CN105400908B - A kind of primer, kit and detection method using pyrosequencing techniques detection channel catfish virus - Google Patents
A kind of primer, kit and detection method using pyrosequencing techniques detection channel catfish virus Download PDFInfo
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Abstract
The invention discloses a kind of primer, kit and detection methods using pyrosequencing techniques detection channel catfish virus.In particular it relates to one group of primer using pyrosequencing techniques detection channel catfish virus, including the amplimer pair as shown in SEQ ID No.2 in sequence table and SEQ ID No.3, and the sequencing primer as shown in SEQ ID No.4 in sequence table.Invention additionally discloses the detection kits containing these primers.The present invention detects channel catfish virus using pyrosequencing techniques, has the advantages that high-throughput, fast and accurately, can accurately carry out short dna sequence analysis, sequencing procedure time-consuming is short, convenient for building normalizing operation process.
Description
Technical field
The present invention relates to a kind of primer, kit and detections using pyrosequencing techniques detection channel catfish virus
Method belongs to molecular biology field.
Background technique
Channel catfish virus (Channel catfish virus, CCV) is a kind of double-stranded DNA virus for having cyst membrane, again
Cheng Channel-catfish Simplex Virus Type I (Ictalurid herpesvirus 1), the newest disease of the 8th report of International Commission on Virus Classification
Malicious categorizing system is classified as herpetoviridae, Channel-catfish Tobamovirus.Channel catfish virosis (Channel caused by the virus
Catfish virus disease, CCVD) it is a kind of acute fatal sexually transmitted disease, it can lead to channel catfish aquaculture industry
Heavy losses, China are classified as two classes of " People's Republic of China (PRC) enter the territory animal one, two class infectious diseases, parasitic disease register "
Infectious disease.Channel catfish and Qi Ta Channel-catfish are susceptible to CCV, and virus only educate by sense dye Channel-catfish juvenile fish and fry, rigid hatching in natural water body
The seedling death rate is up to 100%.This disease is mainly in North America prevalence, China's large-scale farming channel catfish in recent years, but at present also not
There is the definite report of CCV.After CCVD is popular, remaining fish is often stealthy carrier, generally without clinical symptoms.Establish the fast of CCV
Speed detection early warning technology, reinforces channel catfish seed and at fish quarantine and the monitoring of breeding water body environment, to pre- diseases prevention
Poison infection, effectively cutting viral transmission, ensure that the healthy and sustainable development of channel catfish aquaculture is of great significance.
So far, the standard method of CCV detection mainly with cell is separately cultured virus, then with neutralization test, exempt from
The means such as epidemic disease fluorescence, ELISA or PCR are identified that the judgement to the fish infection CCV of no clinical symptoms is with serological side
Method, by measuring fish CCV antibody.Serological method sensibility is lower；PCR method is although sensitive, but still has false positive appearance
Possibility；Other methods cannot achieve the purpose that carry out Rapid Confirmation on a molecular scale.
Pyrosequencing techniques are the enzyme cascade chemiluminescence reactions in the same reaction system by 4 kinds of enzymatics, are suitable for
To the sequencing analysis of short sequence, it is not necessarily to fluorescent dye primer or nucleic acid probe, is not necessarily to electrophoresis, quick with analysis, accurate,
The characteristics of high sensitivity, economy, automation and real-time detection.This method surveys PCR product on the basis of regular-PCR
Sequence, improves the specificity of detection, and gives gold standard-gene order of molecular diagnosis, reduces due to PCR sensitivity
Property the high and false positive results that occur, improve the accuracy of detection.Pyrosequencing techniques can quickly and accurately carry out short
DNA sequence analysis, flux is high, easy to operate, convenient for building normalizing operation process, thus quite by the joyous of domestic and international researcher
It meets, is widely used to pathogenic microorganism Rapid identification.
Summary of the invention
The technical problem to be solved in the present invention is to provide one group to utilize pyrosequencing techniques detection channel catfish virus
Primer.
The invention solves another technical problem be to provide it is a kind of using pyrosequencing techniques detect channel catfish
The kit of Channel-catfish virus.
The invention solves third technical problem be to provide it is a kind of using pyrosequencing techniques detect channel catfish
The detection method of Channel-catfish virus.
To achieve the above object, cardinal principle of the invention is to pass through archaeal dna polymerase (DNA using pyrosequencing techniques
Ploymerase), adenosine triphosphate sulfurylase (ATP sulfurylase), luciferase (luciferase) and bis phosphoric acid
The single-stranded enzyme with sequencing primer of 4 kinds of enzymatic DNA to be sequenced of enzyme (apyrase) cascades chemiluminescence reaction, reaction substrate 5 '-
Adjacent acyl sulfate (adenosine5 ' phosphosulfate, APS) and fluorescein.In each round sequencing reaction, it is added a kind
DNTP, if the dNTP and template are matched, polymerase can be incorporated into primer strand and release the burnt phosphorus of equimolar number
Acid groups (PPi).Sulfurylase is catalyzed APS and PPi and forms ATP, and the fluorescein that the latter drives luciferase to mediate is glimmering to aoxidizing
The conversion of light element, issues the visible light signal directly proportional to ATP amount, and optical signals CCD camera detects and by PyrogramTMInstead
It should be peak, the nucleotide number mixed in the peak height of each optical signal and reaction is directly proportional.ATP and the dNTP being not incorporated into are by double phosphorus
Sour enzyme degradation, is quenched optical signal, and regeneration reaction system, a kind of dNTP under being then added.Final sequence order to be measured can be from
It reacts and is read in the signal peak of light intensity.
The present invention provides one group of primers using pyrosequencing techniques detection channel catfish virus, including amplification to draw
Object to and sequencing primer:
Amplimer pair: upstream primer: 5 '-GGCTTGGGCTTGATGGAC-3 ' (SEQ ID No.2)
Downstream primer: 5 '-Biotin-GAGGAGGACAACGCGACTG-3 ' (SEQ ID No.3)
Sequencing primer: 5 '-CTTGGGCTTGATGGA-3 ' (SEQ ID No.4)
Wherein, 5 ' end label biotins of downstream primer.
Channel catfish virus specific primers and Pyrosequencing primer of the present invention are according to channel catfish
The conservative region sequence of virus protein kinases (protein kinase, PK) gene, passes through Assay Design SW software design
, to amplify the single band of specificity, further according to the specific nucleotide sequence (specific target sequence for including in amplification region
Shown in SEQ ID No.1) Pyrosequencing primer is designed to determine channel catfish virus.Channel catfish virus PK gene
Target sequence: CCGGTCGTGT CGGTGTCGGT GGCTGTGTCG GCTTCCGTTT CACCG (SEQ ID No.1)；Its PCR
Expanding fragment length is 144bp.
The present invention also provides a kind of kit using pyrosequencing techniques detection channel catfish virus, the kits
Primer sets including above-mentioned detection channel catfish virus, i.e. amplimer to and sequencing primer.Further, the kit is also
Including completion PCR reaction and pyrosequencing material requested and reagent, such as DNA extraction reagent (can be according in the prior art
Disclosed method is voluntarily prepared, and the kit of commercialization purchase also can be used)；PCR reaction reagent (can be according to the prior art
Disclosed in method voluntarily prepare, also can be used commercialization purchase kit)；Single-stranded template reagent preparation；Pyrophosphoric acid is surveyed
Sequence reagent (can voluntarily be prepared according to method disclosed in the prior art, the kit of commercialization purchase also can be used) etc.,
Above-mentioned material and reagent can be hybrid packed, can also individually pack, it is preferable that for individually packaging.
The present invention also provides a kind of detection method using pyrosequencing techniques detection channel catfish virus, the party
Method includes:
(1) sample to be tested DNA is extracted as template, and extracting method well known in the prior art or commercialization examination can be used
Agent box extracts；
(2) using above-mentioned amplimer to progress pcr amplification reaction；
(3) if the reaction of channel catfish virus specific PCR is the positive, i.e. amplified fragments are 144bp, then recycle PCR amplification
Product prepares pyrosequencing single-stranded template, then using above-mentioned sequencing primer carry out pyrosequencing, if aim sequence with
CCGGTCGTGT CGGTGTCGGT GGCTGTGTCG GCTTCCGTTT CACCG (SEQ ID No.1) specific target sequence one
It causes, then contains channel catfish virus in sample to be tested；If the reaction of channel catfish virus specific PCR is feminine gender, sample is determined
Product are non-channel catfish virus.
It is and existing the invention has the advantages that the present invention detects channel catfish virus using pyrosequencing techniques
There is technology to compare, which has the advantages that high-throughput, fast and accurately, can accurately carry out short dna sequence analysis, be sequenced
Process time-consuming is short, convenient for building normalizing operation process.PCR product can be directly used for being sequenced, and be not required to carry out product purification etc. two
Secondary processing, easy to operate, required sample size is few.
The invention will be further described with specific embodiment with reference to the accompanying drawings of the specification, all to disclose according to the present invention
The equivalent replacement of any this field that content is done, all belongs to the scope of protection of the present invention.
Detailed description of the invention
Fig. 1 is that the present invention carries out agarose gel electrophoresis results to channel catfish virus PCR increasing product；Wherein M is
DNA Marker, 1 is channel catfish virus, and 2 be negative control.
Fig. 2 is the result that the present invention carries out pyrosequencing to channel catfish virus PCR increasing product.
Fig. 3 is the result that the present invention carries out agarose gel electrophoresis to the pcr amplification product of sample tissue DNA；Wherein M is
DNA Marker, 1 is the Channel-catfish fish tissues sample of channel catfish virus infection, and 2 be normal Channel-catfish fish sample, and 3 be negative control.
Fig. 4 is sequencing result of the present invention to channel catfish virus infection Channel-catfish fish tissues DNA pcr amplification product.
Fig. 5 is the sensitivity Detection result that the present invention carries out pyrosequencing to channel catfish virus PCR increasing product.
Wherein, A-G is followed successively by channel catfish viral template from 10-1It is diluted to 10-7The pyrosequencing of pcr amplification product detects knot
Fruit.
Fig. 6 is the agarose gel electrophoresis results that the present invention carries out PCR amplification specific detection to channel catfish virus.
Wherein M is DNA Marker, and 1 for channel catfish virus, 2 is goldfish hematopoietic necrosis virus, and 3 be Koi herpesvirus, 4
It is soft-shelled turtle irido virus for popular hematopoietic necrosis virus, 5,6 be negative control.
Specific embodiment
Embodiment 1 detects the design of the primer of channel catfish virus
By to the channel catfish virus announced in US National Bioinformatics Institute NCBI gene pool (GenBank)
Gene order compares and analyzes, according to the specific sequence of CCV PK gene design specific PCR primers to and pyrosequencing
Primer filters out one group of best primer and carries out subsequent PCR reaction and examining order.
The present invention is special according to the channel catfish virus of the specific regions sequence design of channel catfish virus PK gene
Property primer the single band of specificity can be amplified for channel catfish viral DNA, pcr amplified fragment length is 144bp.Again
Pyrosequencing is designed according to the specific nucleotide sequence (shown in specific target sequence SEQ ID No.1) for including in amplification region
Primer is to determine channel catfish virus.The target sequence of channel catfish virus PK gene: CCGGTCGTGT CGGTGTCGGT
GGCTGTGTCG GCTTCCGTTT CACCG(SEQ ID No.1)。
The present invention is by screening, the channel catfish virus specific PCR amplification primer pair and sequencing primer designed point
Not are as follows:
Amplimer pair: upstream primer (CCVF): 5 '-GGCTTGGGCTTGATGGAC-3 ' (SEQ ID No.2)；
Downstream primer (CCVR): 5 '-Biotin-GAGGAGGACAACGCGACTG-3 ' (5 ' end label biotin) (SEQ
ID No.3)；
Sequencing primer (CCVS): 5 '-CTTGGGCTTGATGGA-3 ' (SEQ ID No.4).
2 kit forms of embodiment
The kit include embodiment 1 design detection channel catfish virus primer sets, i.e., amplimer to and survey
Sequence primer.The kit further includes completing PCR reaction and pyrosequencing material requested and reagent, such as DNA extracts reagent
(PyroMark PCR Kit, QIAGEN are public for (DNeasy Blood&Tissue Kit, QIAGEN company), PCR reaction reagent
Department), single-stranded template reagent preparation (the coated magnetic bead of Streptavidin, GE company), pyrosequencing reagent (PyroMark
Gold Q96 SQA Reagents, denaturation buffer, dcq buffer liquid, QIAGEN company) etc., above-mentioned material and reagent can be with
It is hybrid packed, it can also individually pack, it is preferable that for individually packaging.
The foundation of 3 detection method of embodiment
1, the extraction of channel catfish viral DNA
With CCV (preservation of this laboratory) for raw material, using DNeasy Blood&Tissue Kit (QIAGEN company, article No.
69506) DNA, kit operating procedure are extracted are as follows:
(1) 20 μ l Proteinase Ks are drawn to the bottom of 1.5ml centrifuge tube.(2) virus of 200 μ l genomes to be extracted is added
Liquid (virus titer 107TCID50/ 0.1ml) into centrifuge tube.(3) 200 μ l Buffer AL are added into sample, be vortexed vibration
Swing 15s mixing.(4) 56 DEG C of incubation 10min.(5) rapid centrifugation, removal remain in the drop in 1.5ml centrifuge tube lid.(6)
The ethyl alcohol (96-100%) of 200 μ l (sample is isometric) is added, vortex oscillation 15s is mixed.After oscillation, rapid centrifugation is gone
Except the drop remained in 1.5ml centrifuge tube lid.(7) mixture for obtaining step 6 is transferred to QIAamp Mini centrifuge tube
Column.Lid is buckled, 6000g is centrifuged 1min.(8) QIAamp Min centrifugal column is put into a new clean 2ml reception pipe, it will
The collecting pipe that filtrate was used in connection with abandons.(9) QIAamp Mini centrifugal column is carefully opened, 500 μ l Buffer AW1 are added.
Lid is covered tightly, 6000g is centrifuged 1min.QIAamp Mini centrifugal column is transferred in a new 2ml collecting pipe, filtrate is connected
It is abandoned with used collecting pipe.Filtrate is abandoned, it is careful to open QIAamp Mini centrifugal column, 500 μ l Buffer AW2 are added.Lid
Tight lid, maximum (top) speed are centrifuged 3min.(10) QIAamp Mini centrifugal column is transferred to a new 1.5ml collecting pipe.Carefully
Centrifugal column is opened, 50 μ l Buffer AE or distilled water is added.(15-25 DEG C) incubation 1min of room temperature, then 6000g is centrifuged
1min.- 20 DEG C of refrigerators save backup.
2, PCR reaction system and condition
Using the CCV DNA of extraction as template, carried out with PyroMark PCR Kit (QIAGEN company, article No. 978703)
PCR expands target fragment, and upstream and downstream primer is that embodiment 1 designs obtained CCVF and CCVR primer, and PCR reaction system is as follows:
Response procedures are as follows: 95 DEG C of initial denaturation 15min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of annealing 30s are recycled altogether
45 times；Then 10min is re-extended for 72 DEG C.
3, agarose gel electrophoresis
PCR product is subjected to agarose gel electrophoresis, takes 0.6g agarose, is added in 30ml 1 × TAE electrophoretic buffer
Heat sufficiently melts, and GoldView is addedTMElectrophoresis is added in 10000 times of DNA dyestuff diluted 2 μ l of solution, glue in electrophoresis tank
Buffer makes liquid level not have gel just；Take 5 μ l pcr amplification products respectively and after 1 μ 6 × Loading of l Buffer mixing,
Point sample；10V/cm constant pressure electrophoresis stops electrophoresis until bromophenol blue indicator is migrated in the middle part of gel, gel is placed in ultraviolet solidifying
Electrophoresis result is observed under glue imaging system, electrophoresis result is shown in Fig. 1.
4, pyrosequencing single-stranded template is prepared
The PCR product and 30 μ l ddH of biotin are marked with using 20 μ l2O mixing after again with 200 μ g Streptavidin packets
The magnetic bead of quilt mixes, and 1400rpm concussion is incubated for 15min on the underlying oscillator of room temperature, will be in conjunction with magnetic bead with vacsorb pump after
PCR product pick up, then pass sequentially through 70% ethyl alcohol 5s；Denaturation buffer 5s；Dcq buffer liquid 5s is finally moved to every in advance
Hole is added above the PSQ96 orifice plate for the combination buffer that 45 μ l contain 0.3mol/L sequencing primer, discharges magnetic bead, 96 orifice plates are put
Enter after heating 2min in 80 DEG C of baking ovens, taking-up is cooled to room temperature.
5, pyrosequencing
It is reacted on pyrosequencing instrument (PYROMARK Q96), at 20-28 DEG C, used base is added suitable
Sequence is 15 (ATGC), and every wheel sequencing detection runing time is 65min, and primer strand extends with the addition of different dNTP, with
The combination of nucleic acid, CCD camera detect the optical signal of sending, and the special pyrosequencing result of channel catfish virus is shown in Fig. 2,
The sequence of reading is CGGTCGTGT CGGTGTCGGT GGCTGTGTCG GCTTCCGTTT CACCG (SEQ ID No.1).
6, result judgement
The electrophoresis result of PCR product shows to have amplified 144bp target fragment, consistent with standard；By pyrosequencing,
The aim sequence of measurement is consistent with the specific target sequence of channel catfish virus PK gene, is CGGTCGTGT CGGTGTCGGT
GGCTGTGTCG GCTTCCGTTT CACCG (SEQ ID No.1), therefore can determine that as channel catfish virus.
Embodiment 4 utilizes pyrosequencing techniques test sample
1 part of channel catfish virus infection Channel-catfish fish tissues sample and 1 part of Zheng Chang Channel-catfish fish tissues sample are taken, using embodiment
3 method detects sample.
1, the extraction of channel catfish tissue sample DNA
Channel catfish tissue sample is extracted using DNeasy Blood&Tissue Kit (QIAGEN company, article No. 69506)
Product genomic DNA, -20 DEG C of refrigerators save backup.Referring to 3 step 1 of embodiment.
2, PCR reaction system and condition
PCR reaction system and condition referring to embodiment 3 corresponding steps.With the channel catfish tissue sample DNA of extraction
For template, PCR is carried out with PyroMark PCR Kit (QIAGEN company, article No. 978703), expands target fragment.
3, agarose gel electrophoresis
Electrophoresis method referring to embodiment 3 step 3.The electrophoresis result of the present embodiment is shown in Fig. 3.
4, pyrosequencing single-stranded template is prepared
Method referring to embodiment 3 step 4.
5, pyrosequencing
It is reacted on pyrosequencing instrument (PYROMARK Q96), at 20-28 DEG C, used base is added suitable
Sequence is 15 (ATGC), and every wheel sequencing detection runing time is 65min, and primer strand extends with the addition of different dNTP, with
The combination of nucleic acid, CCD camera detect the optical signal of sending, infect CCV Channel-catfish fish tissues DNA pcr amplification product pyrophosphoric acid
Sequencing result is shown in Fig. 4, and the sequence of reading is CGGTCGTGT CGGTGTCGGT GGCTGTGTCG GCTTCCGTTT CACCG
(SEQ ID No.1)。
6, result judgement
The electrophoresis result of PCR product shows that the Channel-catfish fish tissues of channel catfish virus infection have amplified the piece of 144bp mesh
Section, it is consistent with the testing result of CCV；By pyrosequencing, aim sequence and the channel catfish virus PK gene of measurement
Specific target sequence is consistent, is CGGTCGTGT CGGTGTCGGT GGCTGTGTCG GCTTCCGTTT CACCG (SEQ ID
No.1), therefore it can determine that No. 1 sample is channel catfish virus infection.Zheng Chang Channel-catfish fish tissues do not amplify the purpose piece of 144bp
Section, therefore can determine that immaculate Cha Wei Channel-catfish virus in No. 2 samples.
Embodiment 5 detects the sensitivity experiments of channel catfish virus using pyrosequencing techniques
Using the CCV DNA extracted in embodiment 3 as template, 10 doubling dilutions are successively carried out to 10-7, while it is right to set feminine gender
According to.Sensitivity Detection is carried out using the step in embodiment 3.
1, PCR reaction system and condition
PCR reaction system and condition referring to embodiment 3 corresponding steps.Using diluted viral DNA as template, use
PyroMark PCR Kit (QIAGEN company, article No. 978703) carry out PCR amplification, reaction system, response procedures referring to
Embodiment 3.
3, agarose gel electrophoresis
Electrophoresis method referring to embodiment 3 step 3.
4, pyrosequencing single-stranded template is prepared
Method referring to embodiment 3 step 4.
5, pyrosequencing
It is reacted on pyrosequencing instrument (PYROMARK Q96), at 20-28 DEG C, used base is added suitable
Sequence is 15 (ATGC), and every wheel sequencing detection runing time is 65min, and primer strand extends with the addition of different dNTP, with
The combination of nucleic acid, CCD camera detect the optical signal of sending, and CCV DNA pcr amplification product pyrosequencing result is shown in figure
5, the sequence of reading is CGGTCGTGT CGGTGTCGGT GGCTGTGTCG GCTTCCGTTT CACCG (SEQ ID No.1).
6, result judgement
Electrophoresis result shows CCV DNA profiling from 10-1It is diluted to 10-7The target fragment of 144bp, band are still amplified afterwards
Brightness gradually weakens；By pyrosequencing, CCV DNA profiling is diluted to 10-6, CCV aim sequence and the PK gene of measurement
Specific target sequence is consistent, is CGGTCGTGT CGGTGTCGGT GGCTGTGTCG GCTTCCGTTT CACCG (SEQ ID
No.1)；It is diluted to 10-7When, specific target sequence is not measured through pyrosequencing.This example demonstrates that the utilization that the present invention establishes
The method sensibility that pyrosequencing techniques detect channel catfish virus is stronger, can be diluted to 10-6, i.e. 20TCID50。
Embodiment 6 detects the specificity experiments of channel catfish virus using pyrosequencing techniques
Goldfish hematopoietic necrosis virus (GFHNV), Koi herpesvirus (KHV), popular blood forming organ are extracted respectively
The DNA (being that this laboratory saves) of necrosis virus (EHNV) and soft-shelled turtle irido virus (STIV), with what is extracted in embodiment 3
CCV DNA is positive control, while setting negative control.Specific detection is carried out using the step in embodiment 3.
1, the extraction of viral sample DNA
Viral DNA, kit operation step are extracted using DNeasy Blood&Tissue Kit (QIAGEN company, 69506)
Suddenly referring to the step 1 of embodiment 3.
2, PCR reaction system and condition
Using extraction virus and control viral sample DNA as template, with PyroMark PCR Kit (QIAGEN company,
978703) PCR amplification is carried out, reaction system, response procedures are referring to the step 2 of embodiment 3.
3, agarose gel electrophoresis
Electrophoresis method referring to embodiment 3 step 3.The electrophoresis result of the present embodiment is shown in Fig. 6.
4, pyrosequencing single-stranded template is prepared
Method referring to embodiment 3 step 4.
5, pyrosequencing
It is reacted on pyrosequencing instrument (PYROMARK Q96), at 20-28 DEG C, used base is added suitable
Sequence is 15 (ATGC), and every wheel sequencing detection runing time is 65min, and primer strand extends with the addition of different dNTP, with
The combination of nucleic acid, CCD camera detect the optical signal of sending, CCV DNA pcr amplification product pyrosequencing result such as Fig. 2
Shown, the sequence of reading is CGGTCGTGT CGGTGTCGGT GGCTGTGTCG GCTTCCGTTT CACCG (SEQ ID
No.1)。
6, result judgement
Electrophoresis result shows that GFHNV, KHV, EHNV and STIV do not amplify the target fragment of 144bp；CCV is amplified
144bp target fragment, it is consistent with standard；By pyrosequencing, the CCV aim sequence of measurement and the particular target sequence of PK gene
Column are consistent, are CGGTCGTGT CGGTGTCGGT GGCTGTGTCG GCTTCCGTTT CACCG (SEQ ID No.1).This implementation
The method using pyrosequencing techniques detection channel catfish virus that example illustrates that the present invention establishes has good specificity
And accuracy.Sequencing primer that those skilled in the art can provide according to the present invention, target sequence building are for detecting spot fork
The kit of Wei Channel-catfish virus.The kit can be used for being used for quickly detecting channel catfish virus, have high-throughput, low cost
The characteristics of, product can be directly used for being sequenced, and not need to carry out secondary treatment, operation is extremely easy, and required sample size is small, has good
Good application prospect.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair
The restriction of embodiments of the present invention.For those of ordinary skill in the art, may be used also on the basis of the above description
To make other variations or changes in different ways.Here all embodiments can not be exhaustive.It is all to belong to this hair
The obvious changes or variations that bright technical solution is extended out are still in the scope of protection of the present invention.
Claims (3)
1. one group of primer using pyrosequencing techniques detection channel catfish virus, it is characterised in that: including such as sequence table
Amplimer pair shown in middle SEQ ID No.2 and SEQ ID No.3, and surveyed as shown in SEQ ID No.4 in sequence table
Sequence primer, wherein 5 ' end label biotins of primer shown in SEQ ID No.3.
2. a kind of kit using pyrosequencing techniques detection channel catfish virus, which is characterized in that the kit
Primer including one group described in claim 1 using pyrosequencing techniques detection channel catfish virus.
3. kit according to claim 2, which is characterized in that the kit further includes that extract reagent, PCR anti-by DNA
Answer reagent, single-stranded template reagent preparation and pyrosequencing reagent.
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| CN101240341A (en) * | 2008-03-14 | 2008-08-13 | 东南大学 | DNA sequencing method by using thiooligonucleotide probe |
| CN102703609A (en) * | 2012-07-04 | 2012-10-03 | 中国水产科学研究院黄海水产研究所 | Onsite quick detection kit of channel catfish virus and detection method thereof |
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| CN101240341A (en) * | 2008-03-14 | 2008-08-13 | 东南大学 | DNA sequencing method by using thiooligonucleotide probe |
| CN102703609A (en) * | 2012-07-04 | 2012-10-03 | 中国水产科学研究院黄海水产研究所 | Onsite quick detection kit of channel catfish virus and detection method thereof |
| EP2722399A1 (en) * | 2012-10-18 | 2014-04-23 | Roche Diagniostics GmbH | Method for preventing high molecular weight products during amplification |
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