CN107043831A - Ana 1 aviadenovirus A types and 2 type Real time PCR detection primers, probe and kit - Google Patents
Ana 1 aviadenovirus A types and 2 type Real time PCR detection primers, probe and kit Download PDFInfo
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Abstract
The present invention provides Ana 1 aviadenovirus A types and the type real-time fluorescence quantitative PCR detection primer of Ana 1 aviadenovirus 2, probe and kit, and the primer and probe sequence is as shown in SEQ ID NO.1 6, with very high specificity and sensitivity.Currently both at home and abroad there is not yet the research report of the primer and probe of the dual TaqMan real-time fluorescence quantitative PCR detection methods of detection can be carried out to Ana 1 aviadenovirus A types and the type of Ana 1 aviadenovirus 2 simultaneously, foundation of the invention can fill up domestic and international association area blank.
Description
Technical field
The present invention provides Ana 1 aviadenovirus A types and the type real-time fluorescence quantitative PCR detection primer of Ana 1 aviadenovirus 2, probe and reagent
Box, belongs to veterinary field.
Background technology
Classified according to International Commission on Virus Classification, Adenoviridae Adenoviridae divides into 5 category, be respectively richness AT glands
TobamovirusAtadenovirus, AviadenovirusAviadenovirus, fish AdenovirusIchtadenovirus, mammal
AdenovirusMastadenovirusWith sialidase TobamovirusSiadenovirus.Wherein richness AT Adenoviruses have 5 kinds, point
Wei not cow adenovirus D typesBovine adenovirus D, ovine adenovirus D typesOvine adenovirus D, kangaroo adenovirus
A typesPossum adenovirus A, snake adenovirus A typesSnake adenovirus A and Ana 1 aviadenovirus A typesDuck adenovirus A（Abbreviation DAdV-A）.
Adenoviral gene group total length is about 33 kd, and genome is linear dsdna；The viruslike particle is the core clothing without cyst membrane
Shell is in icosahedral symmetry, and the nucleocapsid of wherein adenovirus has 3 major structural proteins, respectively hexon（Hexon）、
Fibrin (Fiber) and penton protein（Penton）.Wherein, Hexon albumen is that Adenoviridae respectively belongs to viral topmost
Structural proteins, each Hexon protein surfaces carry the species-specific antigen determinant of the main category of virus, are that virus neutralization is anti-
The target of body, it is pathogenic closely related with virus containing the protective antigen gene cluster that virus is main.
The type of Ana 1 aviadenovirus 2（Duck adenovirus 2, DAd V-2）It is to be found in recent years using viral metagenomics
A kind of duck source New-type adenovirus, the disease occurred mainly in 20~30 age in days ducks, using liver and splenomegaly, bleeding to be main special
Levy, the death rate is about 7%.The discovery of nucleotide homology com-parison and analysis, the type of Ana 1 aviadenovirus 2（GR plants）Separated with Ana 1 aviadenovirus A types
The nucleotide homology of strain is about 50.0%.By being found to its phylogenetic analysis based on Hexon albumen, all duck adenopathies
Malicious A types separation strains are in the sub- branch of identical in genetic evolution, and with ovine adenovirus D types（OAV287 plants）It is in richness
AT Adenovirus genetic evolutions branch.But the type of Ana 1 aviadenovirus 2（GR plants）With Phelps plants of fowl adenovirus A（Ji Yuan）With P29 plants
（Goose source）In same genetic evolution branch, Aviadenovirus genetic evolution branch is belonged to.
Real time fluorescence quantifying PCR method（Real time PCR）It is a kind of in DNA amplification reaction, with fluorescence chemical thing
Quality detection surveys each PCR（PCR）The method of product total amount after circulation.By internal reference or outer ginseng method to be measured
The method that specific dna sequence in sample carries out quantitative analysis.Real-time fluorescence quantitative PCR is in PCR amplification procedures, by glimmering
Optical signal, is detected in real time to PCR processes.Due to the exponential time base expanded in PCR, the Ct values of template and the starting of the template
There is linear relationship in copy number.Fluorescence probe method is that product is detected with the fluorescence labeling probe of sequence specific, and sonde method goes out
Now so that the specificity of quantitative PCR technique is greatly improved than Standard PCR technology, conventional at present has TaqMan probe, FRET miscellaneous
Hand over probe (fluorescence resonance energy transmission probe) and molecular beacon molecular Beacon.TaqMan probe method refers to that PCR expands
A specific fluorescence probe is it is possible to additionally incorporate during increasing while pair of primers is added, the probe is only tied with template specificity
Close, its binding site is between two primers.5 ' ends of probe are marked with fluorescent reporter group, such as FAM, VIC, ROX, JOE,
3 ' ends are marked with fluorescent quenching group, such as Eclipse, TAMRA.Real-Time Fluorescent Quantitative PCR Technique detect cause of disease when not only
Can be with the presence or absence of qualitative detection cause of disease, but also can be with the number of quantitative analysis of virus content, in etiology nucleic acid detection technique
It is widely used.Multiple real time fluorescence quantifying PCR is a kind of special quantitative fluorescent PCR form, and the characteristics of it is protruded is once
Real-time fluorescence quantitative PCR reaction can detect a variety of cause of diseases simultaneously, and inspection is differentiated to cause of disease complexity or the cause of disease that there is Multi-genotype
Survey largely effective.
Currently, there are 2 kinds of Adenoviridaes in duck group but belong to the cause of disease of different Adenoviruses：Ana 1 aviadenovirus A types（Category
In Adenoviridae richness AT Adenoviruses）With the type of Ana 1 aviadenovirus 2（Belong to Adenoviridae Aviadenovirus）.Therefore, energy is set up
Enough detection methods simultaneously to Ana 1 aviadenovirus A types and the type of Ana 1 aviadenovirus 2 in duck group, both simplified operation programs, cost-effective, again
To 2 kinds of Adenoviridaes in duck group but the cause of diseases of different Adenoviruses can be belonged to（Ana 1 aviadenovirus A types and the type of Ana 1 aviadenovirus 2）'s
Epidemiology survey and the related aetology pathogenesis of development are respectively provided with important meaning.But, currently both at home and abroad not yet
The dual TaqMan real-time fluorescence quantitative PCR detection methods of detection can be carried out to Ana 1 aviadenovirus A types and the type of Ana 1 aviadenovirus 2 simultaneously by seeing
The research report of primer and probe, foundation of the invention can fill up domestic and international association area research blank.
The content of the invention
The present invention provides Ana 1 aviadenovirus A types and the type real-time fluorescence quantitative PCR detection primer of Ana 1 aviadenovirus 2, probe and reagent
Box, setting up can be while to the detection method of Ana 1 aviadenovirus A types and the type of Ana 1 aviadenovirus 2 in duck group, simplifying procedures, saving
Cost.
To achieve the above object, using following technical scheme：
Ana 1 aviadenovirus A types and the type real-time fluorescence quantitative PCR detection primer of Ana 1 aviadenovirus 2 and probe, including it is as follows：
For detecting Ana 1 aviadenovirus A types：
Sense primer DAdV-A-F：5 '-TCCTCCTCATGGTTTTATG-3 ',
Anti-sense primer DAdV-A-R：5 '-CGAAGATTGCAGTAGTATTAG-3 ',
Fluorescence probe DAdV-A-P：5’- CCTGACACTGGCACGGCTAT-3’；
For detecting the type of Ana 1 aviadenovirus 2：
Sense primer DAdV-2-F：5’- CGATGATGCAGATGATAAC-3’；
Anti-sense primer DAdV-2-R：5’- GATCGCTCTGAGTTTAGG-3’；
Fluorescence probe DAdV-2-P：5’- ACCAGAACATACTCCAGAATCGTGA-3’；
Wherein, the fluorescence probe DAdV-A-P of Ana 1 aviadenovirus A types 5 ' ends are marked with fluorescent reporter group ROX, the type of Ana 1 aviadenovirus 2
Fluorescence probe DAdV-2-P 5 ' end be marked with fluorescent reporter group FAM；, the fluorescence probe DAdV-A-P of Ana 1 aviadenovirus A types
3 ' ends and the type of Ana 1 aviadenovirus 2 fluorescence probe DAdV-2-P 3 ' ends mark quenching group Eclipse.
For the kit for the real time fluorescence quantifying PCR method for detecting Ana 1 aviadenovirus A types and the type of Ana 1 aviadenovirus 2, including institute
The primer and probe stated.
Dual TaqMan real-time fluorescence quantitative PCRs reaction system is：
Dual real-time fluorescence quantifying PCR method reaction condition is：95 DEG C of s of pre-degeneration 60；95 DEG C of 10s, 56 DEG C of annealing 10
S, 72 DEG C of 15 s of extension, 40 circulations.
After real-time fluorescence quantitative PCR reaction terminates, result of the test is analyzed, selects ROX passages to see there is positive expansion at 605nm
Increase signal, show there is the infection of Ana 1 aviadenovirus A types in detection sample；FAM passages are selected to see there is positive amplification letter at 520nm
Number, show there is the infection of the type of Ana 1 aviadenovirus 2 in detection sample；If selecting to select FAM passages at ROX passages and 520nm at 605nm
Seeing has positive amplification signal, shows there is Ana 1 aviadenovirus A types and the infection of the type of Ana 1 aviadenovirus 2 in sample.
The present invention provides Ana 1 aviadenovirus A types and the type real-time fluorescence quantitative PCR detection primer of Ana 1 aviadenovirus 2, probe and reagent
Box, with advantages below and effect：
1st, detect simultaneously：The method of foundation can be examined to Ana 1 aviadenovirus A types in duck group and the infection of the type of Ana 1 aviadenovirus 2 simultaneously
Survey, simplify procedures, cost-effective.After real-time fluorescence quantitative PCR reaction terminates, result of the test is analyzed, is selected at 605nm
ROX passages, which are shown in, positive amplification signal, shows there is the infection of Ana 1 aviadenovirus A types in detection sample；FAM is selected to lead at 520nm
Road, which is shown in, positive amplification signal, shows there is the infection of the type of Ana 1 aviadenovirus 2 in detection sample；If at 605nm select ROX passages and
Select FAM passages to see there is positive amplification signal at 520nm, show there is Ana 1 aviadenovirus A types and the type sense of Ana 1 aviadenovirus 2 in sample
Dye.
2nd, detect quick, efficient：The detection method need not carry out conventional agarose gel electrophoresis detection, react after terminating i.e.
The carry out result judgement that can be carried by real-time fluorescence quantitative PCR machine.90 min are only needed from nucleic acid extraction to result judgement, and
96 sample detections can be once carried out simultaneously.
3rd, it is quantitative accurate：By preparing standard items, drawing standard curve, according to Ana 1 aviadenovirus A types in detection measuring samples
With the Ct values of the type of Ana 1 aviadenovirus 2, directly infect it Ana 1 aviadenovirus A types and the type of Ana 1 aviadenovirus 2 carries out accurate quantitative analysis.
4th, sensitivity is high：Minimum detectable 8.37 copies of Ana 1 aviadenovirus A types；The type of Ana 1 aviadenovirus 2 minimum detectable 6.92
Individual copy, more conventional PCR detection sensitivities improve 100 times.
5th, high specificity：With the common transmittable disease such as E. coli isolated from ducks, Riemerlla anatipestifer, the killing property fowl of duck source in duck group more
The reactionless signal of Pasteurella, duck plague virus and Muscovy duck parvovirus, only to Ana 1 aviadenovirus A types（605nm ROX passages）With
The type of Ana 1 aviadenovirus 2（520nm FAM passages）There is fluorescence signal in detection.
85 parts of clinical censorship duck source pathological material of diseases are carried out with the dual real-time fluorescence of Ana 1 aviadenovirus A types and the infection of the type of Ana 1 aviadenovirus 2
Quantitative PCR detection, corresponding DNA is extracted with commercialization Viral nucleic acid extraction reagent box, is quantified according to dual real-time fluorescence is set up
PCR system and condition are detected.As a result find, select ROX passages to see there is 6 parts of Sample Positive amplified signals, table at 605nm
There is the infection of Ana 1 aviadenovirus A types in bright 6 parts of detections sample, positive rate is 7.06%；FAM passages are selected to see there are 2 parts at 520nm
Sample Positive amplified signal, shows there is the infection of Ana 1 aviadenovirus A types in 2 parts of detection samples, positive rate is 2.35%；Wherein also have 1
Part sample selects selection FAM passages at ROX passages and 520nm to have amplified signal at 605nm, shows to deposit in 1 part of sample
Infected in Ana 1 aviadenovirus A types and the type of Ana 1 aviadenovirus 2, positive rate is 1.17%.
Brief description of the drawings
The amplification kinetic curve of Fig. 1 Ana 1 aviadenovirus A type real time fluorescence quantifying PCR methods.
The standard curve of Fig. 2 Ana 1 aviadenovirus A type real time fluorescence quantifying PCR methods.
The specific test of Fig. 3 Ana 1 aviadenovirus A type real time fluorescence quantifying PCR methods.
The amplification kinetic curve of the type real time fluorescence quantifying PCR method of Fig. 4 Ana 1 aviadenoviruses 2.
The standard curve of the type real time fluorescence quantifying PCR method of Fig. 5 Ana 1 aviadenoviruses 2.
The specific test of the type real time fluorescence quantifying PCR method of Fig. 6 Ana 1 aviadenoviruses 2.
Embodiment
The present invention will be further described for example below.
Embodiment 1
1st, correlation test cause of disease
Experiment with killing property bar cause of disease Ana 1 aviadenovirus A types, the type of Ana 1 aviadenovirus 2, E. coli isolated from ducks, Riemerlla anatipestifer, duck source fowl more
Family name bacillus, duck plague virus and Muscovy duck parvovirus are identified and preserved by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute.
, primer and probe design
According to the Ana 1 aviadenovirus A types and the specific Hexon coding sequences of the type of Ana 1 aviadenovirus 2 retrieved in GenBank,
Design is specific for Ana 1 aviadenovirus A types and the primer and probe of the type of Ana 1 aviadenovirus 2, and its sequence is as follows：
Ana 1 aviadenovirus A types（DAdV-A）：
Sense primer DAdV-A-F：5’- TCCTCCTCATGGTTTTATG-3’；
Anti-sense primer DAdV-A-R：5’- CGAAGATTGCAGTAGTATTAG-3’；
Fluorescence probe DAdV-A-P：5’- CCTGACACTGGCACGGCTAT-3’；
The type of Ana 1 aviadenovirus 2（DAdV-2）：
Sense primer DAdV-2-F：5’- CGATGATGCAGATGATAAC-3’；
Anti-sense primer DAdV-2-R：5’- GATCGCTCTGAGTTTAGG-3’；
Fluorescence probe DAdV-2-P：5’- ACCAGAACATACTCCAGAATCGTGA-3’；
Wherein, the fluorescence probe DAdV-A-P of Ana 1 aviadenovirus A types 5 ' ends are marked with fluorescent reporter group ROX, the type of Ana 1 aviadenovirus 2
Fluorescence probe DAdV-2-P 5 ' end be marked with fluorescent reporter group FAM；, the fluorescence probe DAdV-A-P of Ana 1 aviadenovirus A types
3 ' ends and the type of Ana 1 aviadenovirus 2 fluorescence probe DAdV-2-P 3 ' ends mark quenching group Eclipse.
Above-mentioned primer cures biotechnology in precious day（Beijing）Co., Ltd synthesizes.
, Ana 1 aviadenovirus A types and the type of Ana 1 aviadenovirus 2 dual real-time fluorescence quantitative PCR detecting method foundation
The structure of 3.1 Ana 1 aviadenovirus A type positive criteria products
According to Ana 1 aviadenovirus A types（JX2016 plants）Hexon protein-coding regions genetic traits, utilize primer-design software Oligo (versions
This v7.37) specific primer is designed, primer sequence is：DAdV-A-F2：5 '-ATGATAAATTTGTACGTGTTATTG-3 ' and
DAdV-A-R2：5 '-ACGCAGCATCAATTCCAGCTC -3 ', for expanding about 719 bp'sHexonGenetic fragment, primer
Biotechnology is cured by precious day（Beijing）Co., Ltd synthesizes.
Ana 1 aviadenovirus A types are extracted with commercialization Viral nucleic acid extraction reagent box（JX2016 plants）Nucleic acid DNA, according to 2 ×
TransTaq-T PCR SuperMix (+dye) specification enters performing PCR reaction, and reaction system reference reagent box specification is prepared,
Reaction system is 50 μ L, wherein the μ L of 2 × TransTaq-T PCR SuperMix reaction solutions 25, upstream and downstream primer（DAdV-A-F2
And DAdV-A-R2,10 μM）Each 1 μ L, the μ L of nucleic acid DNA 1 extracted, supplement the μ L of sterile deionized water 22 to the μ L of final volume 50.Reaction
Condition is：94 DEG C of min of pre-degeneration 4；94 DEG C of 50 s, 54 DEG C of 35 s, 72 DEG C of 45 s, 35 circulations；After circulation terminates
72 DEG C of 10 min of extension.PCR primer is identified with 1.0% agarose gel electrophoresis, is reclaimed and tried using Ago-Gel
Agent box carries out gel extraction to specific purpose fragment.According to pEASY-T1 Simple Cloning Kit clone's connection reagents
Box specification willHexonGene fragment clone is on pEASY-T1 carriers, and random 8 single bacteriums of picking fall within ampicillin（Contain
Measure as 100 μ g/mL）After the h of LB fluid nutrient mediums culture 14 of resistance, corresponding matter is extracted using the small extraction reagent kit of rapid plasmid
Grain.Primer when being expanded using PCR（DAdV-A-F2 and DAdV-A-R2）Enter performing PCR identification to the plasmid of extraction with condition, will
The positive recombinant plasmid filtered out send precious day doctor biotechnology（Beijing）Co., Ltd is sequenced.By sequencing result on NCBI
Carry out BLAST analysis checking, positive recombinant plasmid expected from Pass Test as real-time fluorescence quantitative PCR positive criteria product
（T- DAdV-A）, it is placed in -20 DEG C after packing and saves backup.
The structure of the type positive criteria product of 3.2 Ana 1 aviadenovirus 2
According to the type of Ana 1 aviadenovirus 2（FJ20171 plants）Hexon protein-coding regions genetic traits, utilize primer-design software Oligo
(version v7.37) designs specific primer, and primer sequence is：DAdV-2-F2：5′- CGAAACAAATTCAGACAGACT -3′
And DAdV-2-R2：5 '-AGTCTTCATACCCATCGTTATT -3 ', for expanding about 988 bp'sHexonGenetic fragment, draws
Thing cures biotechnology by precious day（Beijing）Co., Ltd synthesizes.
The type of Ana 1 aviadenovirus 2 is extracted with commercialization Viral nucleic acid extraction reagent box（FJ20171 plants）Nucleic acid DNA, according to 2 ×
TransTaq-T PCR SuperMix (+dye) specification enters performing PCR reaction, and reaction system reference reagent box specification is prepared,
Reaction system is 50 μ L, wherein the μ L of 2 × TransTaq-T PCR SuperMix reaction solutions 25, upstream and downstream primer（DAdV-2-F2
And DAdV-2-R2,10 μM）Each 1 μ L, the μ L of nucleic acid DNA 1 extracted, supplement the μ L of sterile deionized water 22 to the μ L of final volume 50.Reaction
Condition is：94 DEG C of min of pre-degeneration 4；94 DEG C of 50 s, 54 DEG C of 35 s, 72 DEG C of 45 s, 35 circulations；Circulation terminates
72 DEG C extend 10 min afterwards.PCR primer is identified with 1.0% agarose gel electrophoresis, reclaimed using Ago-Gel
Kit carries out gel extraction to specific purpose fragment.According to pEASY-T1 Simple Cloning Kit clone's connection examinations
Agent box specification willHexonGene fragment clone is on pEASY-T1 carriers, and random 8 single bacteriums of picking fall within ampicillin
（Content is 100 μ g/mL）After the h of LB fluid nutrient mediums culture 14 of resistance, extract corresponding using the small extraction reagent kit of rapid plasmid
Plasmid.Primer when being expanded using PCR（DAdV-2-F2 and DAdV-2-R2）Enter performing PCR mirror to the plasmid of extraction with condition
It is fixed, send precious day to cure biotechnology the positive recombinant plasmid filtered out（Beijing）Co., Ltd is sequenced.Sequencing result is existed
BLAST analysis checkings are carried out on NCBI, positive recombinant plasmid expected from Pass Test is marked as the positive of real-time fluorescence quantitative PCR
Quasi- product（T- DAdV-2）, it is placed in -20 DEG C after packing and saves backup.
The optimization of 3.3 reaction conditions and the foundation of standard curve
The real-time fluorescence quantitative PCR for preparing 20 μ L according to Premix Ex Taq (Probe qPCR) kit specification is anti-
System is answered, in different annealing temperature（56,58,60,62 and 64 DEG C）, primer concentration（2.5-20 μM）And concentration and probe concentration（1.25-
20 μM）Lower progress real-time fluorescence quantitative PCR reaction, is optimized to reaction condition.With Ana 1 aviadenovirus A type positive criteria products（T-
DAdV-A）With the type positive criteria product of Ana 1 aviadenovirus 2（T- DAdV-2）For template, ROX passages are selected at 605nm, in 520nm
Place's selection FAM passages.Expanded with the reaction condition after optimization, obtain amplification kinetic curve.With standard items starting copies
Several common logarithms（lgC）For abscissa, with period threshold value（Cycle threshold, Ct values）For ordinate, bid is derived
Almost linear regression equation（Standard curve）.
As a result：The dual real-time fluorescence quantifying PCR method optimal reaction system of optimization（20 μL）Such as table 1 below.
Dual TaqMan real-time fluorescence quantitative PCRs reaction system after the optimization of table 1
As a result：The dual real-time fluorescence quantifying PCR method optimum reaction condition of optimization is：95 DEG C of s of pre-degeneration 60；95 ℃
10s, 56 DEG C of annealing 10 s, 72 DEG C of 15 s of extension, 40 circulations.
The judgement of experimental result：
After real-time fluorescence quantitative PCR reaction terminates, result of the test is analyzed, selects ROX passages to see there is positive amplification letter at 605nm
Number, show there is the infection of Ana 1 aviadenovirus A types in detection sample；FAM passages are selected to see there is positive amplification signal, table at 520nm
There is the infection of the type of Ana 1 aviadenovirus 2 in bright detection sample；If selecting selection FAM passages at ROX passages and 520nm to see at 605nm
There is positive amplification signal, show there is Ana 1 aviadenovirus A types and the infection of the type of Ana 1 aviadenovirus 2 in sample.
3.4 Ana 1 aviadenovirus A type sensitivity tests
Positive criteria product is determined using trace dna analyzer（T- DAdV-A）Concentration, calculate its copy number for 8.37 × 107
Copy/μ L, 10 times of serial dilutions are carried out to it, serial dilution is carried out altogether（Plasmid content is respectively 8.37 × 106, 8.37 × 105,
8.37×104, 8.37 × 103, 8.37 × 102, 8.37 × 101With 8.37 × 100Copy/μ L）, it is placed in -20 DEG C of preservations after packing
It is standby.Respectively with above-mentioned different dilution factor positive criteria products（T- DAdV-A）As template, carried out with the reaction condition after optimization
Amplification, obtains amplification kinetic curve（See Fig. 1）.With the common logarithm of standard items starting copy number（lgC）For abscissa, to follow
Number of rings threshold value（Cycle threshold, Ct values）For ordinate, standard linear regression equation is derived（Standard curve）（See figure
2）, obtain its sensitivity tests data.
It will be seen from figure 1 that the real time fluorescence quantifying PCR method lowest detection that the present invention is set up is limited to 8.37 copies/μ L.
With plasmid content in each standard items（C）Common logarithm（lgC）For abscissa, with period threshold value（Cycle threshold,
Ct values）For ordinate, Ana 1 aviadenovirus A type real-time fluorescence quantitative PCR standard curves are obtained（Fig. 2）, obtain slope of standard curve
For -3.516, Y intercept is 35.38, and coefficient correlation is 0.996, and amplification efficiency is 99.9%, meets experiment and is expected.
The type sensitivity tests of 3.5 Ana 1 aviadenovirus 2
Positive criteria product is determined using trace dna analyzer（T- DAdV-2）Concentration, calculate its copy number for 6.92 × 107
Copy/μ L, 10 times of serial dilutions are carried out to it, serial dilution is carried out altogether（Plasmid content is respectively 6.92 × 106, 6.92 × 105,
6.92×104, 6.92 × 103, 6.92 × 102, 6.92 × 101With 6.92 × 100Copy/μ L）, it is placed in -20 DEG C of preservations after packing
It is standby.Respectively with above-mentioned different dilution factor positive criteria products（T- DAdV-2）As template, carried out with the reaction condition after optimization
Amplification, obtains amplification kinetic curve（See Fig. 3）.With the common logarithm of standard items starting copy number（lgC）For abscissa, to follow
Number of rings threshold value（Cycle threshold, Ct values）For ordinate, standard linear regression equation is derived（Standard curve）（See figure
4）, obtain its sensitivity tests data.
From fig. 4, it can be seen that the real time fluorescence quantifying PCR method lowest detection that the present invention is set up is limited to 6.92 copies/μ L.
With plasmid content in each standard items（C）Common logarithm（lgC）For abscissa, with period threshold value（Cycle threshold,
Ct values）For ordinate, Ana 1 aviadenovirus A type real-time fluorescence quantitative PCR standard curves are obtained（Fig. 5）, obtain slope of standard curve
For -3.241, Y intercept is 33.99, and coefficient correlation is 0.998, and amplification efficiency is 100%, meets experiment and is expected.
3.6 specific detection
With the dual real-time fluorescence quantitative PCR condition after optimization, to aquatic bird, other Common genes group nucleic acid types are DNA's respectively
Encountered pathogenic, such as E. coli isolated from ducks, Riemerlla anatipestifer, duck source eggs crack detection, duck plague virus and the tiny disease of kind duck
Poison carries out real-time fluorescence quantitative PCR detection, using Ana 1 aviadenovirus A types and the type of Ana 1 aviadenovirus 2 as positive control.Use viral genome
DNA/RNA extracts kits extract the nucleic acid DNA of corresponding virus, according to Premix Ex Taq (Probe qPCR) kit
Specification is detected with the reaction condition after optimization, evaluates the specificity for the real time fluorescence quantifying PCR method set up.
It can be seen from figure 3 that with the dual real-time fluorescence quantitative PCR condition after optimization respectively to other Common genes group cores of aquatic bird
Acids type is DNA encountered pathogenic, such as E. coli isolated from ducks, Riemerlla anatipestifer, duck source eggs crack detection disease, duck plague disease
Poison, Muscovy duck parvovirus carry out real-time fluorescence quantitative PCR detection, from ROX fluorescence signal passages, rarely seen to have Ana 1 aviadenovirus A types special
Specific fluorescent signals, the type of passage Ana 1 aviadenovirus 2 also has no Positive fluorescence signal.
As seen from Figure 6, with the dual real-time fluorescence quantitative PCR condition after optimization respectively to other Common genes group cores of aquatic bird
Acids type is DNA encountered pathogenic, such as E. coli isolated from ducks, Riemerlla anatipestifer, duck source eggs crack detection, duck plague virus
Real-time fluorescence quantitative PCR detection is carried out with Muscovy duck parvovirus, it is rarely seen to have the type of Ana 1 aviadenovirus 2 special from FAM fluorescence signal passages
Property fluorescence signal, passage Ana 1 aviadenovirus A types also have no Positive fluorescence signal.
The above results show, the dual real-time fluorescence quantitative PCR high specificity of foundation, to other Common genes of aquatic bird
The encountered pathogenic that group nucleic acid type is DNA, such as E. coli isolated from ducks, Riemerlla anatipestifer, duck source eggs crack detection, duck plague
Virus and Muscovy duck parvovirus are showed no non-specific cross jamming.
The detection of 3.7 clinical samples
The dual real-time fluorescence that 85 parts of clinical censorship duck source pathological material of diseases carry out Ana 1 aviadenovirus A types and the infection of the type of Ana 1 aviadenovirus 2 is quantified
PCR is detected, corresponding DNA is extracted with commercialization Viral nucleic acid extraction reagent box, according to setting up dual real-time fluorescence quantitative PCR body
System and condition are detected.As a result find, select ROX passages to see there are 6 parts of Sample Positive amplified signals at 605nm, show 6 parts
There is the infection of Ana 1 aviadenovirus A types in detection sample, positive rate is 7.06%；FAM passages are selected to see there are 2 parts of sample sun at 520nm
Property amplified signal, show 2 parts detection samples in exist Ana 1 aviadenovirus A types infection, positive rate is 2.35%；Wherein there is 1 part of sample
Select selection FAM passages at ROX passages and 520nm to have amplified signal at 605nm, show there is duck gland in 1 part of sample
Virus type A and the infection of the type of Ana 1 aviadenovirus 2, positive rate is 1.17%.
Related positive real-time fluorescence quantitative PCR is reacted into the product utilization Ago-Gel QIAquick Gel Extraction Kit after terminating
Gel extraction is carried out to specific purpose fragment.Said according to pEASY-T1 Simple Cloning Kit clone's connection kits
Bright book willHexonGene fragment clone is on pEASY-T1 carriers, and random 8 single bacteriums of picking fall within ampicillin（Content is
100 μg/mL）After the h of LB fluid nutrient mediums culture 14 of resistance, corresponding plasmid is extracted using the small extraction reagent kit of rapid plasmid.
Primer when being expanded using PCR（DAdV-2-F2 and DAdV-2-R2）Enter performing PCR identification to the plasmid of extraction with condition, will screen
The positive recombinant plasmid gone out send precious day doctor biotechnology（Beijing）Co., Ltd is sequenced.Sequencing result is carried out on NCBI
BLAST analysis checkings, are corresponding Ana 1 aviadenovirus A types and the type Hexon genetic fragments of Ana 1 aviadenovirus 2, coincidence rate is 100%.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>Ana 1 aviadenovirus A types and 2 type Real time PCR detection primers, probe and kit
<130> 10
<160> 10
<170> PatentIn version 3.3
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Claims (2)
1. Ana 1 aviadenovirus A types and the type real-time fluorescence quantitative PCR detection primer of Ana 1 aviadenovirus 2 and probe, it is characterised in that：It is described to draw
Thing and probe include as follows：
For detecting Ana 1 aviadenovirus A types：
Sense primer DAdV-A-F：5 '-TCCTCCTCATGGTTTTATG-3 ',
Anti-sense primer DAdV-A-R：5 '-CGAAGATTGCAGTAGTATTAG-3 ',
Fluorescence probe DAdV-A-P：5’- CCTGACACTGGCACGGCTAT-3’；
For detecting the type of Ana 1 aviadenovirus 2：
Sense primer DAdV-2-F：5 '-CGATGATGCAGATGATAAC-3 ',
Anti-sense primer DAdV-2-R：5 '-GATCGCTCTGAGTTTAGG-3 ',
Fluorescence probe DAdV-2-P：5’- ACCAGAACATACTCCAGAATCGTGA-3’；
Wherein, the fluorescence probe DAdV-A-P of Ana 1 aviadenovirus A types 5 ' ends are marked with fluorescent reporter group ROX, the type of Ana 1 aviadenovirus 2
Fluorescence probe DAdV-2-P 5 ' end be marked with fluorescent reporter group FAM；, the fluorescence probe DAdV-A-P of Ana 1 aviadenovirus A types
3 ' ends and the type of Ana 1 aviadenovirus 2 fluorescence probe DAdV-2-P 3 ' ends mark quenching group Eclipse.
2. a kind of kit for being used to detect the real time fluorescence quantifying PCR method of Ana 1 aviadenovirus A types and the type of Ana 1 aviadenovirus 2, it is special
Levy and be：The kit includes the primer and probe described in claim 1.
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| CN108842000A (en) * | 2018-07-06 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | For identifying the primer sets of DAdV-3 and DAdV-A |
| CN108842004A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | Distinguish real-time fluorescence quantitative PCR primer and its differentiating method and the application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN108842005A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | The MGB probe and its mating primer and method of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B are detected simultaneously |
| CN108950070A (en) * | 2018-07-28 | 2018-12-07 | 福建省农业科学院畜牧兽医研究所 | Identify the PCR primer and its discrimination method and application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
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