CN106868212A - The universal PCR detection primers of I subgroup aviadenovirus and detection method - Google Patents
The universal PCR detection primers of I subgroup aviadenovirus and detection method Download PDFInfo
- Publication number
- CN106868212A CN106868212A CN201710159937.7A CN201710159937A CN106868212A CN 106868212 A CN106868212 A CN 106868212A CN 201710159937 A CN201710159937 A CN 201710159937A CN 106868212 A CN106868212 A CN 106868212A
- Authority
- CN
- China
- Prior art keywords
- aviadenovirus
- subgroup
- fadv
- pcr
- detection method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000701802 Aviadenovirus Species 0.000 title claims abstract description 57
- 238000001514 detection method Methods 0.000 title claims abstract description 50
- 238000006243 chemical reaction Methods 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 21
- 238000012408 PCR amplification Methods 0.000 claims abstract description 12
- 230000000692 anti-sense effect Effects 0.000 claims abstract description 9
- 239000012634 fragment Substances 0.000 claims abstract description 6
- 239000007795 chemical reaction product Substances 0.000 claims abstract description 3
- 108020004414 DNA Proteins 0.000 claims description 37
- 241001465754 Metazoa Species 0.000 claims description 7
- 241000807592 Fowl adenovirus Species 0.000 claims description 6
- 238000012423 maintenance Methods 0.000 claims description 6
- 108020005202 Viral DNA Proteins 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 238000005498 polishing Methods 0.000 claims description 2
- 230000003139 buffering effect Effects 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 claims 1
- 230000003612 virological effect Effects 0.000 abstract description 7
- 241000271566 Aves Species 0.000 abstract description 4
- 241000700605 Viruses Species 0.000 description 14
- 210000001519 tissue Anatomy 0.000 description 14
- 239000002574 poison Substances 0.000 description 13
- 231100000614 poison Toxicity 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 210000000056 organ Anatomy 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 230000009182 swimming Effects 0.000 description 9
- 241001634261 Fowl adenovirus 8b Species 0.000 description 7
- 241000703540 Fowl aviadenovirus 11 Species 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 7
- 210000004185 liver Anatomy 0.000 description 7
- 230000000762 glandular Effects 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- 210000004072 lung Anatomy 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 210000003205 muscle Anatomy 0.000 description 6
- 239000013642 negative control Substances 0.000 description 6
- 210000000952 spleen Anatomy 0.000 description 6
- 210000002784 stomach Anatomy 0.000 description 6
- 208000011580 syndromic disease Diseases 0.000 description 6
- 241001634265 Fowl adenovirus 8a Species 0.000 description 5
- 210000002216 heart Anatomy 0.000 description 5
- 208000006758 Marek Disease Diseases 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 108020000999 Viral RNA Proteins 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 208000006454 hepatitis Diseases 0.000 description 3
- 231100000283 hepatitis Toxicity 0.000 description 3
- 210000003000 inclusion body Anatomy 0.000 description 3
- 230000017448 oviposition Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 241000701242 Adenoviridae Species 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 108700010877 adenoviridae proteins Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 235000013330 chicken meat Nutrition 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000003628 erosive effect Effects 0.000 description 2
- 210000004317 gizzard Anatomy 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 235000013594 poultry meat Nutrition 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- NTIZESTWPVYFNL-UHFFFAOYSA-N Methyl isobutyl ketone Chemical compound CC(C)CC(C)=O NTIZESTWPVYFNL-UHFFFAOYSA-N 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 238000009361 aviculture Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000009585 enzyme analysis Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 108700004029 pol Genes Proteins 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of universal PCR detection primers of I subgroup aviadenovirus and detection method, belong to avian viral detection technique field.The universal PCR detection primers of I subgroup aviadenovirus of the invention are made up of sense primer and anti-sense primer；As shown in SEQ ID NO.1, the anti-sense primer is as shown in SEQ ID NO.2 for the sense primer.I subgroup aviadenovirus detection method, including carry out pcr amplification reaction using the described universal PCR detection primers of I subgroup aviadenovirus.It is the positive when 494bp fragments occurs in the pcr amplification reaction product, is otherwise feminine gender.The inventive method is that can detect 5 kinds such as I subgroup aviadenovirus A, B, C, D, E totally 12 universal methods of serotype, with versatility is good, high specificity, sensitiveness it is high, easy to operate, quick and precisely the features such as.
Description
Technical field
The present invention relates to avian viral detection technique field, more particularly to a kind of universal PCR detections of I subgroup aviadenovirus
Primer and detection method.
Background technology
I subgroup aviadenovirus（Group I fowl aviadenovirus, FAdV）Belong to Adenoviridae
（Adenoviridae）Aviadenovirus（genus Aviadenovirus）Member, is the relatively conservative double-stranded DNA disease of genome
Poison, mainly causes birds inclusion body hepatitis（Inclusion body hepatitis, IBH）, hydropericardium syndrome
（Hydropericardium syndrome, HPS）With gizzard erosion disease（Gizzard erosion, GE）Deng.
FAdV is divided into 5 kinds of A, B, C, D, E totally 12 serotype, including 1-8a, 8b-11, in addition, also includes
The tentative specieses such as Ana 1 aviadenovirus, dove adenovirus and turkey adenovirus（Document：Saif Y M. disease of poultry [M]：12nd edition Su Jing
Good, Gao Fu, Suo Xun translate Beijing：Chinese agriculture publishing house, 2012. 289-334.）.
Since 2015, the death of FAdV Infectives is broken out in the province such as China Shandong, Jiangsu, Hebei, Henan, and the death rate has
Up to 80%, be at present national fashion trend, heavy losses are caused to aviculture.
Popular report strain domestic at present includes FAdV-1（A kinds）、FAdV-2（D kinds）、FAdV-4（C kinds）、FAdV-8a（E
Kind）、FAdV-8b（E kinds）、FAdV-10（C kinds）And FAdV-11（D kinds）Serotype（Document：Niu Dengyun, Shen Yuan, Wang Rui,
Deng China I group I fowl adenovirus Molecule Epidemiology Investigation [J] Chinese poultry resources in 2015,2016,38 (9): 65-68.
Document：State records and builds, Diao Youxiang, Cheng Yanli, waits pathogenic [J] the China veterinary science of the type of I group I fowl adenovirus serum of 10
Report, 2013,33 (08): 1179-83.）.
Because 5 kind nucleotide homologies of FAdV A-E are between 52%-72%, and domestic report FAdV polymerase chains
Reaction（Polymerase chain reaction, PCR）Detection method is generally for single serotype（Document：Yuan Wanzhe,
Walk slowly like a woman, Chen Ping waits the foundation and the animal quarantine of application [J] China, 2016,33 of the type PCR detection method of aviadenovirus 4
(05): 72-74.）, it is impossible to general detection, or detected using Nested Polymerase Chain Reaction for Hexon GFPs（Document：Suo Nan
Drolma, the foundation of the group I fowl adenovirus sleeve type PCR detection methods of increasing bar I and application [J] China animal and veterinary, 2013,
40(08): 30-33.）, process is cumbersome, time-consuming（4-5 hours, in addition it is longer）, there is limitation when clinical detection is carried out.
The content of the invention
In order to make up the deficiencies in the prior art, to solve to detect that I subgroup aviadenovirus process is cumbersome, time-consuming in the prior art
Long and detection can only be directed to the problem of single serotype, draw the invention provides a kind of universal PCR detections of I subgroup aviadenovirus
Thing and detection method, the inventive method are that can detect 5 kinds such as I subgroup aviadenovirus A, B, C, D, E totally 12 serotype
Universal method, with versatility is good, high specificity, sensitiveness it is high, easy to operate, quick and precisely the features such as.
The technical scheme is that：
A kind of universal PCR detection primers of I subgroup aviadenovirus, the primer is made up of sense primer and anti-sense primer；It is described
As shown in SEQ ID NO.1, the anti-sense primer is as shown in SEQ ID NO.2 for sense primer.
A kind of I subgroup aviadenovirus detection method, methods described includes that I subgroup aviadenovirus described in use is universal
PCR detection primers carry out pcr amplification reaction.
Preferably, the reaction system of the amplified reaction includes PCR premixed liquids, DNA profiling, the sense primer
With the anti-sense primer.
Further, reaction system described in every 25 μ L includes PCR premixed liquid 2 × Taq PCR StarMix, 3 μ of 12.5 μ L
The DNA profiling of L, the sense primer of 1 μ L, the anti-sense primer of 1 μ L, using sterilizing ultra-pure water polishing to 25 μ L.
Preferably, the reaction condition of the pcr amplification reaction is：First, the reaction system is in 94-95 DEG C of bar
5-10 min are maintained under part carries out predegeneration；Then, the reaction system maintains s, 50.4-61 DEG C of dimension of 30-45 in 94-95 DEG C
30-45 s, 72 DEG C of maintenance 30-40 s are held, is circulated 30-40 times；Extend 5-10 min at last 72 DEG C.
Preferably, it is the positive when 494bp fragments occurs in the pcr amplification reaction product, is otherwise feminine gender.
Preferably, the DNA profiling is by affected animal tissue extraction.
Further, animal tissue is taken, adds PBS to be fully ground, -20 DEG C of multigelations repeatedly, are centrifuged,
Supernatant is taken, supernatant is extracted into aviadenovirus DNA with GeneJET Viral DNA/RNA Purification kits.
Beneficial effects of the present invention are：
1st, versatility；The present invention gathers according to each serotype strain complete genome DNAs of relevant FAdV issued in GenBank
Synthase sequence, by sequence alignment analysis, chooses the fragment guarded in gene and designs and synthesizes pair of primers, to FAdV-1
（A kinds）、FAdV-4（C kinds）, FAdV-2 and FAdV-11（D kinds）, FAdV-8a and FAdV-8b（E kinds）Deng 4 kinds, 6 kinds of serotypes
Virus general can detect, meanwhile, in theory to including B kinds（FAdV-5）Also can be special in 6 kinds of serotypes of other interior
Property detection, amplified production is 494 bp.
2nd, specificity is strong with sensitiveness；The I universal PCR detection method of subgroup aviadenovirus that the present invention sets up is normal to other
See fowl DNA virus（Egg-laying reduction syndrome virus, Marek's disease poison, bird pox virus）It is reactionless, high specificity；Minimum detection
Measure is 2.8 × 102Copy viral DNA, sensitiveness is strong.
3rd, strong applicability；Although I subgroup aviadenovirus is with liver viral level highest, the designed primer of the present invention and inspection
Survey method virus attack poison with poison the organs and tissues DNA extracts such as SPF hearts, liver, spleen, lung, kidney, glandular stomach, muscle of dying enter performing PCR inspection
Survey, the DNA fragmentation that size is 494bp is amplified, with excellent clinical detection applicability.
4th, quick and precisely；Detection can be completed in the present invention in 80 min, and by verification experimental verification, rate of accuracy reached is arrived
100%。
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1：The PCR method specific test electrophoresis result figure of I subgroup aviadenovirus is detected, wherein M represents DL2000
DNA Marker (precious bioengineering (Dalian) Co., Ltd product)；N is negative control；It is comprehensive that swimming lane 1-4 is respectively egg drop reduction
Simulator sickness virus（EDSV）, Marek's disease poison（MDV）, bird pox virus（APV）, the type of I subgroup aviadenovirus 4（FAdV-4）Amplification knot
Really；
Fig. 2：The PCR method sensitivity tests electrophoresis result figure of I subgroup aviadenovirus is detected, wherein swimming lane 1 is containing I subgroup fowl gland
The standard positive plasmid of viral dna polymerase Gene Partial segment（2.8×106/ 3 μ l of copy）；Swimming lane 2-7 is to do positive control
On the basis of it is continuous 10 times dilution templates；N represents negative control；M represents DL2000 DNA Marker；
Fig. 3：Attack and poison the organs and tissues such as SPF hearts, liver, spleen, lung, kidney, glandular stomach, muscle PCR detection electrophoresis result figures of dying with poison, wherein
Swimming lane 1-3:Lungs detect product；4-6:Muscle detects PCR primer；7-9:Kidney detects PCR primer；10-12:Glandular stomach is examined
Survey PCR primer；13-15:Spleen detects PCR primer；16-18:Liver detects PCR primer；19-21:Heart detection PCR is produced
Thing；M. DL2000 DNA Marker are represented.Result shows that the PCR detection method that this research is set up is in all internal organs for gathering
Tissue can be detected.（Note：Every 3 swimming lanes are an organs and tissues testing result, be sequentially universal primer of the present invention,
FAdV-F1/R1 primers, FAdV-F2/R2 primers）；
Fig. 4：PCR detects FAdV-4 virus results；M. DL2000 DNA molecular criterias；N. negative control；1. PCR detections
FAdV-4 products；
Fig. 5：Detect the PCR method versatility experiment electrophoresis result figure of I subgroup aviadenovirus, M. DL2000 DNA molecule marks
It is accurate；N. negative control；Swimming lane 1-6 is followed successively by FAdV-1（A kinds）、FAdV-4（C kinds）, FAdV-2 and FAdV-11（D kinds）、FAdV-
8a and FAdV-8b（E kinds）4 kinds, 6 kinds of serotype detection products.
Specific embodiment
Embodiment 1
Design of primers and synthesis：According to the 12 serotype strain virus genom DNA pol genes of FAdV A-E kinds announced
Sequence（GenBank accession number： AC_000014.1、KT862805.1、KT862807.1、GU188428.1、NC_021221.1、
KT862808.1、KT862810.1、KT862810.1、KT862811.1、AF083975.2、KT717889.1、
KM096545.1）, sequence analysis comparison is carried out using Lasergene MegAlign softwares, conservative region is screened, design general
Primer, sequence is：
SEQ ID NO.1：GTGGTRTCCATGTTGGT
SEQ ID NO.2：ATGTACGCYTCCGCCCTC.
Samples sources：FAdV is attacked and is poisoned the organs and tissues such as SPF hearts, liver, spleen, lung, kidney, glandular stomach, muscle of dying with poison.
Main agents：2 × Taq PCR StarMix are purchased from GenStar companies；GeneJET Viral DNA/RNA
Purification Kit are purchased from Thermo Scientific；DL2000 DNA Marker are purchased from precious bioengineering (Dalian)
Co., Ltd.
Key instrument：PTC-220 types PCR amplification instrument is purchased from BIO-RAD companies；The micro high speed refrigerated centrifuges of Micro 17R
Machine, NanoDrop2000 ultramicrospectrophotometers are purchased from Thermo Scientific；Alpha gel imaging systems are purchased from
Protein Simple companies（Former Alpha Innotech companies）.
Determine that the optimal conditions of pcr amplification reaction is by condition optimizing experiment：First, the reaction system is in 94 DEG C of bars
5 min are maintained under part carries out predegeneration；Then, reaction system maintains 40 s, 55 DEG C of 40 s of maintenance, 72 DEG C of maintenances 30 in 94 DEG C
S, circulates 35 times；Extend 10min at last 72 DEG C.
All experiments are carried out under the optimal conditions.
The detection method of I subgroup aviadenovirus, comprises the following steps that：
（1）Sample treatment：
Core, liver, spleen, lung, kidney, glandular stomach, each 100 mg of organs and tissues such as muscle, being separately added into 1 ml PBSs is carried out
It is fully ground, -20 DEG C of multigelations 3 times, 8000rpm is centrifuged 10 min, the μ l of supernatant 500 is taken, for extracting aviadenovirus
DNA。
（2）The extraction of aviadenovirus DNA：By the sample after treatment, with GeneJET Viral DNA/RNA
Purification Kit extract aviadenovirus DNA.
（3）Using the PCR reaction systems of 25 μ l, 2 × Taq PCR are separately added into 0.2 ml PCR reaction tubes
The μ l of StarMix 12.5, each μ l of 1 μ l, DNA template 3 of upstream and downstream primer, cumulative volume is added to sterilizing ultra-pure water, is mixed.
（4）PCR amplification conditions are：First, the reaction system maintains 5 min to carry out predegeneration under the conditions of 94 DEG C；So
Afterwards, reaction system maintains 40 s, 55 DEG C of 40 s of maintenance, 72 DEG C of 30 s of maintenance in 94 DEG C, circulates 35 times；Extend at last 72 DEG C
10min。
（5）5 μ l PCR primers are taken in electrophoresis on 1% Ago-Gel, gel imaging system observation electrophoresis result, the heart, liver,
The organs and tissues such as spleen, lung, kidney, glandular stomach, muscle extract DNA and expand to 494 bp products, are judged to I subgroup aviadenovirus positive.
To verify the subgroup aviadenovirus high specificity of primer detection I of the present invention, sensitiveness is strong, applicability is good, versatility is good,
Four groups of experiments are designed, each test procedure is carried out according to above step.
Test group one：Detect the specificity verification of the PCR method of I subgroup aviadenovirus
The variable of this group experiment is viral species, and viral species include in the experiment of this group：Egg-laying reduction syndrome virus（EDSV）、
Marek's disease poison（MDV）, bird pox virus（APV）, the type of I subgroup aviadenovirus 4（FAdV-4）.
As shown in figure 1, M represents that (precious bioengineering (Dalian) Co., Ltd produces DL2000 DNA Marker in Fig. 1
Product)；N is negative control；Swimming lane 1-4 is respectively egg-laying reduction syndrome virus（EDSV）, Marek's disease poison（MDV）, fowl pos disease
Poison（APV）, the type of I subgroup aviadenovirus 4（FAdV-4）Amplification.
Result shows that the inventive method detects I subgroup aviadenovirus high specificity.
Test group two：Detect the sensitiveness checking of the PCR method of I subgroup aviadenovirus
Variable in the experiment of this group is DNA profiling concentration, 6 concentration is devised altogether, as a result as shown in Figure 2.Swimming lane 1 is in Fig. 2
Standard positive plasmid containing I subgroup aviadenovirus DNA polymerase gene partial segments（2.8×106/ 3 μ l of copy）；Swimming lane 2-7
It is the continuous 10 times templates of dilution；N represents negative control；M represents DL2000 DNA Marker.
Result shows that the detection least concentration of the inventive method is 2.8 × 102/ 3 μ l of copy, sensitiveness is strong.
Test group three：Detect the applicability checking of the PCR method of I subgroup aviadenovirus
Organs and tissues of the variable of this group experiment belonging to DNA profiling, take the DNA moulds attacked and poison SPF chicken different organs tissues of dying with poison
Plate, is detected, as a result as shown in Fig. 31,4,7,10,13,16,19.
In addition, according to report document（Document：Meulemans G, Boschmans M, Berg T P, et al.
Polymerase chain reaction combined with restriction enzyme analysis for
Detection and differentiation of fowl adenoviruses [J] .Avian pathology, 2001,30
(6)：655-660. documents：Zhao J, Zhong Q, Zhao Y, et al. Pathogenicity and Complete
Genome Characterization of Fowl Adenoviruses Isolated from Chickens
Associated with Inclusion Body Hepatitis and Hydropericardium Syndrome in
China [J]. PloS one, 2015, 10(7): e0133073.）2 pairs of primers of synthesis, organs and tissues are carried out with the present invention
Viral Determination experiment.
The primer sequence that document is recorded is as follows：
FAdV-F1：CAARTTCAGRCAGACGGT
FAdV-R1：TAGTGATGMCGSGACATCAT；
FAdV-F2：TGCTCGTTGTGGATGGTGAA
FAdV-R2：CTCCGTGTTGGGCTGGTC.
Primer synthesizes and the completion of purifying student on commission's work bioengineering (Shanghai) limited company.
Determination test group one is to be detected using the carrying out of FAdV-F1/R1 primers, as a result as 2 in Fig. 3,5,8,11,
14th, shown in 17,20.
Determination test group two is to be detected using the carrying out of FAdV-F2/R2 primers, as a result as 3 in Fig. 3,6,9,12,
15th, shown in 18,21.
Fig. 3 shows that the DNA profiling acquired in all organs and tissues of primer pair of the present invention can be detected, existing FAdV-
F1/R1 primers and FAdV-F2/R2 primers be only capable of detecting the DNA profiling acquired in specific organs and tissues, primer of the present invention
Strong applicability.
Test group one, the positives reference substance of test group two are to contain the type of I subgroup aviadenovirus 4（FAdV-4）Archaeal dna polymerase
The standard positive plasmid of Gene Partial fragment, the 494bp products expanded by PCR are connected to carrier pMD18-T（Precious bioengineering
(Dalian) Co., Ltd product）It is prepared from, concentration is 2.8 × 106/ 3 μ l of copy（NanoDrop2000 ultramicron spectrophotometrics
Meter quantified, according to formula copy number=（6.02×1023）×（ng/μL×10-9）/（DNA length×660）Calculate dense
Degree, then dilutes）.Expanded FAdV-4 fragments nucleotides sequence is classified as SEQ ID NO.3.
SEQ ID NO.3：
gtggtatcca tgttggtcgc gaaggccccg tagagggcgt tgctgagcat tttcgagatg 60
gatctcatga cctcattttt ctcttgatcg gctttttcct tggccgcgat gttcttgctg 120
acgtaatccg cacaaatggt cttccactcg gggaagacaa tgttcatgtc gtcatgcagg 180
gcggtgactt tccacccgcg gttgtgaagc gtgatgatgt ctaggacggt gacgacctcg 240
tcgtagagca cctcgttggc ccatacgagg cgaccgcctc tgcggttgca cagcggaggg 300
agggtatcga gctgttcggg gggcggcggg taggcttcta ttttgaggat ggaaggtttg 360
atgcgtgcgt cgaagtagct gatgggtgcg ggctggagca atatagcgtt cagctcgtcc 420
acgtgtgccg ccgtgaactt gggatcgaga ggcataccgt ggggcatggg gtgggtgagg 480
gcggaggcgtacat 494。
With FAdV-4 serotype genomic DNAs template, PCR amplifications electrophoresis detection is as shown in Figure 4.It can be seen that, it is designed
Primer specific can expand the single goal gene that size is 494 bp.
Test group four：Detect the versatility checking of the PCR method of I subgroup aviadenovirus
Variable in the experiment of this group is that the variable of this test group is the serum type of I subgroup aviadenovirus, I used by this test group
Subgroup aviadenovirus type includes：FAdV-1（A kinds）、FAdV-4（C kinds）, FAdV-2 and FAdV-11（D kinds）, FAdV-8a with
FAdV-8b（E kinds）4 kinds, 6 kinds of serotypes.Result is as shown in Figure 5.
As shown in Figure 5, using the inventive method, FAdV-1（A kinds）、FAdV-4（C kinds）, FAdV-2 and FAdV-11（D
Kind）, FAdV-8a and FAdV-8b（E kinds）4 kinds, 6 kinds of serotypes can be detected, and the inventive method versatility is good.
In theory, I 5 kinds of subgroup aviadenovirus A-E totally 12 serotypes it is amplifiable go out 494 bp products, but
It is domestic at present to report only FAdV-1（A kinds）, FAdV-4 and FAdV-10（C kinds）, FAdV-8a and FAdV-8b（E kinds）And FAdV-11
（D kinds）Serotype, other have no report including 6 serotypes including B kinds, simultaneously as laboratory can obtain I
Subgroup aviadenovirus kind and serotype are limited, including above-mentioned except FAdV-10（C kinds）5 serotypes and FAdV-2 serum in addition
Type virus, therefore, this method only demonstrates FAdV-1（A kinds）、FAdV-4（C kinds）, FAdV-2 and FAdV-11（D kinds）、FAdV-
8a and FAdV-8b（E kinds）Deng 4 kinds, 6 kinds of serotypes.But in theory to including B kinds（FAdV-5）In 6 kinds of blood of other interior
Clear type virus also can specific detection, amplified production is 494 bp.
SEQUENCE LISTING
<110>Shandong Province Binzhou animal and veterinary research institute
<120>The universal PCR detection primers of I subgroup aviadenovirus and detection method
<130> 2017
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213>Artificial sequence
<400> 1
gtggtrtcca tgttggt 17
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
atgtacgcyt ccgccctc 18
<210> 3
<211> 494
<212> DNA
<213>I subgroup aviadenovirus FAdV-4
<400> 3
gtggtatcca tgttggtcgc gaaggccccg tagagggcgt tgctgagcat tttcgagatg 60
gatctcatga cctcattttt ctcttgatcg gctttttcct tggccgcgat gttcttgctg 120
acgtaatccg cacaaatggt cttccactcg gggaagacaa tgttcatgtc gtcatgcagg 180
gcggtgactt tccacccgcg gttgtgaagc gtgatgatgt ctaggacggt gacgacctcg 240
tcgtagagca cctcgttggc ccatacgagg cgaccgcctc tgcggttgca cagcggaggg 300
agggtatcga gctgttcggg gggcggcggg taggcttcta ttttgaggat ggaaggtttg 360
atgcgtgcgt cgaagtagct gatgggtgcg ggctggagca atatagcgtt cagctcgtcc 420
acgtgtgccg ccgtgaactt gggatcgaga ggcataccgt ggggcatggg gtgggtgagg 480
gcggaggcgt acat 494
Claims (8)
1. universal PCR detection primers of a kind of I subgroup aviadenovirus, it is characterised in that：The primer is by sense primer and downstream
Primer is constituted；As shown in SEQ ID NO.1, the anti-sense primer is as shown in SEQ ID NO.2 for the sense primer.
2. a kind of I subgroup aviadenovirus detection method, it is characterised in that：Methods described includes that described in usage right requirement 1 I is sub-
The universal PCR detection primers of group I fowl adenovirus carry out pcr amplification reaction.
3. I subgroup aviadenovirus detection method as claimed in claim 2, it is characterised in that：The reaction system of the amplified reaction
Including PCR premixed liquids, DNA profiling, the sense primer and the anti-sense primer.
4. I subgroup aviadenovirus detection method as claimed in claim 3, it is characterised in that：Reaction system described in every 25 μ L includes
PCR premixed liquid 2 × Taq PCR StarMix of 12.5 μ L, the DNA profiling of 3 μ L, the sense primer of 1 μ L, the anti-sense primer of 1 μ L,
Using sterilizing ultra-pure water polishing to 25 μ L.
5. the I subgroup aviadenovirus detection method as described in claim 3 or 4, it is characterised in that：The pcr amplification reaction it is anti-
The condition is answered to be：First, the reaction system maintains the 5-10 min to carry out predegeneration under the conditions of 94-95 DEG C；Then, the reaction
System maintains s, 50.4-61 DEG C of 30-45 to maintain 30-45 s, 72 DEG C of maintenance 30-40 s in 94-95 DEG C, circulates 30-40 times；Most
Extend 5-10 min at 72 DEG C afterwards.
6. I subgroup aviadenovirus detection method as claimed in claim 2, it is characterised in that：The pcr amplification reaction product occurs
It is the positive during 494bp fragments, is otherwise feminine gender.
7. I subgroup aviadenovirus detection method as claimed in claim 2, it is characterised in that：The DNA profiling is by affected animal group
Knit extraction.
8. I subgroup aviadenovirus detection method as claimed in claim 7, it is characterised in that：Animal tissue is taken, PBS bufferings are added
Liquid is fully ground, and repeatedly, centrifugation takes supernatant, by supernatant GeneJET Viral DNA/ to -20 DEG C of multigelations
RNA Purification kits extract aviadenovirus DNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710159937.7A CN106868212B (en) | 2017-03-17 | 2017-03-17 | General PCR detection primer and detection method for I subgroup avian adenovirus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710159937.7A CN106868212B (en) | 2017-03-17 | 2017-03-17 | General PCR detection primer and detection method for I subgroup avian adenovirus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106868212A true CN106868212A (en) | 2017-06-20 |
| CN106868212B CN106868212B (en) | 2021-01-22 |
Family
ID=59172065
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710159937.7A Active CN106868212B (en) | 2017-03-17 | 2017-03-17 | General PCR detection primer and detection method for I subgroup avian adenovirus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106868212B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531663A (en) * | 2018-07-06 | 2018-09-14 | 福建省农业科学院畜牧兽医研究所 | The universal detection primers of Ana 1 aviadenovirus DAdV-3 and DAdV-A and its application |
| CN109182597A (en) * | 2018-09-03 | 2019-01-11 | 广州维伯鑫生物科技有限公司 | It is a kind of to detect I, II, III groups of multiple fluorescence quantitative PCR kits of aviadenovirus and its detection method simultaneously |
| CN110373499A (en) * | 2019-07-24 | 2019-10-25 | 安徽科技学院 | A kind of universal two warm formula PCR primers and method of I group I fowl adenovirus of quick detection |
| CN110551849A (en) * | 2019-08-29 | 2019-12-10 | 中国动物卫生与流行病学中心 | Group I avian adenovirus detection primer, detection kit, virus detection method and application |
| CN111218526A (en) * | 2019-12-09 | 2020-06-02 | 温氏食品集团股份有限公司 | PCR primer pair and probe for rapidly identifying I subgroup serum 11 type avian adenovirus and application thereof |
| CN112410471A (en) * | 2020-12-18 | 2021-02-26 | 西藏职业技术学院 | Specific primer of avian adenovirus group I nested PCR, detection method and application thereof |
| CN113564280A (en) * | 2021-08-04 | 2021-10-29 | 山东农业大学 | RAA primer for detecting 12 serotypes of avian adenovirus group I and detection method thereof |
| CN113981145A (en) * | 2021-11-16 | 2022-01-28 | 广州蔚捷生物医药科技有限公司 | Detection method and detection kit for group I avian adenovirus A, B, C, D and E |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105603124A (en) * | 2016-02-05 | 2016-05-25 | 乾元浩生物股份有限公司 | LAMP primer pair used for I subgroup fowl adenovirus detection and detection method thereof |
| CN106399585A (en) * | 2016-08-31 | 2017-02-15 | 中国农业大学 | Universal PCR primers and method for detecting group I aviadenovirus and detection kit |
| 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 |
-
2017
- 2017-03-17 CN CN201710159937.7A patent/CN106868212B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105603124A (en) * | 2016-02-05 | 2016-05-25 | 乾元浩生物股份有限公司 | LAMP primer pair used for I subgroup fowl adenovirus detection and detection method thereof |
| CN106399585A (en) * | 2016-08-31 | 2017-02-15 | 中国农业大学 | Universal PCR primers and method for detecting group I aviadenovirus and detection kit |
| 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 |
Non-Patent Citations (4)
| Title |
|---|
| RRAUE等: "Hexon based PCRs combined with restriction enzyme analysis for rapid detection and differentiation of fowl adenoviruses and egg drop syndrome virus", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 文艳玲等: "Ⅰ群禽腺病毒SYBR GreenⅠ荧光PCR 检测方法的建立", 《中国兽医科学》 * |
| 李天芝等: "基于hexon基因的Ⅰ群禽腺病毒PCR快速检测方法的建立和应用", 《养禽与禽病防治》 * |
| 邱丽叶等: "禽腺病毒荧光定量PCR检测方法的建立与初步应用", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531663A (en) * | 2018-07-06 | 2018-09-14 | 福建省农业科学院畜牧兽医研究所 | The universal detection primers of Ana 1 aviadenovirus DAdV-3 and DAdV-A and its application |
| CN109182597A (en) * | 2018-09-03 | 2019-01-11 | 广州维伯鑫生物科技有限公司 | It is a kind of to detect I, II, III groups of multiple fluorescence quantitative PCR kits of aviadenovirus and its detection method simultaneously |
| CN109182597B (en) * | 2018-09-03 | 2022-01-04 | 广州维伯鑫生物科技股份有限公司 | Multiplex fluorescence quantitative PCR kit for simultaneously detecting avian adenovirus group I, II and III and detection method thereof |
| CN110373499A (en) * | 2019-07-24 | 2019-10-25 | 安徽科技学院 | A kind of universal two warm formula PCR primers and method of I group I fowl adenovirus of quick detection |
| CN110551849A (en) * | 2019-08-29 | 2019-12-10 | 中国动物卫生与流行病学中心 | Group I avian adenovirus detection primer, detection kit, virus detection method and application |
| CN111218526A (en) * | 2019-12-09 | 2020-06-02 | 温氏食品集团股份有限公司 | PCR primer pair and probe for rapidly identifying I subgroup serum 11 type avian adenovirus and application thereof |
| CN111218526B (en) * | 2019-12-09 | 2024-01-09 | 温氏食品集团股份有限公司 | PCR primer pair and probe for rapidly identifying I subgroup serum 11 type avian adenovirus and application thereof |
| CN112410471A (en) * | 2020-12-18 | 2021-02-26 | 西藏职业技术学院 | Specific primer of avian adenovirus group I nested PCR, detection method and application thereof |
| CN113564280A (en) * | 2021-08-04 | 2021-10-29 | 山东农业大学 | RAA primer for detecting 12 serotypes of avian adenovirus group I and detection method thereof |
| CN113981145A (en) * | 2021-11-16 | 2022-01-28 | 广州蔚捷生物医药科技有限公司 | Detection method and detection kit for group I avian adenovirus A, B, C, D and E |
| CN113981145B (en) * | 2021-11-16 | 2022-08-12 | 广州蔚捷生物医药科技有限公司 | Detection method and detection kit for group I avian adenovirus A, B, C, D and E |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106868212B (en) | 2021-01-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106868212A (en) | The universal PCR detection primers of I subgroup aviadenovirus and detection method | |
| CN106636471B (en) | Multiplex PCR detection kit for simultaneously detecting WSSV, AHPND, EHP and IHHNV of prawns | |
| CN106947838B (en) | African swine fever virus non-structural gene real-time fluorescence LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method | |
| CN107299155A (en) | A kind of primer and probe of goose astrovirus real-time fluorescence quantitative PCR detection | |
| CN107841575B (en) | Nano multiplex PCR method for distinguishing four serotype avian adenovirus group I | |
| Bezerra et al. | Detection of avian group D rotavirus using the polymerase chain reaction for the VP6 gene | |
| CN107385111A (en) | The real-time fluorescence quantitative PCR detection primer and its kit of a kind of goose astrovirus | |
| CN110241261A (en) | A kind of RT-RAA detection method of avian infectious bronchitis virus | |
| CN105886667A (en) | Detection kit for porcine epidemic diarrhea virus and detection method thereof | |
| CN106399585A (en) | Universal PCR primers and method for detecting group I aviadenovirus and detection kit | |
| CN102643931B (en) | Reverse transcription-polymerase chain reaction (RT-PCR) method for verifying avian infectious bronchitis virus (IBV) epidemic strains and vaccine strains | |
| CN110438260A (en) | A kind of African swine fever virus nucleic acid test strips detection kit | |
| CN110699485A (en) | RPA primer pair, probe, kit and detection method for rapidly detecting Marek's disease virus | |
| CN110373496A (en) | Kit and detection method for the detection of 8 type aviadenovirus of I subgroup serum | |
| Steyer et al. | A diagnostic method based on MGB probes for rapid detection and simultaneous differentiation between virulent and vaccine strains of avian paramyxovirus type 1 | |
| CN105950785A (en) | Ternary fluorescence RT-PCR detection kit of avian influenza virus, new castle disease virus and infectious bronchitis virus, primers and probes | |
| Zhai et al. | A loop-mediated isothermal amplification coupling with a lateral flow dipstick for rapid and specific detection of fowl adenovirus serotype-4 | |
| CN101575649B (en) | Quick detection technology for H9 type avian influenza virus | |
| CN108411041A (en) | A kind of fluorescence quantitative RT-PCR kit of the novel avian reovirus of detection and application | |
| CN107190103A (en) | Multiple PCR primer group, kit and the method for three kinds of fishes virus are detected simultaneously | |
| CN108998575B (en) | Establishment of double PCR detection method for chicken parvovirus and chicken newcastle disease virus | |
| Davidson | Biotic concerns in generating molecular diagnosis matrixes for 4 avian viruses with emphasis on Marek’s disease virus | |
| CN107604101A (en) | One breeding pigeon New-type adenovirus real-time fluorescence quantitative PCR detection kit | |
| CN109439799A (en) | It is a kind of for detecting the sleeve type PCR primer and kit of pig acute diarrhea coronavirus | |
| CN104388593A (en) | Taqman Real-time PCR (polymerase chain reaction) kit for detecting porcine circovirus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |