CN110373499A - A kind of universal two warm formula PCR primers and method of I group I fowl adenovirus of quick detection - Google Patents
A kind of universal two warm formula PCR primers and method of I group I fowl adenovirus of quick detection Download PDFInfo
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- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- 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
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- C12Q1/701—Specific hybridization probes
Abstract
The invention discloses a kind of universal two warm formula PCR primers of quickly detection I group I fowl adenovirus and methods, which contains a pair of of specific primer, and has versatility between detection I group I fowl adenovirus different serotypes.Test proves that the method for the present invention effectively prevents the shortcoming of conventional three warm formula PCR, can most complete amplified reaction in 15 minutes fastly, and have many advantages, such as that high sensitivity, high specificity, cmot are good, at low cost.The present invention causes the quick diagnosis of epidemic disease for the infection of I group I fowl adenovirus, and carries the avian health status investigation research of I group I fowl adenovirus.
Description
Technical field
The present invention relates to technical field of molecular biology, and in particular to a kind of to use polymerase chain reaction (PCR) technology
The quickly primer and method of detection I group I fowl adenovirus.
Background technique
Aviadenovirus (Fowl Aviadenovirus, FAdV) belongs to Adenoviridae, Aviadenovirus.FAdV genome is
Unimolecule, threadiness, double-stranded DNA, size is between 26kb~45kb.It can be 3 by FAdV points according to group specific antigen difference
Group: I crowds, II crowd and III crowd.I crowds of FAdV include separating the adenovirus obtained from chicken, turkey, goose and other birds, and it includes 12
A serotype (1~7,8a, 8b, 9~11), is divided into 5 gene kinds: A(serum 1 type further accordance with gene genetic relationship), B(blood
Clear 5 type), C(serum 4,10 types), 2,3,9,11 type of D(serum) and E(serum 6,7,8a, 8b type).Fowl group Infective I crowds
The clinical pathological symptoms such as inclusion body hepatitis, hydropericardium, gizzard erosion are shown as after FAdV, are not only caused under breeding performonce fo animals
Drop, and part serotype FAdV causes the death rate up to 10%~80%.Since especially in June, 2015, more than 20, China
4 type epidemic infection of I groups of FAdV serum has been broken out in province, causes serious economic loss to aviculture.Yin Yanbo is to 2007-
The 86 plants of aviadenovirus serotype identification results separated in the main culture zone clinic pathological material of disease in China in 2017 show China's prevalence
I groups of FAdV predominant serotypes are 4,8a, 8b and 11 types.Research shows that FAdV both can horizontal transmission, can also vertical transmission, give the disease
Former prevention and control bring huge challenge.
Currently, mainly including that routine based on Virus Isolation and virus neutralization tests is examined to the detection means of FAdV
Disconnected method, immunological method, molecule hybridizing method, ring isothermal amplification technique and polymerase chain reaction (PCR) etc..Wherein PCR
Method is wherein most quick and sensitive the Methods of Detection of Pathogens, is had broad application prospects in the context of detection of disease.Two temperature
Formula PCR is a kind of special shape of PCR, is more simply and rapidly to detect skill made of development on the basis of conventional three temperature formula PCR
Art.In two warm formula PCR, annealing and extension carry out at the same temperature, and optimum temperature is usually than the annealing temperature of Standard PCR
Height, therefore the specificity of reaction is not only increased, and save the time-consuming for the cooling that heats up repeatedly, improve the expansion of target gene
Increasing Efficiency.Most of fluorescent quantitative PCR detection methods all use two warm formula PCR methods, the exhibition in terms of reaction speed and sensitivity
Show certain superiority, however fluorescent quantitative PCR instrument is expensive, reagent cost is high and needs professional training personnel, far not
And the application popularization of Standard PCR instrument and regular-PCR method.
106,435,031 106,399,585 106,868,212 107841575 A of A, CN of A, CN of A, CN of Publication No. CN,
The PCR method of I group FAdV of the Chinese patent application of 109136407 A of CN detection is conventional three temperature formula PCR, there is annealing
In place of the deficiencies of temperature is not high, the reaction time is longer.Authorize the Chinese patent application of 104017903 B of Publication No. CN H3
The warm formula RT-PCR detection kit of subtype avian influenza virus two and its primer pair, shorten PCR amplification time-consuming to a certain extent.
Researcher establishes a variety of epidemic disease the Methods of Detection of Pathogens with two warm formula round pcrs, but so far there are no that detection I group I fowl adenovirus is general
The report of the warm formula PCR method of type two.In addition, in presently disclosed data, first is that because reaction volume is excessive and/or amplified fragments
Too long, so that two temperature formula PCR are denaturalized and anneal, extension is held time longer；Second is that because of annealing elongating temperature and archaeal dna polymerase
(70~75 DEG C) of optimal reaction temperature appoint there are gaps, reduce the amplification efficiency and specificity of polymerase.
Summary of the invention
To solve above-mentioned defect, the object of the present invention is to provide a kind of the general of quickly detection I group I fowl adenovirus
The warm formula PCR primer of type two and method.The universal two temperature formula PCR that the present invention devises a pair of quickly detection I group I fowl adenovirus draws
Object, not only versatility is good between 12 serotype I group I fowl adenovirus for the PCR primer, but also detects the special of I group I fowl adenovirus
Property is strong.The two warm formula PCR method reaction volumes using the primer are only 10uL, amplified fragments only 235bp, using common rTaq
Archaeal dna polymerase, anneals and elongating temperature may be up to 72 DEG C, thus reaction time consumption is short, and the two warm formula PCR method sensibility
Height, cmot is good, testing cost is low.The present invention is suitable for high-volume sample and quickly detects and carry I group I fowl adenovirus
The research of avian health status investigation.
The technical solution used in the present invention is:
Universal two warm formula PCR primers of a pair of quickly detection I group I fowl adenovirus (FAdV-I) are announced according to the website GenBank
I group's 5 gene kinds of FAdV 12 serotype gene orders, devised in 52/55KDa gene conserved sequence region
A pair of of versatility primer.The primer pair includes upstream primer 5 '-GACGGTGGTCGAGYTGAGCATGA-3 ' (such as sequence table
Shown in SEQ ID No.1) and downstream primer 5 '-CGCCTGGGTCAAACCGAACAT-3 ' (such as sequence table SEQ ID No.2 institute
Show), in which: the Y in upstream primer is to annex base, Y=C/T.
The present invention also provides the universal two warm formula PCR methods of I crowds of FAdV of quickly detection a kind of, and the method includes making
Two warm formula pcr amplification reactions are carried out with the universal PCR primer of I crowds of FAdV.
The reaction system of the warm formula PCR of universal the two of the I group I fowl adenovirus is 10uL, including 10 × rTaq buffer
1uL, 2.5mmol/L dNTP 1uL, 5U/uL rTaq archaeal dna polymerase 1uL, sample DNA template 1uL, 10umol/L upstream are drawn
Object 1.5uL~3uL, 10umol/L downstream primer 1.5uL~2.5uL, the upstream primer and downstream primer usage ratio are 1:
1, with sterilizing ddH2O complements to 10uL.
The warm formula PCR method of universal the two of the I group I fowl adenovirus, to reduce reaction system evaporation, in amplified reaction body
After the completion of system prepares, 2.5uL paraffin oil or vegetable oil are added on the outside of the inner circle of PCR reaction tube pipe lid, pipe lid is formed when being closed
Oiliness fluid-tight.
The reaction condition of the warm formula PCR of universal the two of the I group I fowl adenovirus are as follows: firstly, the reaction system is at 94 DEG C
1min is maintained to carry out initial denaturation；Then, the reaction system is in 94 DEG C of 3~5s of maintenance, 68~72 DEG C of 8~10s of maintenance, circulation 35
~40 times；It is saved at last 4 DEG C.
It is the positive when 235bp band occur in universal two warm formula PCR products of the I group I fowl adenovirus, band does not occur
Or there is primer dimer band of the segment less than 100bp only for feminine gender.
The sample DNA template is extracted from animal tissue or cloacal swab.
The beneficial effects of the present invention are:
1, quickly: the method for the present invention is since reaction volume is small, annealing elongating temperature is high, amplified fragments are short thus fast in heating and cooling
Rate is that amplified reaction can be completed in 15 minutes in 5 DEG C/sec PCR amplification instrument.
2, versatility is good: 12 serotype 52/55KDa of I group FAdV that the present invention is announced according to the website GenBank
Gene order, select conserved sequence region design and synthesize pair of primers, to FAdV-1(A kind), FAdV-5(B kind), FAdV-4
(C kind), FAdV-11(D kind), FAdV-8(E kind) 5 totally kinds of 5 kinds of serotypes general can detect, according to inter-species gene genetic close
System and primer location sequence high consistency, tri- kinds of C, D, E of other 7 serotypes are theoretically detectable.
3, sensibility is good: the method for the present invention detection limit 100.3 EID50/ 0.1mL, lowest detection copy number are 1.39
×101Copies/uL(, that is, 13.9copies/uL), it is shown that excellent sensibility.
4, high specificity: the method for the present invention is to other common fowl DNA virus (egg-laying reduction syndrome virus, Marek's
Virus, infections chicken cloacal bursa virus, Chicken Infectious Anemia Virus), RNA virus (newcastle disease virus, infectious laryngotracheitis of chicken
Virus, avian infectious bronchitis virus, H9 type avian influenza virus, J subgroup avian leucosis virus) and bacterium (Escherichia coli, bar
Family name bacillus, salmonella) it cannot amplify 235bp segment, this method high specificity.
5, cmot is good: the method for the present invention attacks I crowds of FAdV and poisons the heart for dying SPF chicken, liver, spleen, lung, kidney, gland with poison
Stomach, small intestine, cecal tonsil, tracheae, thymus gland, pancreas, the bursa of farbricius, brain, cloacal swab totally 14 kinds of tissue DNA extracts into
Row detection, can amplify the segment of 235bp, have good detection cmot.
6, identify gene kind: the 235bp fragment products direct Sequencing of the method for the present invention amplification simultaneously carries out phylogenetic evolution
12 serotypes of I crowds of FAdV can be divided into 5 gene kinds of corresponding A~E by tree analysis, and then can identify positive strain
Affiliated gene kind.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, the accompanying drawings in the following description is only some realities of the invention
Example is applied, it for those of ordinary skill in the art, without creative efforts, can also be according to these attached drawings
Obtain other attached drawings.
Fig. 1 is that the position that oily liquids is added in PCR pipe in the present invention is indicated by black arrow.
Fig. 2 is the dosage optimization result of primer designed by the present invention: wherein M indicates DL2000 DNA Marker, swimming lane 1-
5 be respectively 10umol/L upstream primer and each 2.5uL, 2uL, 1.5uL, 1uL, 0.5uL testing result of downstream primer.
Fig. 3 is simultaneous elongating temperature optimum results of annealing in the method for the present invention: wherein M indicates DL2000 DNA Marker, swimming
Road 1-5 is respectively anneal simultaneous 68 DEG C of elongating temperature, 69 DEG C, 70 DEG C, 71 DEG C, 72 DEG C of testing results.
Fig. 4 is versatility testing result of the method for the present invention to I groups of FAdV different serotypes: wherein M indicates DL2000
DNA Marker, swimming lane 1-5 are respectively I groups of FAdV serotype FAdV-1(A kinds), FAdV-5(B kind), FAdV-4(C kind),
FAdV-11(D kind), FAdV-8a(E kind) testing result.
Fig. 5 is the sensitivity Detection result of the method for the present invention: wherein M indicates that DL2000 DNA Marker, swimming lane 1-5 divide
It Wei not I groups of FAdV CH/AHMC/2015 strains 103.3EID50/0.1mL、102.3EID50/0.1mL、101.3EID50/0.1mL、
100.3EID50/0.1mL、100.03EID50/ 0.1mL nucleic acid diluted concentration testing result.
Fig. 6 is the sensitivity Detection result of 3 pairs of document primed methods: wherein M indicates DL2000 DNA Marker, swimming lane
1-6 is respectively 1 primed method of document to I groups of FAdV CH/AHMC/2015 strains 104.3EID50/0.1mL、103.3EID50/0.1mL、
102.3EID50/0.1mL、101.3EID50/0.1mL、100.3EID50/0.1mL、100.03EID50The detection of/0.1mL nucleic acid diluted concentration
As a result；Swimming lane 7-12 is respectively 2 primed method of document to I groups of FAdV CH/AHMC/2015 strains 104.3EID50/0.1mL、
103.3EID50/0.1mL、102.3EID50/0.1mL、101.3EID50/0.1mL、100.3EID50/0.1mL、100.03EID50/0.1mL
Nucleic acid diluted concentration testing result；Swimming lane 13-18 is respectively 3 primed method of document to I groups CH/AHMC/2015 plants of FAdV
104.3EID50/0.1mL、103.3EID50/0.1mL、102.3EID50/0.1mL、101.3EID50/0.1mL、100.3EID50/0.1mL、
100.03EID50/ 0.1mL nucleic acid diluted concentration testing result.
Fig. 7 is that the method for the present invention detects minimum nucleic acid copies qualification result: wherein M indicates DL2000 DNA Marker,
Swimming lane 1-10 is respectively plasmid copy number 1.39 × 108copies/uL、1.39×107copies/uL、1.39×106copies/
uL、1.39×105copies/uL、1.39×104copies/uL、1.39×103copies/uL、1.39×102copies/
uL、1.39×101copies/uL、1.39×100copies/uL、1.39×10-1Copies/uL testing result.
Fig. 8 is the specific detection result of the method for the present invention: wherein M indicates that DL2000 DNA Marker, swimming lane 1 are sun
Property control, swimming lane 2-8 be respectively egg-laying reduction syndrome virus, marek virus, infections chicken cloacal bursa virus, chicken infect
Property anemia virus, Escherichia coli, Pasteurella, salmonella detection of nucleic acids result；Swimming lane 9-13 is respectively chicken infectivity throat gas
Pipe inflammation virus, avian infectious bronchitis virus, newcastle disease virus, H9 type avian influenza virus, J hypotype avian leukosis virus core
Testing result after sour reverse transcription.
Fig. 9 is the cmot testing result of the method for the present invention: wherein M indicates DL2000 DNA Marker, swimming lane 1-
14 be respectively that CH/AHMC/2015 plants of FAdV attack that poison the brain for dying SPF chicken, thymus gland, tracheae, pancreas, the bursa of farbricius, glandular stomach, caecum with poison flat
Peach body, small intestine, spleen, lungs, kidney, liver, heart, cloacal swab extract viral nucleic acid testing result.
Figure 10 is that the gene constructed phylogenetic evolution tree of the method for the present invention amplified fragments analyzes result: examined in FAdV-1- institute,
FAdV-5- poultry disease prevention and control monitoring key lab, province examines institute in FAdV-4-, institute is examined in FAdV-11-, examining in FAdV-8a-
Measured sequence is compared with the 12 235 primer amplified region sequences (191nt) of serotype strain 52/55KDa gene announced
And construct phylogenetic evolution tree.
Specific embodiment
Technical solution of the present invention is described in detail combined with specific embodiments below, but following embodiments are only used for
Improving eyesight, rather than limit the scope of the invention.Experimental method used in following embodiment unless otherwise specified, is
Conventional method；Material, reagent used etc., are commercially available unless otherwise specified.
The universal detection primer design of 1 I crowds of FAdV of embodiment and synthesis
12 serotype strain virus 5 2/55KDa gene order (12 blood of I group FAdV A~E kind announced according to GenBank
Clear type strain GenBank accession number be respectively as follows: U46933, KT862805, KT862807, KM096544, NC_021221,
HE603116, HE603117, KT862810, KT862811, AF083975, HE602019, KT862812), use Lasergene
MegAlign software carries out Multiple Sequence Alignment analysis conservative region and as specific detection target, by designed a large amount of
After primer carries out artificial synthesized and experiment sieving, selects effect best and the highest I groups of FAdV versatilities of annealing temperature detect and draw
Object, and it is named as 235 primers, primer sequence are as follows: upstream primer 5 '-GACGGTGGTCGAGYTGAGCATGA-3 ', downstream primer
5 '-CGCCTGGGTCAAACCGAACAT-3 ', wherein representing C/T with base Y is annexed.The raw work in 235 primer commission Shanghai is raw
Object Engineering Co., Ltd is synthesized and is purified.
The optimization of the warm formula PCR method of 2 I crowds of FAdV of embodiment universal two is established
(1) strain and reagent material
I crowds of FAdV 1,4,8a, 11 serotype standard strains be purchased from China Veterinery Drug Inspection Office (middle inspection institute), 4 type CH/ of serum
AHMC/2015 plants (GenBank accession number: MG148335), that Serotype 5 FAdV is that poultry disease prevention and control monitor Anhui Province's emphasis is real
It tests room separation to save, SPF hatching egg is purchased from Beijing Cimmeria Wei Tong Bioisystech Co., Ltd, E.Z.N.A.TM Viral DNA
Kit is purchased from OMEGA company, and virus genom DNA/RNA extracts kit is purchased from Tiangeng biochemical technology Co., Ltd, PCR pipe purchase
From Axygen Co., Ltd；PrimeScript™ II 1st Strand cDNA Synthesis Kit,10×Taq
Buffer, dNTP, rTaq archaeal dna polymerase are purchased from TaKaRa company.
(2) by OMEGA company E.Z.N.A.TM Viral DNA Kit kit operating instruction carry out I groups of FAdV serum 1,
4,5,8a, 11 types and CH/AHMC/2015 separation strains virus genom DNA extract, and are finally dissolved in 50uL sterilizing ddH2In O, -20
DEG C refrigerator saves backup.
The optimization of (3) two warm formula PCR method reaction conditions
Total reaction volume is 10uL, comprising: 10 × rTaq buffer 1uL, 2.5mmol/L dNTP 1uL, 5U/uL rTaq
Archaeal dna polymerase 1uL, 10umol/L upstream primer and each 1.5uL of downstream primer, template DNA 1uL, with sterilizing ddH2O is complemented to
10uL.To effectively prevent liquid evaporation in PCR pipe, (specific location is shown in attached drawing 1) adds 2.5uL paraffin on the outside of the inner circle of pipe lid
Oil closes upper tube cap, PCR pipe is pressed consolidation in PCR instrument.It carries out amplification reaction system optimization: 5 test groups is arranged in parallel,
Annealing elongating temperature is set into 68 DEG C, 69 DEG C, 70 DEG C, 71 DEG C, 72 DEG C respectively, to determine best annealing elongating temperature.It is arranged in parallel 5
Upstream primer and downstream primer dosage are set 0.5uL, 1uL, 1.5uL, 2uL, 2.5uL by a test group respectively, are most preferably drawn with determination
Object dosage.4 test groups are arranged in parallel, respectively plus 2.5uL paraffin oil, bean plant oil, corn on the outside of the inner circle of lid
Vegetable oil, peanut plant oil, to determine the best oily liquids for preventing liquid evaporation.9 test groups are arranged in parallel, when by being denaturalized
Between set 5s, 3s, 1s respectively, annealing extension of time sets 10s, 8s, 6s respectively, to determine best denaturation and annealing extension of time model
It encloses.3 test groups are arranged in parallel, PCR cycle number are set to 30,35,40 times, to determine optimum cycle number.It will be each
Test group reaction product carries out 1% agarose gel electrophoresis analysis, and electrophoresis result criterion: 235bp band occurs in PCR product
When for the positive, do not occur band or only occur segment less than 100bp primer dimer band for feminine gender.
(4) optimize reaction condition result
The results show that best primer (10umol/L) dosage is that 1.5uL~2.5uL(is shown in attached drawing 2), simultaneous elongating temperature range of annealing
At 68~72 DEG C (see attached drawings 3).Optimize reaction condition are as follows: in 94 DEG C of initial denaturation 1min；Then, 94 DEG C of 3~5s of denaturation, 68~
72 DEG C of annealing and extension maintain 8~10s, recycle 35~40 times；It is saved at last 4 DEG C.Paraffin is added on the outside of the inner circle of pipe lid
After oil or three vegetable oils, reaction volume evaporation is reduced within the scope of 0.5uL~1.5uL, and amplification efficiency is not significantly affected.
Although this method amplification rear electrophoresis shows the primer dimer band less than 100bp, the judgement of testing result is not influenced.
The versatility verifying of the warm formula PCR method of 3 I crowds of FAdV of embodiment universal two
For the versatility for the warm formula PCR method of I group FAdV bis- that the verifying optimization of embodiment 2 is established, we supervise from Chinese veterinary medicament
Examine had purchased I crowds of FAdV 1,4,8a, 11 serotype standard strains, in conjunction with poultry disease prevention and control monitoring Anhui Province's emphasis experiment
Room separation save I crowds of FAdV of 1 plant of Serotype 5, i.e., we obtain FAdV-1(A kind), FAdV-5(B kind), FAdV-4(C kind),
FAdV-11(D kind), FAdV-8a(E kind) 5 serotypes of totally 5 gene kinds.Utilize OMEGA company E.Z.N.A.TM
Viral DNA Kit kit extracts 5 gene kinds, 5 serotype DNA, the I group FAdV established with the optimization of embodiment 2
Two warm formula PCR methods are detected, and as the result is shown: 5 serotype testing results of above-mentioned 5 gene kinds are the positive
(see attached drawing 4).Test result shows that the warm formula PCR method versatility of I crowds of FAdV universal two is good.To I groups of FAdV A~E kinds 12
The 52/55KDa gene genetic relationship and sequence alignment analysis of serotype show, gene C kind, D kind, each serotype contained by E kind it
Between it is highly conserved, and the upstream primer and the position of downstream primer are consistent in I groups of FAdV 52/55KDa gene order height
Position, theoretically, other 7 serotype strains for adhering to 3 gene kinds separately can also be amplified 235bp product.
Proliferation time of the warm formula PCR method of 4 I crowds of FAdV of embodiment universal two in different PCR instruments
Since the warming and cooling rate of different PCR instruments is different, pcr amplification reaction time-consuming is also different, therefore we are in 5 different product
It is time-consuming that the warm formula PCR method amplified reaction of I group FAdV bis- that embodiment 2 is established is detected in the PCR instrument of board or model, while considering ring
Influence of the border temperature to amplified reaction time-consuming, environment temperature control is at 25 DEG C or so when experiment.Reaction condition are as follows: 94 DEG C of initial denaturations
1min；94 DEG C of denaturation 3s, 70 DEG C of annealing extend 8s, recycle 35 times；It is saved at last 4 DEG C.The results show that PCR instrument heating and cooling speed
Degree is faster, and reaction time consumption is fewer, in German Eppendorf board Mastercycler Epgradient S and Mastercycler
In 6325 model PCR instrument of Pro S, amplified reaction is most fast, and (see Table 1 for details) can be completed in 15min.Warming and cooling rate about 1
DEG C/PCR instrument of sec on amplified reaction time-consuming in 30min or so.
The sensitivity Detection of the warm formula PCR method of 5 I crowds of FAdV of embodiment universal two
Using OMEGA company E.Z.N.A.TM Viral DNA Kit kit to 105.3EID50The I group FAdV CH/ of/0.1mL
AHMC/2015 strain virus liquid carries out viral nucleic acid extraction, and the DNA of extraction is carried out 10 times and is serially diluted 6 concentration, i.e.,
104.3EID50/0.1mL、103.3EID50/0.1mL、102.3EID50/0.1mL、101.3EID50/0.1mL、100.3EID50/0.1mL
With 100.03EID50/ 0.1mL, and the DNA that serial dilutions will be extracted, with therein 103.3EID50/0.1mL、102.3EID50/
0.1mL、101.3EID50/0.1mL、100.3EID50/0.1mL、100.03EID50Totally 5 diluted concentrations extract DNA conduct to/0.1mL
Template, the warm formula PCR method of the I group FAdV bis- established with the optimization of embodiment 2 are detected, reaction condition are as follows: 94 DEG C of initial denaturations
1min；94 DEG C of denaturation 5s, 68 DEG C of annealing extend 10s, recycle 35 times；It is saved at last 4 DEG C.Electrophoretogram result is shown: I crowds of FAdV
Two warm formula PCR method sensibility are up to 100.3EID50/ 0.1mL(105Times diluted concentration), it is detailed in attached drawing 5.
In addition, according to 3 documents (document 1:G ü nes A, Marek A, Grafl B, et al. Real-time
PCR assay for universal detection and quantitation of all five species of
fowl adenoviruses (FAdV-A to FAdV-E)[J]. Journal of virological methods,
2012,183:147-153. document 2: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. document 3: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.) I group FAdV primers of disclosed detection and method, draw for 3 pairs in synthesis document
Object (see Table 2) carries out sensibility comparative test with the present invention.Primer synthesis and the raw limited public affairs of work bioengineering in purifying commission Shanghai
Department completes.3 pairs of primer PCR amplification methods are illustrated to carry out by respective document respectively, to CH/AHMC/2015 plants of nucleic acid of I crowds of FAdV
It is serially diluted 6 concentration for 10 times to be detected respectively, test result is shown, the detection sensitivity of document 1 and document 2 reaches
101.3EID50/ 0.1mL(104Times diluted concentration), the detection sensitivity of document 3 is up to 103.3EID50/ 0.1mL(102It dilutes again dense
Degree), it is detailed in attached drawing 6.3 pairs of document primers and method detection sensitivity are below I crowds of FAdV universal two established by the present invention
Warm formula PCR primer and method show that the method for the present invention sensibility is very good.
The minimum nucleic acid copies identification of detection of the warm formula PCR method of 6 I crowds of FAdV of embodiment universal two
Using poultry disease prevention and control monitoring, key lab, Anhui Province has been constructed containing I groups of 235 purposes of FAdV CH/AHMC/2015 strain
The pUC57 plasmid of gene (as shown in sequence table SEQ ID No.3), converts TOP10 Escherichia coli, and identification positive bacteria carries out shaking bacterium
Proliferation carries out plasmid extraction with OMEGA company E.Z.N.A.TM Plasmid Mini Kit I kit, and plasmid is extracted in measurement
Concentration be 4.5 ug/mL, according to plasmid overall length 2945bp(, that is, 2710+235bp) and copy number calculation formula (plasmid quality/
Plasmid average molecular weight × molecular number), being converted into plasmid copy number is about 1.39 × 109copies/uL.It is minimum for determination
It detects nucleic acid copies, extracted plasmid is carried out 10 times and is serially diluted 10 concentration, i.e., 1.39 × 108copies/uL、1.39
×107copies/uL、1.39×106copies/uL、1.39×105copies/uL、1.39×104copies/uL、1.39×
103copies/uL、1.39×102copies/uL、1.39×101copies/uL、1.39×100copies/uL、1.39×10-1 copies/uL.10 diluted concentrations are respectively as template, the warm formula PCR method of I group FAdV bis- established with the optimization of embodiment 2
It is detected, 10 × rTaq buffer 1uL, 2.5mmol/L dNTP 1uL, 5U/uL rTaq archaeal dna polymerase 1uL, upstream
Primer and each 2uL of downstream primer, template plasmid 1uL, use ddH2O complements to 10uL.Reaction condition are as follows: 94 DEG C of initial denaturation 1min；
94 DEG C of denaturation 5s, 68 DEG C of annealing extend 10s, recycle 40 times；It is saved at last 4 DEG C.Electrophoretogram result is shown: I groups of bis- temperature of FAdV
Formula PCR method lowest detection copy number is 1.39 × 101Copies/uL(, that is, 13.9/uL), it is detailed in attached drawing 7.
The specific detection of the warm formula PCR method of 7 I crowds of FAdV of embodiment universal two
To determine that embodiment 2 optimizes the specificity for the warm formula PCR method of I group FAdV bis- established, poultry disease prevention and control are monitored and are pacified
Common fowl DNA virus (egg-laying reduction syndrome virus, marek virus, the avian infectious method that emblem key lab, province saves
Family name capsule virus, Chicken Infectious Anemia Virus) and bacterium (Escherichia coli, Pasteurella, salmonella) utilize OMEGA company
E.Z.N.A.TMViral DNA Kit kit extracts cause of disease DNA, to common fowl RNA virus (chicken infectious laryngotracheitis
Poison, avian infectious bronchitis virus, newcastle disease virus, H9 type avian influenza virus, J hypotype avian leukosis virus) utilize Tiangeng
Biochemical technology Co., Ltd virus genom DNA/RNA extracts kit extracts viral RNA, utilizes TaKaRa company
PrimeScript II 1st Strand cDNA Synthesis Kit carries out reverse transcription cDNA, by 7 kinds of cause of diseases of extraction
The bis- temperature formula PCR method of I group FAdV that the DNA and reverse transcription cDNA of 5 kinds of RNA virus is established as template, with the optimization of embodiment 2 into
Row detection, reaction condition are as follows: 94 DEG C of initial denaturation 1min；94 DEG C of denaturation 5s, 68 DEG C of annealing extend 10s, recycle 35 times；Last 4 DEG C
Lower preservation.As the result is shown: positive control is set up, and 12 plants of poultry encountered pathogenic testing results are negative (see attached drawing 8), test knot
Fruit shows that the warm formula PCR method specificity of I crowds of FAdV universal two is strong.
The cmot detection of the warm formula PCR method of 8 I crowds of FAdV of embodiment universal two
Existing research shows that I crowds of FAdV cause a disease strain in liver content highest, for primer and I crowds of FAdV designed by the verifying present invention
The cmot of two warm formula PCR methods, we acquire CH/AHMC/2015 plants of FAdV attack poison with poison the heart for dying SPF chicken, liver,
Spleen, lungs, kidney, glandular stomach, small intestine, cecal tonsil, tracheae, thymus gland, pancreas, the bursa of farbricius, brain, cloacal swab totally 14
Kind tissue, takes each tissue 0.2g respectively, is separately added into 1mL PBS(0.01mol/L, pH7.4) and a steel ball, use QIAGEN
Company's T issueLyser II type tissue refiner, by the 30 frequency oscillation 5min of concussion in 1 second, 10 000rpm are centrifuged later
3min obtains supernatant, presses OMEGA company E.Z.N.A. to 14 kinds of tissue supernatantsTMViral DNA Kit kit carries out disease
Malicious DNA is extracted, and the warm formula PCR method of the I group FAdV bis- established with the optimization of embodiment 2 is detected, as the result is shown: 14 kinds of tissues are equal
The DNA fragmentation that size is 235bp is amplified, it is as a result all positive (see attached drawing 9).Wherein liver specimens amplified band is most bright,
Indicate the viral level highest of liver.Test result shows that the warm formula PCR method of established I group FAdV bis- has excellent clinic
Detect adaptability.
The sequencing of 9 the method for the present invention amplified fragments gene of embodiment and phylogenetic evolution tree are analyzed
Optimize the warm formula PCR method of the I group FAdV bis- established with embodiment 2 to 5 serotype diseases of 5 gene kinds in embodiment 4
Poison carries out PCR amplification, and amplified production directly serves Hai Shenggong bioengineering Co., Ltd and carries out purifying and bidirectional sequencing.It will be surveyed
Fixed 5 strain sequences and the 12 235 primer amplified region sequences (191nt) of serotype strain 52/55KDa gene announced,
Multiple Sequence Alignment and building phylogenetic evolution tree are carried out using 6.06 software of MEGA.The results show that according to 235 primer amplifications
The phylogenetic evolution tree of regional sequence (191nt) building can divide 5 strains of I crowds of FAdV and 12 reference serum types
To 5 gene kinds of corresponding A~E (see attached drawing 10).It can be seen that expanding positive product direct Sequencing and comparing analysis energy
Identify the affiliated gene kind of I groups of FAdV strains.
Sequence table
<110>Anhui Science and Technology College
<120>the universal two warm formula PCR primers and method of a kind of I group I fowl adenovirus of quickly detection
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>upstream primer (artificial synthesized)
<400> 1
gacggtggtc gagytgagca tga 23
<210> 2
<211> 21
<212> DNA
<213>downstream primer (artificial synthesized)
<400> 2
cgcctgggtc aaaccgaaca t 21
<210> 3
<211> 235
<212> DNA
<213>aviadenovirus (fowl adenovirus)
<400> 3
gacggtggtc gagttgagca tgaagtacgc caagttggaa gcgaagaacg gctacccgtc 60
catggctcag atggccaagg cccaggaatt tttctaccgg gtgatgcagg aggtgctgga 120
cttgggggtg cagttaggcg tgtacaacaa caggccggta accttccgtc agaagcggat 180
gagcgagatt ccgcagatga ctgacgccga gtacatgttc ggtttgaccc aggcg 235
Claims (6)
1. a kind of universal two warm formula PCR primers pair for quickly detecting I group I fowl adenovirus, it is characterised in that: the primer
It is formed to by upstream primer and downstream primer；The sequence of the upstream primer be SEQ ID No.1 shown in, the downstream primer
Sequence is shown in SEQ ID No.2.
2. a kind of universal two warm formula PCR methods of quickly detection I group I fowl adenovirus, it is characterised in that: the method includes making
The universal PCR primer of the I group I fowl adenovirus described in claim 1 is to carrying out two warm formula pcr amplification reactions.
3. quickly detecting universal two warm formula PCR methods of I group I fowl adenovirus as claimed in claim 2, it is characterised in that: described
Amplified reaction volume is 10uL, including 10 × rTaq buffer 1uL, 2.5mmol/L dNTP 1uL, 5U/uL rTaq DNA
Polymerase 1uL, sample DNA template 1uL, 10umol/L upstream primer 1.5uL~2.5uL, 10umol/L downstream primer 1.5uL~
2.5uL, the upstream primer and downstream primer usage ratio are 1:1, with sterilizing ddH2O complements to 10uL.
4. quickly detecting universal two warm formula PCR methods of I group I fowl adenovirus as claimed in claim 2, it is characterised in that:
After the completion of PCR reaction tube reaction system is prepared, 2.5uL paraffin oil or vegetable oil are added on the outside of the inner circle of pipe lid.
5. quickly detecting universal two warm formula PCR methods of I group I fowl adenovirus as claimed in claim 2, it is characterised in that: described
Amplification reaction condition are as follows: reaction system initial denaturation 1min under the conditions of 94 DEG C；Then in 94 DEG C of denaturation 3~5s, 68~72
DEG C annealing and extend 8~10s, amplification cycles number be 35~40 times；Finally kept at 4 DEG C.
6. quickly detecting universal two warm formula PCR methods of I group I fowl adenovirus as claimed in claim 2, it is characterised in that: described
Pcr amplification reaction product electrophoresis detection occurs when 235bp band being the positive, does not occur band or segment only occurs less than 100bp
Primer dimer band be feminine gender.
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