJ2015-1 strain separated extracts；Negative control:
The nucleic acid samples that normal chick embryo allantoic liquid extracts.
Embodiment 1 DNA extracts
1, sample treatment
The process of tissue sample: the tissue of the easily separated FAdV4 such as chicken liver and spleen that takes to die of illness, after shredding with shears according to
1:3 volume ratio adds sterilizing PBS and is fully ground.By the tissue suspension that has been homogenized in-20 DEG C of (or lower temperature)-room temperature multigelations
After 3 times 12,000g is centrifuged 10min, takes supernatant 200 μ L in new sterile centrifugation tube.
Allantoic fluid process: collect virus infect chick embryo allantoic liquid carry out 12,000g is centrifuged 10min, take supernatant 200 μ L in
In new sterile centrifugation tube.Collect simultaneously and be uninfected by the allantoic fluid of Embryo Gallus domesticus as negative sample；FAdV4ZJ2015-1 infects allantois
Liquid is as positive control.
2, DNA extracting
The sample 200 μ L taking above-mentioned process adds in 1.5ml EP pipe, is subsequently adding 0.4mL Digestion Buffer and 20uL
Proteinase K, vibration mixing, room temperature stands 30-60min；Adding 0.2mL BD Buffer, fully mixing is (if produced
White precipitate, 70 DEG C of water-baths stand 10min, solubilized)；Add 400 μ L dehydrated alcohol, the most reverse mixing；By adsorption column
Putting into collecting pipe, move on in adsorption column with pipettor by solution and translucent fibre shape float, room temperature stands two minutes, 12000g
Centrifugal 3min；Abandoning waste liquid, adsorption column adds 500 μ L PW Solution (confirming before using the most illustratively to add isopropanol), 10000g
Centrifugal 1min；Abandon waste liquid in collecting pipe, adsorption column adds 500 μ L Wash Solution and (confirms before using the most illustratively to add nothing
Water-ethanol), 10000g is centrifuged 1min；Abandoning waste liquid in collecting pipe, adsorption column is put back to collecting pipe, 10000g is centrifuged 2min；Will
Adsorption column transfers to clean 1.5mL EP pipe, and toward adsorbed film central authorities add 30 μ L Elution Buffer (use front 60 DEG C pre-
Heat), room temperature stands 5min, 10000g and is centrifuged 1min；Collect eluent and be DNA solution.Ultraviolet spectrophotometer measures
React for PCR after DNA concentration.
Embodiment 2 PCR reacts
1, design of primers
According to the conserved sequence on FAdV4 structural protein Hexon gene, design and synthesized following primer:
FAdV-F is 5'-ATACCAACACGAGCACCTC-3'；
FAdV-R is 5'-TTATCCCTGAACCCGATG-3'；
The DNA fragmentation of anticipated amplification aviadenovirus 403bp.The sequence of described primer such as SEQ ID NO.1 and SEQ ID NO.2
Shown in.
2, PCR reaction
Take the DNA2.5 μ L of extracting, be separately added into primers F AdV-F of 10uM and each 2 μ L of primers F AdV-R of 10uM,
DNTP 4 μ L, 10 × buffer 5 μ L, the template 2.5 μ L of 2.5mM, adds water to 50 μ L；Above-mentioned PCR reaction system warp: 95 DEG C
Denaturation 5min, 95 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, 35 circulations, 72 DEG C of journeys extending 10min
Sequence carries out PCR amplification.
PCR primer is after the agarose gel electrophoresis of 1.5%, and DueRed dyes, and observes under ultraviolet light.Result such as Fig. 1 institute
Showing, FAdV4ZJ2015-1 infects the DNA sample of allantoic fluid extraction and occurs in that bright DNA band in the position of 403bp,
And negative sample does not has band to occur.
3, PCR primer sequence verification
Positive PCR primer is reclaimed in rubber tapping, after the PCR primer of recovery is connected to carrier T, send order-checking company to check order.Sequence
The result that row are analyzed shows, the fragment of amplification is 403bp, and its sequence is as shown in SEQ ID NO.3.Analyze through BLAST and also show
Showing, this sequence is the Hexon gene order of FAdV4.
The specificity verification of embodiment 3 test kit
The DNA and the common avian viral that take the extraction of 3 parts of positive pathological material of diseases from Shandong that this laboratory obtains include other blood
The chicken of the infection such as the clear adenovirus (1 type and 5 types) of type, bird flu virus, newcastle disease virus, avian infectious bronchitis virus
The DNA of embryo allantoic liquid extracting carries out PCR under the same conditions.
Result as in figure 2 it is shown, only extract the target DNA fragment expanding expection size DNA from 3 parts of pathological material of diseases, and from
Other fowl diseases virus selected does not amplifies any DNA band, illustrates that the PCR method set up has high specificity.
The sensitivity Detection of embodiment 4 test kit
The chick embryo allantoic liquid of aviadenovirus 4 type ZJ2015-1 virus strain infection extracts DNA, with FAdV-F and FAdV-R primer PCR
After.Product connecting carrier T, converts escherichia coli, picking is with the escherichia coli expanding propagation of positive recombiant plasmid, extracting restructuring
Plasmid, quantitatively after as the template of PCR sensitivity tests amplification.
Positive recombinant plasmid dna concentration with FAdV4 pcr amplified fragment is 7.2 × 103Ng/ μ L, is carried out 10 times
The laggard performing PCR of serial dilution reacts.
Result such as Fig. 3 shows, template is diluted to 7.2 × 10-4Ng/ μ L, moreover it is possible to see that obvious PCR expands purpose fragment.Therefore,
The aviadenovirus 4 type PCR method that the present invention sets up at least can detect the 0.00072ng in every microlitre that is 7.2 × 10-4Ng's
DNA。
The clinical sample detection of embodiment 5 test kit
The clinical sample that the 7 parts of FAdV4 preserved by laboratory infect, extracts DNA, the reagent developed by the present invention after process
Box detection checking.As shown in Figure 4,7 parts of samples all amplify the DNA band of expection size to result.
Claims (8)
1. primer pair, including forward primer and downstream primer, it is characterised in that the nucleotide sequence of forward primer such as SEQ ID
Shown in NO.1, the nucleotide sequence of downstream primer is as shown in SEQ ID NO.2.
2. the PCR detection kit comprising primer pair as claimed in claim 1.
3. primer as claimed in claim 1 is to the application in preparation detection aviadenovirus 4 type test kit.
4. the method detecting aviadenovirus 4 type, it is characterised in that including:
(1) STb gene of testing sample is extracted；
(2) set up PCR reaction system, with described STb gene as template, use the primer described in claim 1 to carrying out PCR
Amplification；
(3) pcr amplification product is carried out detected through gel electrophoresis；
As occurred the band of 403bp in amplified production, then containing aviadenovirus 4 type in explanation testing sample, otherwise, explanation is treated
Without aviadenovirus 4 type in test sample product.
5. method as claimed in claim 4, it is characterised in that described testing sample is birds embryo allantoic liquid or in vitro pathologic group
Knit.
6. method as claimed in claim 5, it is characterised in that described birds is chicken.
7. method as claimed in claim 4, it is characterised in that described PCR reaction system includes: PCR reaction buffer
Final concentration of 1 ×, each 0.2mM of ExTaq enzyme 1.0U, dNTP, each 0.4mM of upstream and downstream primer.
8. method as claimed in claim 4, it is characterised in that the condition of PCR reaction is: 94 DEG C of denaturations 5min, 94 DEG C
Degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, 35 circulations, and 72 DEG C extend 10min.
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Cited By (18)
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| CN106443015A (en) * | 2016-09-21 | 2017-02-22 | 扬州大学 | ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting fowl adenovirus antibody based on hexon protein N-terminal conservative area |
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| CN106443015A (en) * | 2016-09-21 | 2017-02-22 | 扬州大学 | ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting fowl adenovirus antibody based on hexon protein N-terminal conservative area |
| 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 |
| CN107102138A (en) * | 2017-04-14 | 2017-08-29 | 杨凌职业技术学院 | Detect indirect ELISA reagent kit and its detection method and its application of I group I fowl adenovirus antibody |
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