CN106755582B - A kind of primer combination and application that adenovirus is detected, is sequenced - Google Patents
A kind of primer combination and application that adenovirus is detected, is sequenced Download PDFInfo
- Publication number
- CN106755582B CN106755582B CN201710004221.XA CN201710004221A CN106755582B CN 106755582 B CN106755582 B CN 106755582B CN 201710004221 A CN201710004221 A CN 201710004221A CN 106755582 B CN106755582 B CN 106755582B
- Authority
- CN
- China
- Prior art keywords
- adenovirus
- primer
- sequencing
- detection
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
Abstract
The present invention provides a kind of detections 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 can specifically detect adenovirus, and application of the detection primer of adenovirus in reagent preparation box is more convenient the detection to adenovirus, and application method is easy and quick；The present invention also provides the sequencing primers of application adenovirus, adenoviral gene group is sequenced for the sequencing primer energy all standing, and application of the sequencing primer in preparation adenovirus sequencing is more convenient the quick sequencing to adenovirus, the type of accurate judgement 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 combination and application.
Background technique
Adenovirus hominis (human adenovirus, HAdV) is a kind of nonencapsulated double-stranded DNA virus, is divided into A-G 7
Serology group, wherein the adenovirus infection of B group at present oneself become acute respiratory disease an important factor for.B group can be divided into B1 again
With B2 subgroup, B1 mainly includes 3,7,16,21,51 types；B2 includes 14,34,35 and 55 types.
Gene order-checking is carried out to the adenovirus strain of China's outburst, facilitates the evolution feature for understanding the virus and prevalence
Trend facilitates prevention and control to 55 type adenovirus.
Currently, not yet it is relatively effective for adenovirus hominis (human adenovirus, HAdV) it is quick detection with
The means of sequencing.Either covering is not comprehensive, and detection inaccuracy is easy to cause erroneous detection and erroneous judgement.
Summary of the invention
The first object of the present invention is to provide a kind of detection primer of adenovirus, and what which can be special detect adenopathy
Poison.
The second object of the present invention is to provide application of the detection primer of above-mentioned adenovirus in detection adenovirus.
The third object of the present invention is that the detection primer for providing above-mentioned adenovirus detects adenoviral agents box in preparation
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 to provide the sequencing primer of above-mentioned adenovirus in adenovirus genomic dna sequencing
Application.
The seventh object of the present invention is to provide the sequencing primer of above-mentioned adenovirus in preparation adenovirus sequencing kit
In application.
The eighth 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, the following technical scheme is adopted:
A kind of detection primer of adenovirus, detection primer include the first primer combination, and the first primer group is combined into 1-12 and draws
Object centering it is one or more, the base sequence of 1-12 primer pair is respectively as shown in SEQ ID No.1-24.
Application of the detection primer of above-mentioned adenovirus in detection adenovirus.
Application of the detection primer of above-mentioned adenovirus in preparation detection adenoviral agents box.
A kind of detection kit of adenovirus, detection kit include above-mentioned detection primer and PCR reaction buffering
Liquid, Taq archaeal dna polymerase, dNTPs and Mg2+At least one of.
A kind of sequencing primer of adenovirus, sequencing primer include that the combination of the second primer and such as above-mentioned the first primer combine,
The combination of second primer includes 13-24 primer pair, and the base sequence of 13-24 primer pair is respectively such as SEQ ID No.25-48 institute
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 preparation adenovirus sequencing kit.
A kind of sequencing kit of adenovirus, sequencing kit include primer pair as claimed in claim 2；And PCR
Reaction buffer, Taq archaeal dna polymerase, ddNTPs, dNTPs and Mg2+At least one of.
Compared with prior art, the invention has the benefit that detection primer that the present invention provides adenovirus can be special
Detect adenovirus, application of the detection primer of adenovirus in reagent preparation box is more convenient the detection to adenovirus, and uses
The simplicity of method and quick；The present invention also provides the sequencing primer of application adenovirus, the sequencing primer energy all standing by adenopathy
Virus gene group sequencing, application of the sequencing primer in preparation adenovirus sequencing, is more convenient the quick sequencing to adenovirus, can help
The type of accurate judgement adenovirus is helped, effect is obvious.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the detection primer electrophoresis result figure that the embodiment of the present invention 1 provides；
Fig. 2 is the result and known array comparison result figure that the present invention is sequenced.
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 invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Invitrogen PureLinkTMViral RNA/DNA Mini Kit has 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, remaining reagent is purchased from traditional Chinese medicines biochemical reagents company.
A kind of adenovirus detection of the embodiment of the present invention, sequencing primer are combined below and application is specifically described.
A kind of detection primer of adenovirus, detection primer include the first primer combination, and the first primer group is combined into 1-12 and draws
Object centering it is one or more, the base sequence of 1-12 primer pair is respectively as shown in SEQ ID No.1-24.
Detection primer is directed to the sequence design of adenovirus, with strong points, specific good, high sensitivity, and testing result is reliable
Accurately.
Application of the detection primer of above-mentioned adenovirus in detection adenovirus.
Using above-mentioned detection primer, in recent years, HAdV-B55 causes in adult especially school and army's special population
Increasingly frequent Acute respiratory infectious disease is popular；The method of applied biochemistry can quickly and effectively detect adenovirus, be
The subsequent targetedly offers such as treatment accurately and reliably testing result.
Further, PCR reaction is carried out by template of adenovirus DNA；
PCR reaction system are as follows: Mix reaction solution, 25 μ L；Upstream primer, 1 μ L；Downstream primer, 1 μ L；DNA profiling, 2 μ L；
DdH2O, 21 μ L；PCR response procedures are as follows: 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 is selected, preferable purpose band can be amplified, avoids amplifying non-specific band interference
Experimental result, 30 circulations can amplify a large amount of segments, and micro template is amplified, and be conducive to detection, and 30 circular responses
Time relatively shortens, and is conducive to fast reaction, quickly takes testing result.
Application of the detection primer of above-mentioned adenovirus in preparation detection adenoviral agents box.
Using above-mentioned detection primer, keep testing result more acurrate reliable.
A kind of detection kit of adenovirus, detection kit include above-mentioned detection primer and PCR reaction buffering
Liquid, Taq archaeal dna polymerase, dNTPs and Mg2+At least one of.
It will test primer to be applied in detection kit, facilitate detection to apply, match, can quickly detect adenovirus,
It is convenient and economical.
A kind of sequencing primer of adenovirus, sequencing primer include that the combination of the second primer and above-mentioned the first primer combine, the
Two primers include 13-24 primer pair, and the base sequence of 13-24 primer pair is respectively as shown in SEQ ID No.25-48.
The design of the sequencing primer, annealing temperature is moderate, primer free dimer, the generation of no mismatching phenomenon.Product length
The length of moderate and each segment also close to, be also beneficial to the progress of sequencing reaction, rapid reaction, 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 for quickly analyzing adenovirus be sequenced, as a result
More credible and reliable, evidence is more abundant.
Further, using adenovirus DNA as template, PCR reaction is carried out；
For sequencing primer pair when sequencing, every a pair of sequencing primer pair is 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 buffering are added in each reaction tube
Liquid, high-fidelity Taq archaeal dna polymerase, dNTPs and Mg2+ primer pair；First reaction tube is additionally added ddATP, and the second reaction tube also adds
Enter ddGTP, third reaction tube is additionally added ddCTP, and the 4th reaction tube is additionally added ddTTP；PCR response procedures are as follows: 98 DEG C, 3min；
98 DEG C, 10s；58-67℃,10s；72 DEG C, 150s；40 circulations；72 DEG C, 5min.
Since the genomic DNA base quantity of adenovirus is few, using first generation sequencing technologies to adenoviral gene group into
Row sequencing；Required data can be fast and accurately obtained, and result is also more reliable and intuitive, it can be directly using the knot read
Fruit；Two generation sequencing technologies are avoided, the post-production to the data of reading is avoided, it is simple and reliable.
Sequencing primer pair is divided into four reaction tubes when use is sequenced, and template DNA is added in each reaction tube, draws
Object to, PCR reaction buffer, high-fidelity Taq archaeal dna polymerase, dNTPs and Mg2+Primer pair；First reaction tube is additionally added
DdATP, the second reaction tube are additionally added ddGTP, and third reaction tube is additionally added ddCTP, and the 4th reaction tube is additionally added ddTTP；It selects
High-fidelity Taq archaeal dna polymerase, it is ensured that during reaction, can preferably guarantee the sequence and template sequence one of amplification
The phenomenon that causing, the sequence of amplification avoided to be distorted；And Mg2+The activity that high-fidelity Taq archaeal dna polymerase can be activated, keeps enzyme
Higher activity；In first reaction tube plus be additionally added ddATP, during reaction carries out, ddATP be connected on reaction chain when
Wait, since also ddATP can not form 3-5 phosphodiester bond, reaction is terminated, due to ddATP be it is random be added to reaction,
So having the position of adenine A that can all have termination in the segment of amplification；Be added ddGTP, ddCTP and ddTTP reaction tube be also
Similarly；Each reaction tube can read the base sequence of the segment entirely expanded by electrophoresis.
Certainly, it also needs to be marked on ddNTPs, generallys use radioactive label or fluorescent marker；From safety
Angle consideration, preferably fluorescent marker.
The ddNTPs of fluorescent marker, four kinds of different ddNTPs mark different fluorescence, after reaction, pass through hair respectively
The amplified fragments of cons electrophoresis, different molecular weight size can be different by identifying to pass through electrophoresis from the sequence of small arrival
The base of fluorescence reading sequence.
Application of the sequencing primer of above-mentioned adenovirus in preparation adenovirus sequencing kit.
A kind of sequencing kit of adenovirus, sequencing kit include above-mentioned sequencing primer pair；And PCR reaction buffering
Liquid, Taq archaeal dna polymerase, ddNTPs, dNTPs and Mg2+At least one of.
Sequencing kit can help quickly to establish sequencing reaction system, quickly read the genome base sequence of adenovirus
Column.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides a kind of detection primer of adenovirus, detection primer includes the first primer combination, the first primer group
It is combined into 1-12 primer pair, the base sequence of 1-12 primer pair is respectively as shown in SEQ ID No.1-24.
The use of above-mentioned adenovirus detection primer, the 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.
2 grades of laboratories (BSL2) of bio-safety using adenovirus sensitive cell line Hep-2 cell to throat swab sample into
Row virus purification.
Experimental method:
1.1 by Hep-2 cell inoculation in 24 orifice plates, inoculum density 1.5 × 105A/hole, culture is for 24 hours；
1.2 are washed cell 3 times using PBS, and the 2% DMEM culture medium of 500 μ l is added in every hole, and patient diagnosed is added in every hole
30 μ l of oropharyngeal swab specimen；
It is persistently cultivated in 1.3 37 DEG C of incubators and observes cytopathy (CPE) situation day by day, when allusion quotation occurs in 75% cell
When type CPE, cell conditioned medium is collected；
1.4 carry out detection of nucleic acids and PCR product are sequenced to identify using PCR method, and positive isolate is dispensed and is saved
In in -80 DEG C of refrigerators.
The extracting method of adenovirus DNA is as follows:
2.1 vial supernatant 500 μ L, 12000rpm are centrifuged 5min, and sample is added in 20 μ L Proteinase Ks 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, mixes well；
2.3 are added 400 dehydrated alcohol in Xiang Guanzhong, and ingredient mixes, at this time it is possible that flocculent deposit, by solution
Adsorption column is added with flocculent deposit, stands 2min；
2.4 12000rpm are centrifuged 2min, abandon waste liquid, adsorption column is put into collecting pipe；
2.5 the rinsing liquid of 700 μ L is added into adsorption column, and 12000rpm revolving speed is centrifuged 1min, waste liquid is abandoned, by adsorption column
It is put into collecting pipe；
2.6 500 μ L rinsing liquids are added into adsorption column, and 12000rpm revolving speed is centrifuged 1min, abandon waste liquid, adsorption column is put
Enter in collecting pipe；
2.7 12000rpm revolving speeds are centrifuged 2min, and adsorption column is placed in room temperature or 50 DEG C of incubators place 3-5min, is dried；
2.8 adsorption columns are put into a clean centrifuge tube, and 50-100 μ L is vacantly added dropwise on adsorbed film through 65 DEG C of water
The eluent for bathing preheating, is placed at room temperature for 5min, and 12000rpm revolving speed is centrifuged 1min；
2.9 centrifugation gained eluents place into adsorption column, are placed at room temperature for 2min, 12000rpm revolving speed is centrifuged 2min, obtains
Adenovirus genomic dna.
PCR reaction detection:
Using the adenovirus DNA of extraction as template, just pcr amplification reaction, above-mentioned 1-12 primer pair carry out simultaneously respectively
Reaction；
PCR reaction system is as follows: 25 μ L of I5-MIX, 1 μ L of upstream primer, downstream primer 1 μ L, ddH221 μ L of O, template
DNA 2μL；
PCR response procedures: 98 DEG C of initial denaturation 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.
(in figure: 1-12 respectively represents 1-12 to primer pair to experimental result, and M is DNA Marker as shown in Figure 1
(DL2000)), 1-12 primer pair can be identified and amplify purpose band, and band is clear, illustrate provided in this embodiment the
1-12 primer pair can detect adenovirus well, and have preferable specificity；Pass through PCR in the case where micro template
Reaction also can quickly detect adenovirus.
Embodiment 2
The present embodiment provides a kind of detection kit of adenovirus, 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 further includes the reagent that can directly carry out PCR reaction, including PCR reaction buffer, Taq
Archaeal dna polymerase, dNTPs and Mg2+At least one of.
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, sequencing primer includes that the combination of the second primer and embodiment 1 mention
The first primer of confession combines, and the combination of the second primer includes 13-24 primer pair, and the base sequence of 13-24 primer pair is respectively such as
Shown in SEQ ID No.25-48.
The use of sequencing primer provided in this embodiment, specific as follows:
The template preparation of sample is sequenced in first part
The extraction for the sample that the template preparation reference implementation example 1 of sample provides and the extracting method of adenovirus DNA.
The sequencing of second part adenovirus DNA, the method is as follows:
For 1.1 1-24 to primer, 4 reaction tubes are respectively set in each pair of primer, be respectively designated as A reaction tube, T reaction tube,
C reaction tube and G reaction tube；
It is added 1 μ L of template adenovirus DNA, high-fidelity Taq archaeal dna polymerase 1 μ L in 1.2 each reaction tubes, 10 ×
5 μ L of buffer buffer, dNTPs (10mM each) 2 μ L, MgSO42 μ L and ddH235 μ L of O, each 0.75 μ of the primer of upstream and downstream
L；
1.3 additionally incorporate the 2.5 μ L of ddATP of double deoxidation in the reaction tube for being named as A, in the reaction tube for being named as T
In additionally incorporate the 2.5 μ L of ddTTP of double deoxidation, the 2.5 μ L of ddCTP of double deoxidation is additionally incorporated in the reaction tube for being named as C,
The 2.5 μ L of ddGTP of double deoxidation is additionally incorporated in the reaction tube for being named as G；The ddTTP, double of the ddATP of double deoxidation, double deoxidation
The ddCTP of deoxidation and the ddGTP of double deoxidation are used32P label；
1.4 carry out PCR reactions, response procedures are as follows: 94 DEG C of initial denaturation 2Min, 94 DEG C of denaturation 30s, 62 DEG C of annealing 30s, and 72
DEG C extend 120s, 40 circulation, 68 DEG C of extension 5min；
4 reaction tube 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 and known adenoviral gene group sequence pass through application software DNAMAN progress sequence alignment, comparison result
As shown in Fig. 2, the result of experiment sequencing is consistent with the base sequence of existing adenovirus by comparing, illustrate provided in this embodiment
The genome of adenovirus can accurately be sequenced in sequencing primer pair, and specificity is higher, and reaction is mild, and the template for concentration of telling somebody what one's real intentions are can also
To be sequenced.
Embodiment 4
The present embodiment provides a kind of sequencing primer of adenovirus, sequencing primer includes the first primer group that embodiment 1 provides
The 1-12 primer pair closed and the combination of the second primer, the second primer include 13-24 primer pair, the base of 13-24 primer pair
Sequence is respectively as shown in SEQ ID No.25-48.
Application method reference implementation example 3, difference is:
The ddATP for the double deoxidation being added in the present embodiment is marked with green fluorescence, the ddTTP red fluorescence mark of double deoxidation
Note, the ddCTP of double deoxidation are marked with yellow fluorescence, and the ddGTP of double deoxidation is marked with blue-fluorescence.
After pcr amplification reaction, reaction product has fluorescence signal identification by Capillary Electrophoresis on Capillary Electrophoresis
Instrument, corresponding different base sequence is automatically converted by the fluorescence signal instrument identified, last different primer pair
The sequence assembly of sequencing at adenovirus genome sequence.
Sequencing result and known adenoviral gene group sequence are by carrying out sequence alignment, sequence alignment using DNAMAN software
As a result as shown in Fig. 2, the result of experiment sequencing is consistent with the base sequence of existing adenoviral gene group by comparing, illustrate this reality
The genome of adenovirus can accurately be sequenced in the sequencing primer pair for applying example offer, and specificity is higher, and reaction is mild, even if lower
The template of concentration can also be sequenced.
Embodiment 5
The present embodiment provides a kind of sequencing kit of adenovirus, sequencing kit includes the primer pair that embodiment 4 provides；
And PCR reaction buffer, Taq archaeal dna polymerase, ddNTPs, dNTPs and Mg2+One of or it is a variety of；DdNTPs includes using
The double deoxidation ddATP of green fluorescence label, the double deoxidation ddTTP of red fluorescence label, 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 provides.
In conclusion the genome of the identification adenovirus of the adenovirus detection primer energy of the offer of the embodiment of the present invention specifically
Sequence, rapidly and efficiently detect adenovirus, multiple primers use simultaneously, achieve the purpose that cross validation, keep result more acurrate
Reliably, using the primer and prepare detection adenovirus kit, can more convenient and quicker detection adenovirus, have preferably answer
Use prospect；The sequencing primer of offer can comprehensively cover the genome of adenovirus, and the fragment length of amplification is reasonable, annealing temperature
Etc. parameters it is relatively mild, reaction condition is mild；Can be quickly obtained and read sequencing result, make to detect it is more accurate, convenient and
Efficiently, being conducive to application in practice, the kit of sequencing primer preparation can more easily be sequenced, and keep cost less expensive,
As a result also more reliable.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
SEQUENCE LISTING
<110>Liu Yuan
<120>the primer combination and application of a kind of adenovirus detection, 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 (6)
1. a kind of detection primer of adenovirus, which is characterized in that the detection primer includes the first primer combination, and described first draws
Object group is combined into 1-12 primer pair, and the base sequence of 1-12 primer pair is respectively as shown in SEQ ID No.1-24.
2. application of the detection primer of adenovirus as described in claim 1 in preparation detection adenoviral agents box.
3. a kind of detection kit of adenovirus, which is characterized in that the detection kit includes inspection as described in claim 1
Survey primer and PCR reaction buffer, Taq DNA polymerase, dNTPs and Mg2+。
4. a kind of sequencing primer of adenovirus, which is characterized in that the sequencing primer includes that the second primer combines and as right is wanted
The combination of the first primer described in asking 1, the second primer combination includes 13-24 primer pair, the base sequence of 13-24 primer pair
Column are respectively as shown in SEQ ID No.25-48.
5. application of the sequencing primer of adenovirus as claimed in claim 4 in preparation adenovirus sequencing kit.
6. a kind of sequencing kit of adenovirus, which is characterized in that the sequencing kit includes drawing as claimed in claim 4
Object pair；And PCR reaction buffer, Taq DNA polymerase, ddNTPs, dNTPs and Mg2+。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710004221.XA CN106755582B (en) | 2017-01-04 | 2017-01-04 | A kind of primer combination and application that adenovirus is detected, is sequenced |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710004221.XA CN106755582B (en) | 2017-01-04 | 2017-01-04 | A kind of primer combination and application that adenovirus is detected, is sequenced |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106755582A CN106755582A (en) | 2017-05-31 |
| CN106755582B true CN106755582B (en) | 2019-08-20 |
Family
ID=58949421
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710004221.XA Active CN106755582B (en) | 2017-01-04 | 2017-01-04 | A kind of primer combination and application that adenovirus is detected, is sequenced |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106755582B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2017
- 2017-01-04 CN CN201710004221.XA patent/CN106755582B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| 人呼吸道腺病毒55型的基因组学与病原学特征研究;刘娟;《中国博士学位论文全文数据库 医药卫生科技辑》;20150115(第01期);第24-26页第1节、第26页第2.1节、第60页第1.3-1.6节、第82-85页附注 |
| 高原地区55 型腺病毒的病原分离及全基因序列分析;王文博 等;《军事医学》;20170630;第41卷(第62期);第453-456页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106755582A (en) | 2017-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106636471B (en) | Multiplex PCR detection kit for simultaneously detecting WSSV, AHPND, EHP and IHHNV of prawns | |
| AU2011227110B2 (en) | Methods, kits and compositions for detection of MRSA | |
| CN107488748B (en) | Composition for detecting 23 respiratory pathogens, kit and detection method thereof | |
| CN107227361A (en) | Primer, probe and detection kit for detecting CYP2C19 gene pleiomorphisms | |
| CN111269995A (en) | Primer group, kit and detection method for detecting pathogen | |
| CN107022641B (en) | Primer for detecting deafness gene and application thereof | |
| CN101712973B (en) | Reactive reagent of nucleic acid amplification by chain replacement at room temperature and nucleic acid amplification method at room temperature thereof | |
| CN102002531A (en) | Toxoplasma gondii detection kit and application thereof | |
| EA008388B1 (en) | Amplification-hybridisation method for detecting and typing human papillomavirus | |
| CN102251061A (en) | Nucleic acid dual fluorescence PCR (Polymerase Chain Reaction) detection kit for influenza A/B virus | |
| CN102010910A (en) | Loop-mediated isothermal amplification technology-based plasmodium genus and species nucleic acid screening method | |
| CN102286639A (en) | Type A H1N1/influenza A virus nucleic acid dual fluorescent polymerase chain reaction (PCR) detection kit | |
| KR20180132603A (en) | A primer set for detecting chlamydia trachomatis, a chlamydia trachomatis detection method using the same, and a reagent kit | |
| Steyer et al. | A diagnostic method based on MGB probes for rapid detection and simultaneous differentiation between virulent and vaccine strains of avian paramyxovirus type 1 | |
| CN102140509B (en) | Gene mutation detection method based on nucleic acid amplification on solid carrier | |
| CN106755582B (en) | A kind of primer combination and application that adenovirus is detected, is sequenced | |
| CN105349666B (en) | Cerebral arterial thrombosis miRNA markers | |
| CN112410465A (en) | Novel coronavirus SARS-CoV-2ORF1ab and N gene constant temperature amplification primer group and kit | |
| CN102212621A (en) | Detection kit and detection method for hepatitis B virus genotyping | |
| CN103060446B (en) | Use of CD157 gene | |
| CN115323075A (en) | RT-RAA primer probe set and kit for detecting infectious bronchitis viruses and genotyping and application of RT-RAA primer probe set and kit | |
| CN102230025B (en) | Kit and method for detecting drug resistance mutation of hepatitis B virus genes | |
| CN110184363B (en) | Primer group and kit for detecting salmonella | |
| CN108531622B (en) | Hybrid membrane strip and detection kit for mycobacterium tuberculosis complex and avian-intracellular mycobacterium tuberculosis complex | |
| CN111197094B (en) | Compositions, kits and methods for genotyping vibrio parahaemolyticus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20190510 Address after: 610083 No. 270 Rongdu Avenue, Jinniu District, Chengdu City, Sichuan Province Applicant after: General Hospital of the Western War Zone of the Chinese People's Liberation Army Address before: 610000 No. 270 Rongdu Avenue, Jinniu District, Chengdu City, Sichuan Province Applicant before: Liu Yuan |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |