CN106755582A - A kind of adenovirus detection, the primer combination and application of sequencing - Google Patents
A kind of adenovirus detection, the primer combination and application of sequencing Download PDFInfo
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Abstract
The invention provides a kind of detection of adenovirus, the primer combination and application of sequencing, belong to molecular biology and field of medical examination.The detection primer that the present invention provides adenovirus special can detect adenovirus, the application of the detection primer of adenovirus in reagent preparation box, be more convenient the detection to adenovirus, and application method is easy and quick；The present invention also provides the sequencing primer of application adenovirus, the sequencing primer energy all standing by the sequencing of adenoviral gene group, application of the sequencing primer in adenovirus sequencing is prepared is more convenient quick sequencing to adenovirus, the accurate type for judging adenovirus can be helped, effect is obvious.
Description
Technical field
The present invention relates to molecular biology and field of medical examination, in particular to a kind of adenovirus detection, sequencing
Primer is combined and applied.
Background technology
Adenovirus hominis (human adenovirus, HAdV) is the nonencapsulated double-stranded DNA virus of a class, is divided into A-G 7
Oneself turns into the key factor of acute respiratory disease at present for serology group, the wherein adenovirus infection of B groups.B groups can be divided into B1 again
With B2 subgroups, B1 is mainly including 3,7,16,21,51 types；B2 includes 14,34,35 and 55 types.
Adenovirus strain to China's outburst carries out gene order-checking, helps to understand the evolution feature and prevalence of the virus
Trend, contributes to the prevention and control to 55 type adenovirus.
At present, also without the relatively effective quick detection for adenovirus hominis (human adenovirus, HAdV) and
The means of sequencing.Or covering is not comprehensive, detect inaccurate, be easily caused flase drop and erroneous judgement.
The content of the invention
The first object of the present invention is to provide a kind of detection primer of adenovirus, and what the primer can be special detects adenopathy
Poison.
The second object of the present invention is application of the detection primer of the adenovirus for providing above-mentioned in adenovirus is detected.
The third object of the present invention is that the detection primer of the adenovirus for providing above-mentioned is preparing detection adenoviral agents box
In application.
The fourth object of the present invention is to provide a kind of detection kit of adenovirus；Quick, the special inspection of kit energy
Measure adenovirus.
The fifth object of the present invention is to provide a kind of sequencing primer of adenovirus.
The sixth object of the present invention is the sequencing primer of the adenovirus for providing above-mentioned in adenovirus genomic dna sequencing
Application.
The seventh object of the present invention is that the sequencing primer of the adenovirus for providing above-mentioned is preparing adenovirus sequencing kit
In application.
The eigth object of the present invention is to provide a kind of sequencing kit of adenovirus.
In order to realize above-mentioned purpose of the invention, spy uses following technical scheme：
A kind of detection primer of adenovirus, detection primer is combined including the first primer, and the first primer is combined as 1-12 and draws
One or more of thing centering, the base sequence of 1-12 primer pairs is respectively as shown in SEQ ID No.1-24.
Application of the detection primer of above-mentioned adenovirus in adenovirus is detected.
Application of the detection primer of above-mentioned adenovirus in detection adenoviral agents box is prepared.
A kind of detection kit of adenovirus, detection kit includes above-mentioned detection primer, and PCR reaction bufferings
Liquid, Taq archaeal dna polymerases, dNTPs and Mg2+At least one of.
A kind of sequencing primer of adenovirus, sequencing primer includes the combination of the second primer and the first primer described above combination,
The combination of second primer includes 13-24 primer pairs, and the base sequence of 13-24 primer pairs is respectively such as SEQ ID No.25-48 institutes
Show.
Application of the sequencing primer of above-mentioned adenovirus in adenovirus genomic dna sequencing.
Application of the sequencing primer of above-mentioned adenovirus in adenovirus sequencing kit is prepared.
A kind of sequencing kit of adenovirus, sequencing kit includes primer pair as claimed in claim 2；And PCR
Reaction buffer, Taq archaeal dna polymerases, ddNTPs, dNTPs and Mg2+In at least one.
Compared with prior art, beneficial effects of the present invention are：The detection primer that the present invention provides adenovirus can be special
Detect adenovirus, the application of the detection primer of adenovirus in reagent preparation box is more convenient the detection to adenovirus, and use
Method it is easy and quick；The present invention also provides the sequencing primer of application adenovirus, the sequencing primer energy all standing by adenopathy
Virus gene group is sequenced, application of the sequencing primer in adenovirus sequencing is prepared, and is more convenient the quick sequencing to adenovirus, can help
The accurate type for judging adenovirus is helped, effect is obvious.
Brief description of the drawings
Technical scheme in order to illustrate more clearly the embodiments of the present invention, below will be attached to what is used needed for embodiment
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, thus be not construed as it is right
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can also be according to this
A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 is the detection primer electrophoresis result figure that the embodiment of the present invention 1 is provided；
Fig. 2 is the result and known array comparison result figure of present invention sequencing.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, are
The conventional products that can be obtained by commercially available purchase.
Invitrogen PureLinkTMViral RNA/DNA Mini Kit have purchased from the silent winged generation your scientific and technological (China) of match
Limit company；Primer and PCR are purchased from Chengdu Qing Ke Zi Xi Bioisystech Co., Ltd with high-fidelity DNA polymerase I5-MIX；It is biochemical
Reagent is purchased from Shanghai Sheng Gong bioengineering Co., Ltd, and remaining reagent is purchased from traditional Chinese medicines biochemical reagents company.
A kind of adenovirus to the embodiment of the present invention is detected below, sequencing primer combination and application are specifically described.
A kind of detection primer of adenovirus, detection primer is combined including the first primer, and the first primer is combined as 1-12 and draws
One or more of thing centering, the base sequence of 1-12 primer pairs is respectively as shown in SEQ ID No.1-24.
, for the sequences Design of adenovirus, with strong points, specificity is good, and sensitivity is high, testing result reliability for detection primer
Accurately.
Application of the detection primer of above-mentioned adenovirus in adenovirus is detected.
Using above-mentioned detection primer, in recent years, HAdV-B55 causes in adult particularly school and army's special population
Increasingly frequently Acute respiratory infectious disease is popular；The method of applied biochemistry, can fast and effectively detect adenovirus, be
Follow-up targetedly treatment etc. provides accurately and reliably testing result.
Further, performing PCR reaction is entered by template of adenovirus DNA；
PCR reaction systems are：Mix reaction solutions, 25 μ L；Sense primer, 1 μ L；Anti-sense primer, 1 μ L；DNA profiling, 2 μ L；
DdH2O, 21 μ L；PCR response procedures are：98 DEG C, 3min；98 DEG C, 10s；57-69 DEG C, 10s；72 DEG C, 45s；30 circulations；72
DEG C, 5min.
57-69 DEG C of annealing temperature of selection, can amplify preferable purpose band, it is to avoid amplify non-specific band interference
Experimental result, 30 circulations can amplify a large amount of fragments, and micro template is amplified, beneficial to detection, and 30 circular responses
Time relatively shortens, and beneficial to fast reaction, quickly takes testing result.
Application of the detection primer of above-mentioned adenovirus in detection adenoviral agents box is prepared.
Using above-mentioned detection primer, make testing result more accurately and reliably.
A kind of detection kit of adenovirus, detection kit includes above-mentioned detection primer, and PCR reaction bufferings
Liquid, Taq archaeal dna polymerases, dNTPs and Mg2+In at least one.
Detection primer is applied in detection kit, convenient detection application is supported the use, energy quick detection adenovirus,
It is convenient and economical.
A kind of sequencing primer of adenovirus, sequencing primer includes that the combination of the second primer and the first above-mentioned primer are combined, the
Two primers include 13-24 primer pairs, and the base sequence of 13-24 primer pairs is respectively as shown in SEQ ID No.25-48.
The design of the sequencing primer, annealing temperature is moderate, without primer dimer, the generation without mismatching phenomenon.Product length
Moderate and each fragment length also close to, be also beneficial to the carrying out of sequencing reaction, reaction is quick, as a result accurately.
Application of the sequencing primer of above-mentioned adenovirus in adenovirus genomic dna sequencing.
The application of the sequencing primer of above-mentioned adenovirus, can help the type of quick analysis adenovirus be sequenced, as a result
More credible and reliable, evidence is more abundant.
Further, with adenovirus DNA as template, performing PCR reaction is entered；
Sequencing primer is to when sequencing, every a pair of sequencing primers are to being provided with the first reaction tube, the second reaction tube, the
Three reaction tubes and the 4th reaction tube, totally four reaction tubes；Template DNA, primer pair, PCR reaction bufferings are added in each reaction tube
Liquid, high-fidelity Taq archaeal dna polymerases, dNTPs and Mg2+ primer pairs；First reaction tube is additionally added ddATP, and the second reaction tube also adds
Enter ddGTP, the 3rd reaction tube is additionally added ddCTP, and the 4th reaction tube is additionally added ddTTP；PCR response procedures are：98 DEG C, 3min；
98 DEG C, 10s；58-67℃,10s；72 DEG C, 150s；40 circulations；72 DEG C, 5min.
Because the genomic DNA base quantity of adenovirus is few, adenoviral gene group is entered using first generation sequencing technologies
Row sequencing；Required data can be fast and accurately obtained, and result is also more reliable and directly perceived, can directly use the knot for reading
Really；Avoid two generation sequencing technologies, it is to avoid the post-production of the data to reading, it is simple and reliable.
Sequencing primer adds template DNA to when sequencing is used, being divided into four reaction tubes, in each reaction tube, draws
Thing to, PCR reaction buffers, high-fidelity Taq archaeal dna polymerases, dNTPs and Mg2+Primer pair；First reaction tube is additionally added
DdATP, the second reaction tube is additionally added ddGTP, and the 3rd reaction tube is additionally added ddCTP, and the 4th reaction tube is additionally added ddTTP；From
High-fidelity Taq archaeal dna polymerases, it is ensured that during reaction, can preferably ensure the sequence and template sequence one of amplification
Cause, it is to avoid the phenomenon of distortion occurs in the sequence of amplification；And Mg2+The activity of high-fidelity Taq archaeal dna polymerases can be activated, enzyme is kept
Activity higher；In first reaction tube plus be additionally added ddATP, during reaction is carried out, ddATP be connected on reaction chain when
Wait, because also ddATP cannot form 3-5 phosphodiester bonds, reaction terminates, due to ddATP be it is random be added to reaction,
So can all there be termination the position for having adenine A in the fragment of amplification；Add ddGTP, ddCTP and ddTTP reaction tube be also
Same；Each reaction tube can just read the base sequence of the fragment of whole amplification by electrophoresis.
Certainly, also need to be marked on ddNTPs, generally use radioactive label or fluorescence labeling；From safety
Angle consideration, preferably fluorescence labeling.
After the ddNTPs of fluorescence labeling, four kinds of different ddNTPs mark different fluorescence, reaction to terminate respectively, by hair
Cons electrophoresis, the amplified fragments of different molecular weight size, by electrophoresis, and can pass through to recognize different with from the order of small arrival
Fluorescence reads the base of sequence.
Application of the sequencing primer of above-mentioned adenovirus in adenovirus sequencing kit is prepared.
A kind of sequencing kit of adenovirus, sequencing kit includes above-mentioned sequencing primer pair；And PCR reaction bufferings
Liquid, Taq archaeal dna polymerases, ddNTPs, dNTPs and Mg2+In at least one.
Sequencing kit can help quickly set up sequencing reaction system, quickly read the genome base sequence of adenovirus
Row.
Feature of the invention and performance are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides a kind of detection primer of adenovirus, and detection primer is combined including the first primer, the first primer sets
It is combined into 1-12 primer pairs, the base sequence of 1-12 primer pairs is respectively as shown in SEQ ID No.1-24.
The use of above-mentioned adenovirus detection primer, specific method is as follows：
The acquisition of sample
Sample：55 type adenovirus sample collections were from Lhasa area's respiratory tract infection case in 2016.
Throat swab sample is entered using adenovirus sensitive cell line Hep-2 cells in 2 grades of (BSL2) laboratories of bio-safety
Row virus purification.
Experimental technique：
1.1 are inoculated in 24 orifice plates Hep-2 cells, inoculum density 1.5 × 105Individual/hole, cultivates 24h；
1.2 wash cell 3 times using PBS, and the 2% of 500 μ l DMEM culture mediums are added per hole, and patient diagnosed is added per hole
The μ l of oropharyngeal swab specimen 30；
Persistently culture and day by day observation of cell lesion (CPE) situation in 1.3 37 DEG C of incubators, when allusion quotation occurs in 75% cell
During type CPE, cell conditioned medium is collected；
1.4 carry out detection of nucleic acids and to PCR primer sequencing identification using PCR method, positive separator is dispensed and is preserved
In in -80 DEG C of refrigerators.
The extracting method of adenovirus DNA is as follows：
20 μ L Proteinase Ks are added sample by μ L, 12000rpm the centrifugation 5min of 2.1 vial supernatant 500 with liquid relief device
In, 65 DEG C of digestion 10-20min；
The combination liquid of 500 μ L is added in 2.2 pipes, is fully mixed；
2.3 add 400 absolute ethyl alcohol, composition to mix in Xiang Guanzhong, now it is possible that flocculent deposit, by solution
Adsorption column is added with flocculent deposit, 2min is stood；
2.4 12000rpm are centrifuged 2min, abandon waste liquid, and adsorption column is put into collecting pipe；
2.5 abandon waste liquid, by adsorption column to the rinsing liquid that 700 μ L are added in adsorption column, 12000rpm rotating speeds centrifugation 1min
It is put into collecting pipe；
2.6 to 500 μ L rinsing liquids are added in adsorption column, 12000rpm rotating speeds centrifugation 1min abandons waste liquid, and adsorption column is put
In entering collecting pipe；
2.7 12000rpm rotating speeds are centrifuged 2min, adsorption column is placed in into room temperature or 50 DEG C of incubators place 3-5min, dry；
2.8 adsorption columns are put into a clean centrifuge tube, to the hanging 50-100 μ L that are added dropwise on adsorbed film through 65 DEG C of water
The eluent of preheating is bathed, room temperature places 5min, 12000rpm rotating speeds centrifugation 1min；
2.9 centrifugation gained eluents place into adsorption column, and room temperature is placed 2min, 12000rpm rotating speeds centrifugation 2min, obtained
Adenovirus genomic dna.
PCR reaction detections：
Adenovirus DNA with extraction is template, just pcr amplification reaction, and above-mentioned 1-12 primer pairs are carried out simultaneously respectively
Reaction；
PCR reaction systems are as follows：The μ L of I5-MIX 25, the μ L of sense primer 1, anti-sense primer 1 μ L, ddH2The μ L of O 21, template
DNA 2μL；
PCR response procedures：98 DEG C of predegenerations 3min, 98 DEG C of denaturation 10s, 62 DEG C of annealing 15s, 72 DEG C of extension 45s, 30 are followed
Ring, 72 DEG C of 5min.
Experimental result is as shown in Figure 1 (in figure：1-12 represents 1-12 to primer pair respectively, and M is DNA Marker
(DL2000)), 1-12 primer pairs can identifys and amplify purpose band, and band is clearly, illustrate the of the present embodiment offer
1-12 primer pairs can well detect adenovirus, and with preferable specificity；Pass through PCR in the case of micro template
Reaction also can quickly detect adenovirus.
Embodiment 2
The present embodiment provides a kind of detection kit of adenovirus, and kit includes the 1st primer provided in embodiment 1
Right, the base sequence of the 1st primer pair is respectively as shown in SEQ ID No.1-2.
Certainly, this kit also includes directly entering the reagent of performing PCR reaction, including PCR reaction buffers, Taq
Archaeal dna polymerase, dNTPs and Mg2+In at least one.
The use of detection kit, the method for reference implementation example 1.
Embodiment 3
The present embodiment provides a kind of sequencing primer of adenovirus, and sequencing primer includes that the combination of the second primer and embodiment 1 are carried
The the first primer combination for supplying, the second primer combination includes 13-24 primer pairs, and the base sequence of 13-24 primer pairs is respectively such as
Shown in SEQ ID No.25-48.
The use of the sequencing primer that the present embodiment is provided, it is specific as follows：
It is prepared by Part I, the template that sample is sequenced
The template of sample prepares extraction and the extracting method of adenovirus DNA of the sample that reference implementation example 1 is provided.
The sequencing of Part II adenovirus DNA, method is as follows：
To primer, each pair primer is respectively provided with 4 reaction tubes to 1.1 1-24, be respectively designated as A reaction tubes, T reaction tubes,
C reaction tubes and G reaction tubes；
Add the μ L of template adenovirus DNA 1, high-fidelity Taq archaeal dna polymerases 1 μ L in 1.2 each reaction tube, 10 ×
Buffer buffer solutions 5 μ L, dNTPs (10mM each) 2 μ L, MgSO42 μ L and ddH2The μ L of O 35, each 0.75 μ of primer of upstream and downstream
L；
The 1.3 μ L of ddATP 2.5 that double deoxidation is it is possible to additionally incorporate in the reaction tube for be named as A, are being named as the reaction tube of T
In it is possible to additionally incorporate the μ L of ddTTP 2.5 of double deoxidation, the μ L of ddCTP 2.5 of double deoxidation are it is possible to additionally incorporate in the reaction tube for be named as C,
The μ L of ddGTP 2.5 of double deoxidation are it is possible to additionally incorporate in the reaction tube for be named as G；It is the ddATP of double deoxidation, the ddTTP of double deoxidation, double
The ddCTP of deoxidation and the ddGTP of double deoxidation are used32P is marked；
1.4 enter performing PCR reaction, and response procedures are：94 DEG C of predegenerations 2Min, 94 DEG C of denaturation 30s, 62 DEG C of anneal 30s, 72
DEG C extend 120s, 40 circulation, 68 DEG C extension 5min；
The reaction tube of the 4 of 1.5 each primer pair individually carries out agarose sugar gel electrophoresis, and then autoradiograph is read
Sequence is taken, the sequence assembly of different primers pair obtains the genome sequence of adenovirus.
Sequencing result carries out sequence alignment, comparison result with known adenoviral gene group sequence by application software DNAMAN
As shown in Fig. 2 the result of experiment sequencing illustrates what the present embodiment was provided by comparing consistent with the base sequence of existing adenovirus
Sequencing primer is to that can accurately be sequenced the genome of adenovirus, and specificity is higher, and reaction is gentle, and the template of concentration of telling somebody what one's real intentions are also may be used
To be sequenced.
Embodiment 4
The present embodiment provides a kind of sequencing primer of adenovirus, and sequencing primer includes the first primer sets that embodiment 1 is provided
The 1-12 primer pairs closed and the combination of the second primer, the second primer include 13-24 primer pairs, the base of 13-24 primer pairs
Sequence is respectively as shown in SEQ ID No.25-48.
Application method reference implementation example 3, difference is：
The ddATP green fluorescent labels of the double deoxidation added in the present embodiment, the ddTTP red fluorescence marks of double deoxidation
Note, the ddCTP of double deoxidation is marked with yellow fluorescence, and the ddGTP of double deoxidation is marked with blue-fluorescence.
After pcr amplification reaction terminates, product has fluorescence signal to recognize by Capillary Electrophoresis on Capillary Electrophoresis
Instrument, pass through recognized fluorescence signal instrument and be automatically converted into corresponding different base sequence, last different primer pair
Genome sequence of the sequence assembly of sequencing into adenovirus.
Sequencing result carries out sequence alignment, sequence alignment with known adenoviral gene group sequence by using DNAMAN softwares
Result is as shown in Fig. 2 the result of experiment sequencing illustrates this reality by comparing consistent with the base sequence of existing adenoviral gene group
The sequencing primer of example offer is applied to the genome of adenovirus can accurately be sequenced, and specificity is higher, reaction is gentle, even if relatively low
The template of concentration can also be sequenced.
Embodiment 5
The present embodiment provides a kind of sequencing kit of adenovirus, and sequencing kit includes the primer pair that embodiment 4 is provided；
And PCR reaction buffers, Taq archaeal dna polymerases, ddNTPs, dNTPs and Mg2+In one or more；DdNTPs includes using
The double deoxidation ddATP of green fluorescent label, the double deoxidation ddTTP of red fluorescence mark, the double deoxidation marked with yellow fluorescence
DdCTP, the double deoxidation ddGTP marked with blue-fluorescence.
The method that the application method reference implementation example 4 of the sequencing kit of adenovirus is provided.
In sum, the genome of the identification adenovirus that the adenovirus detection primer of the offer of the embodiment of the present invention can be special
Sequence, rapidly and efficiently detect adenovirus, multiple primers are used simultaneously, reach the purpose of cross validation, make result more accurate
It is reliable, using the primer and prepare the kit of detection adenovirus, can more convenient and quicker detection adenovirus, with preferably should
Use prospect；The sequencing primer of offer, can comprehensively cover the genome of adenovirus, and the fragment length of amplification is reasonable, annealing temperature
Relatively mild etc. parameter, reaction condition is gentle；Can be quickly obtained and read sequencing result, make detection more accurate, convenient and
Efficiently, application in practice is conducive to, kit prepared by the sequencing primer can more easily be sequenced, and make cost less expensive,
Result is also more reliable.
Embodiments described above is a part of embodiment of the invention, rather than whole embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiment in the present invention, what those of ordinary skill in the art were obtained under the premise of creative work is not made
Every other embodiment, belongs to the scope of protection of the invention.
SEQUENCE LISTING
<110>Liu Yuan
<120>A kind of adenovirus detection, the primer combination and application of sequencing
<130> PA16031461SC
<160> 49
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> human adenovirus
<400> 1
tttgggggtg gagtgttttt gc 22
<210> 2
<211> 23
<212> DNA
<213> human adenovirus
<400> 2
agtgcgggca ataaccaaat gat 23
<210> 3
<211> 21
<212> DNA
<213> human adenovirus
<400> 3
atttgcggtg cttccgatga g 21
<210> 4
<211> 23
<212> DNA
<213> human adenovirus
<400> 4
ggaccccagg tcacgtgaaa tac 23
<210> 5
<211> 21
<212> DNA
<213> human adenovirus
<400> 5
catcaccacc tgagcgaggt t 21
<210> 6
<211> 22
<212> DNA
<213> human adenovirus
<400> 6
rtctgggcga tgtcagcgaa at 22
<210> 7
<211> 21
<212> DNA
<213> human adenovirus
<400> 7
catcaccacc tgagcgaggt t 21
<210> 8
<211> 25
<212> DNA
<213> human adenovirus
<400> 8
atcagtctca acgcactcaa aaagt 25
<210> 9
<211> 23
<212> DNA
<213> human adenovirus
<400> 9
cagatgaggc cggactggta tac 23
<210> 10
<211> 24
<212> DNA
<213> human adenovirus
<400> 10
gtgacatctg aggtggtgag caat 24
<210> 11
<211> 22
<212> DNA
<213> human adenovirus
<400> 11
cgatgtatct ggaggaacgg ac 22
<210> 12
<211> 22
<212> DNA
<213> human adenovirus
<400> 12
aagtaactca gcgtcgacgc ac 22
<210> 13
<211> 25
<212> DNA
<213> human adenovirus
<400> 13
tgcttttaat taaatatgga gtagc 25
<210> 14
<211> 21
<212> DNA
<213> human adenovirus
<400> 14
aggaacatgc agcagaaaag t 21
<210> 15
<211> 25
<212> DNA
<213> human adenovirus
<400> 15
ttctaacacc tgctaccttt ttgat 25
<210> 16
<211> 21
<212> DNA
<213> human adenovirus
<400> 16
acagcggggt atgcaaagtg t 21
<210> 17
<211> 23
<212> DNA
<213> human adenovirus
<400> 17
cttggaagag gttccaaaaa tct 23
<210> 18
<211> 23
<212> DNA
<213> human adenovirus
<400> 18
tggatacctc tgcctctgat tac 23
<210> 19
<211> 25
<212> DNA
<213> human adenovirus
<400> 19
tacttttgga acagtcagct cttac 25
<210> 20
<211> 24
<212> DNA
<213> human adenovirus
<400> 20
tttctaaggt gttctggtgg agta 24
<210> 21
<211> 24
<212> DNA
<213> human adenovirus
<400> 21
caacttctag actggatcct tgtg 24
<210> 22
<211> 21
<212> DNA
<213> human adenovirus
<400> 22
tcttccctct cctctcctgc t 21
<210> 23
<211> 25
<212> DNA
<213> human adenovirus
<400> 23
aaccttgtca taatggagtt gcttc 25
<210> 24
<211> 23
<212> DNA
<213> human adenovirus
<400> 24
gttgcaagtt aagcggatgt gac 23
<210> 25
<211> 23
<212> DNA
<213> human adenovirus
<400> 25
aaaaggagca gtttatgcag act 23
<210> 26
<211> 24
<212> DNA
<213> human adenovirus
<400> 26
ttaaagccaa ctcagatcta gcat 24
<210> 27
<211> 22
<212> DNA
<213> human adenovirus
<400> 27
ccaacgtctg ggtgaacttt ct 22
<210> 28
<211> 21
<212> DNA
<213> human adenovirus
<400> 28
ttcgaccagc aggatgagtc t 21
<210> 29
<211> 22
<212> DNA
<213> human adenovirus
<400> 29
tctaaggcca tttgccattc tc 22
<210> 30
<211> 22
<212> DNA
<213> human adenovirus
<400> 30
gtctcaggac tctggacagc tc 22
<210> 31
<211> 24
<212> DNA
<213> human adenovirus
<400> 31
ggtgatgaaa ttaaagtagg cagt 24
<210> 32
<211> 24
<212> DNA
<213> human adenovirus
<400> 32
ggaacgtatg gagtcttgta gatc 24
<210> 33
<211> 22
<212> DNA
<213> human adenovirus
<400> 33
tgcatgatgg tctttagctg ac 22
<210> 34
<211> 22
<212> DNA
<213> human adenovirus
<400> 34
acttcgctgc tacgtacact gt 22
<210> 35
<211> 22
<212> DNA
<213> human adenovirus
<400> 35
ctcctactgc gcctacatct ac 22
<210> 36
<211> 21
<212> DNA
<213> human adenovirus
<400> 36
agccaaggat gaaaaattga t 21
<210> 37
<211> 25
<212> DNA
<213> human adenovirus
<400> 37
aaaccagata ctaaaatgaa accat 25
<210> 38
<211> 24
<212> DNA
<213> human adenovirus
<400> 38
tgaagtcttt gtaattgacc tcat 24
<210> 39
<211> 20
<212> DNA
<213> human adenovirus
<400> 39
ttgcaggtct gcctacccat 20
<210> 40
<211> 22
<212> DNA
<213> human adenovirus
<400> 40
atgttgacag caatggctga gt 22
<210> 41
<211> 23
<212> DNA
<213> human adenovirus
<400> 41
ttaacaattt tcgctctttc atc 23
<210> 42
<211> 21
<212> DNA
<213> human adenovirus
<400> 42
gtcgcgtgaa ggatatgttt c 21
<210> 43
<211> 24
<212> DNA
<213> human adenovirus
<400> 43
tgaattaaga ctctcctacg gact 24
<210> 44
<211> 24
<212> DNA
<213> human adenovirus
<400> 44
tagtgtgcag tttgagccta tagt 24
<210> 45
<211> 22
<212> DNA
<213> human adenovirus
<400> 45
tacaccctgc tgaagaccct at 22
<210> 46
<211> 23
<212> DNA
<213> human adenovirus
<400> 46
cagatatgag ttttggctgg agt 23
<210> 47
<211> 22
<212> DNA
<213> human adenovirus
<400> 47
caagagcagg acacgctaca gt 22
<210> 48
<211> 23
<212> DNA
<213> human adenovirus
<400> 48
acaaaaaaca gccaatatag cct 23
Claims (10)
1. a kind of detection primer of adenovirus, it is characterised in that the detection primer includes the combination of the first primer, and described first draws
Thing is combined as one or more in 1-12 primer pairs, and the base sequence of 1-12 primer pairs is respectively such as SEQ ID No.1-24
It is shown.
2. application of the detection primer of adenovirus as claimed in claim 1 in adenovirus is detected.
3. application according to claim 2, it is characterised in that including entering performing PCR reaction by template of adenovirus DNA；
PCR response procedures are：98 DEG C, 3min；98 DEG C, 10s；57-69℃,10s；72 DEG C, 45s；30 circulations；72 DEG C, 5min.
4. application of the detection primer of adenovirus as claimed in claim 1 in detection adenoviral agents box is prepared.
5. a kind of detection kit of adenovirus, it is characterised in that the detection kit includes inspection as claimed in claim 1
Survey primer, and PCR reaction buffers, Taq archaeal dna polymerases, dNTPs and Mg2+In at least one.
6. a kind of sequencing primer of adenovirus, it is characterised in that the sequencing primer includes that the second primer is combined and as right will
The first primer described in 1 is asked to combine, the second primer combination includes 13-24 primer pairs, the base sequence of 13-24 primer pairs
Row are respectively as shown in SEQ ID No.25-48.
7. application of the sequencing primer of adenovirus as claimed in claim 6 in adenovirus genomic dna sequencing.
8. application according to claim 7, it is characterised in that including：With adenovirus DNA as template, enter performing PCR reaction；
PCR response procedures are：98 DEG C, 3min；98 DEG C, 10s；58-67℃,10s；72 DEG C, 150s；40 circulations；72 DEG C,
5min。
9. application of the sequencing primer of adenovirus as claimed in claim 6 in adenovirus sequencing kit is prepared.
10. a kind of sequencing kit of adenovirus, it is characterised in that the sequencing kit includes as claimed in claim 6
Primer pair；And PCR reaction buffers, Taq archaeal dna polymerases, ddNTPs, dNTPs and Mg2+In at least one.
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Citations (3)
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| CN103103623A (en) * | 2013-03-01 | 2013-05-15 | 山东维真生物科技有限公司 | Adenovirus chip and application thereof |
| CN105861751A (en) * | 2016-05-16 | 2016-08-17 | 浙江省医学科学院 | Primer pair and fluorescent quantitative PCR kit for detecting mouse adenovirus and application thereof |
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| CN101608242A (en) * | 2009-04-09 | 2009-12-23 | 泰州亲和力生物技术有限公司 | A kind of H3 subtype flu quick-detecting type classifying method based on the RT-LAMP technology |
| CN103103623A (en) * | 2013-03-01 | 2013-05-15 | 山东维真生物科技有限公司 | Adenovirus chip and application thereof |
| CN105861751A (en) * | 2016-05-16 | 2016-08-17 | 浙江省医学科学院 | Primer pair and fluorescent quantitative PCR kit for detecting mouse adenovirus and application thereof |
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