CN105838827A - Virus genome primer and method for detecting virus genome through same - Google Patents
Virus genome primer and method for detecting virus genome through same Download PDFInfo
- Publication number
- CN105838827A CN105838827A CN201610347518.1A CN201610347518A CN105838827A CN 105838827 A CN105838827 A CN 105838827A CN 201610347518 A CN201610347518 A CN 201610347518A CN 105838827 A CN105838827 A CN 105838827A
- Authority
- CN
- China
- Prior art keywords
- primer
- virus
- viral genome
- chain
- 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.)
- Pending
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
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to primer, in particular to a virus genome primer and a method for detecting virus genome through the same. The primer comprises nucleotide sequences shown in SEQ ID NO: 1-SEQ ID NO:8. Through the adoption of the primer, the disadvantage in the prior art that the detection sensitivity is low when the traditional random primer RP is adopted, by analyzing all reference virus genome sequences in a NCBI virus reservoir, oligonucleotide sequences capable of widely recognizing reference virus genome are screened, a virus genome detection technology is constructed on the basis of the sequences, and the primer not only meets the most basic requirement on wide recognition of virus genome, but also achieves the higher target of improving the virus genome detection sensitivity.
Description
Technical field
The present invention relates to a kind of primer, especially relate to a kind of viral genome primer and use its detection virus
The method of genome.
Background technology
2009, Victoria, J.G et al. the most famous magazine Journal of Virology delivers one
Article (the Metagenomic analyses of viruses in stool samples found about newtype enteroviru
From children with acute flaccid paralysis.J Virol, 2009.83 (9): p.4642-51), open
A kind of can non-specific identification and the method for amplification unknown virus genome, the method utilizes random primer
RP be capable of identify that the feature of virulent nucleotide sequence, the case sample of the not clear cause of disease is detected,
Thus the cause of disease of not clear disease is diagnosed, but utilize the method for unknown virus detection of random primer RP
Sensitivity the most on the low side, be only able to detect and at least contain 105The clinical sample of individual virion, this is with regard to pole
The earth limits the method application directly in clinical diagnosis, traces it to its cause, mainly due to random primer
In RP, the most a large amount of dimeric existence, greatly reduce the concentration of effective primer in random primer RP.
In view of this, the special proposition present invention.
Summary of the invention
The present invention provide a kind of can be with the identification of wide spectrum and the primer of amplicon virus genome and comprise this and draw
The kit of thing.
On the other hand, the present invention provides this viral genome primer and this kit in detection viral genome
The application of aspect.
On the other hand, the present invention provides a kind of and uses the viral genome primer virus genomic method of detection.
For solving above-mentioned technical problem, the present invention uses the basic conception of technical scheme to be: a kind of virus base
Because of group primer, including eight nucleotide sequences as shown in SEQ ID NO:1-SEQ ID NO:8.
Annex base N:A/T/C/G；R:A/G；Y:C/T.
The viral genome primer as above application in terms of detection viral genome.
A kind of kit, described kit includes viral genome primer as above.
The application in terms of detection viral genome of a kind of kit as above.
Detect virus genomic method with viral genome primer as above, comprise the steps:
1) viral genome primer as claimed in claim 1 is synthesized；
2) to Sample pretreatment and digestion；
With centrifuge 5 minutes, the filter taking supernatant 0.22 μm filtered, the sample that will obtain
Use the RNase A of TURBO DNase and 50 μ g of the DNase I, 8U of 70U 37 DEG C of temperature
The lower digestion of degree 90 minutes；
3) synthesis of the first chain；
Extract the nucleic acid compositions of the DNA virus/RNA virus comprised in sample, be dissolved into 20 μ L seedless
In the deionized water of enzyme, generate nucleic acid extraction liquid；The nucleic acid extraction liquid that simultaneously will extract, wants by right
Seek the viral genome primer described in 1, the rna gene group of virus is carried out reverse transcription, generates DNA
First chain；
4) synthesis and the PCR of the second chain reacts；
By step 3) obtain virus nucleic acid extraction liquid and the first chain solution, use such as claim 1
Described viral genome primer amplification obtains DNA the second chain；
The DNA double chain liquid that DNA the first chain and DNA the second chain form is stripped；
DNA double chain liquid SEQ ID NO:9 primer after extracting is carried out PCR reaction；
5) clone of PCR primer and sequencing；
It is connected to PCR primer on carrier build clone bank, has the clone of insertion also by blue hickie screening
Check order；
Blue hickie screening is a kind of method of recombinant screen: be the hereditary feature screening restructuring according to carrier
Son, such as α-complementary, antibiotic resistance gene etc..The many carriers currently used are all colibacillary with one
The short section of DNA, wherein has the regulating and controlling sequence of beta-galactosidase gene (lacZ) and first 146
Amino acid whose coding information.Inserting a MCS (MCS) in this code area, it is also
Do not destroy frame, but a few amino acid can be made to be inserted into the aminoterminal of beta galactosidase and not affect
Function, this carrier is applicable to the host cell of codified beta galactosidase C end portion sequence.Therefore,
Though host and plasmid-encoded fragment all do not have an enzymatic activity, but they simultaneously in the presence of, can formed, there is enzyme
Learn the protein of activity.So, lacZ gene lack the host cell of nearly operator section with
Complementation is achieved, referred to as α-complementary between the plasmid of complete nearly operator section.Produced by α-complementary
Raw LacZ+ bacterium, under the effect of derivant IPTG, produces indigo plant in the presence of chromogenic substrate X-Gal
Look bacterium colony, thus readily identified.But, after foreign DNA is inserted into the MCS of plasmid,
Almost invariably cause the n terminal fragment without α-complementary ability so that with the bacterium of recombinant plasmid
Form white colony.The screening of this recon, is also called blue hickie screening.As screened then with blue hickie
The 37 DEG C of incubators of calcification bacterium flat board converted through connecting product are inverted after cultivating 12-16hr, have recombinant plasmid
Bacterium forms white colony.
6) process of data and analysis；
Nucleotide sequence order-checking obtained is sheared, and removes the sequence of carrier part, and rejecting does not contains
There is the nucleotide sequence of SEQ ID NO:9 primer sequence, compare with database, find and most mate
After virus kind/strain, all of sequence information of comprehensive analysis, it is judged that the viral species contained in this sample.
Further, described step 4) in PCR reaction condition as follows: 95 DEG C of 5min of 1 circulation,
95 DEG C of 15sec, 50 DEG C of 15sec, 72 DEG C of 2min of 35 circulations, 72 DEG C of 7min of 1 circulation.
After using technique scheme, the present invention compared with prior art has the advantages that employing
Virus genomic primer of the present invention, overcomes and uses tradition random primer RP in prior art
Detection sensitivity is low, the virus of low concentration in clinical sample is screened the shortcoming of weak effect, by analyzing
All reference virus sequences in NCBI virus base, filter out and can extensively identify reference virus genome
Oligonucleotide sequence, and on the basis of this sequence, establish a kind of viral genome detection technique, it is possible to
The virus genome sequence that in clinical sample, concentration is lower detected, both met and extensively identified viral genome
Most basic needs, again reach improve detect virus genomic sensitivity higher target.
Accompanying drawing explanation
Fig. 1 is the comparison diagram of viral genome primer of the present invention and random primer RP sensitivity.
Detailed description of the invention
Below in conjunction with specific embodiments and the drawings, the present invention is further explained explanation.
The present invention is on the basis of tradition arbitrarily primed polymerase chain reaction (rPCR), by the present invention
The viral genome primer (PVGPs) of screening replaces the random primer RP in rPCR, detects viral genome
Method, concrete technical scheme is as follows:
1, synthesis viral genome primer
The present invention relates to eight groups of nucleotide primers comprising merger base altogether, often group primer synthetic quantity is
5～10OD (see table), according to the TE solution of its synthetic quantity respective volume be dissolved into 100 μMs dense
The primer solution of degree, according to often organizing the primer bar number that primer comprises, calculates in PVGPs mixed liquor and should contain
There is the volume often organizing primer, by these eight groups of primer solution according to last volume mixture arranged of following table, i.e. originally
The PVGPs of invention.In PVGPs of the present invention, the concentration of every primer is equal (being 2.78 μMs),
Detailed process is as shown in table 1 below:
The synthesis of table 1 viral genome primer and preparation
2, Sample pretreatment and digestion
In order to remove cell component, bacterial component and the larger particles material in sample as far as possible, need sample
Originally pre-treatment is carried out.With centrifuge at the highest rotating speed, such as 13,000 rev/min, centrifugal 5 minutes,
The filter (Millipore) taking supernatant 0.22 μm filters.
In order to be removed by DNA and RNA of smudge cells and bacterial genomes, need sample is carried out
DNase/RNase cocktail digestion process.Use the DNase I (Takara), the TURBO of 8U of 70U
The RNase A (Takara) of DNase (Invitrogen) and 50 μ g digests 90 minutes at a temperature of 37 DEG C.
3, viral nucleic acid extraction and the reverse transcription of RNA virus
By postdigestive sample according to PureLink Viral RNA/DNA Mini Kit (Invitrogen, virus
DNA/RNA purification kit) product description operation, extract in sample the DNA virus comprised
The nucleic acid compositions of/RNA virus, and be finally dissolved in the deionized water of 20 μ L rnase-frees, generate core
Acid extract.
To the RNA virus in detection sample, need according to SuperScript III RT kit (Invitrogen)
Product description, use the PVGPs of the present invention that the rna gene group of virus is carried out reverse transcription, raw
Become cDNA chain (the first chain), then use E.coli RNase H (Invitrogen) and cDNA chain 37
DEG C effect 20 minutes, removes RNA chain therein, generates cDNA liquid.
4, the synthesis of the second chain and PCR (PCR)
Use nucleic acid extraction liquid and the cDNA solution of virus, use Klenow fragment (Invitrogen)
Reagent, uses PVGPs synthetic DNA second chain of the present invention.The DNA double chain generated uses phenol
Chloroformic solution is stripped, and is resuspended in the deionized water of 20 μ L rnase-frees, generates DNA double chain liquid.
PCR reaction needs the product description according to AmpliTaq Gold (Invitrogen), uses SEQ
DNA double chain liquid is expanded by ID NO:9 primer, and reaction condition is as follows: 95 DEG C 5 of 1 circulation
Min, 95 DEG C of 15sec, 50 DEG C of 15sec, 72 DEG C of 2min of 35 circulations, 72 DEG C 7 of 1 circulation
min。
Described SEQ ID NO:9 primer is GACCATCTAGCGACCTCCAC.
5, the clone of PCR primer and sequencing
PCR primer according toThe specification of Quick Gel Extraction Kit (Invitrogen),
Cut glue and reclaim the fragment between 100bp～1000bp；According toTA cloning vector
(Invitrogen) specification, is connected to PCR primer in carrier build clone bank.Transformed competence colibacillus is thin
Bacterium DH5 α bacterial cells (Takara), has the clone of insertion to check order by blue hickie screening.
6, the process of data and analysis
Nucleotide sequence order-checking obtained is sheared, and removes the sequence of carrier part, and rejecting does not contains
There is the nucleotide sequence of SEQ ID NO:9 sequence, use blast program
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) and US National Biotechnology Information center
(NCBI) nr/nt database is compared, and finds the viral kind/strain mated most, the most comprehensively analyzes
All of sequence information, it is judged that the viral species contained in this sample.
Viral genome primer PVGPs of the present invention and random primer RP is used said method, to difference
Concentration mode virus, as a example by poliovirus (human poliovirus) vaccine strain, enters respectively
Row detection, it was found that as it is shown in figure 1, in high concentration virus group (1 × 106PFU/100 μ L and 1
×105PFU/100 μ L), two kinds of primers are to there is not significance difference in the recognition capability of poliovirus
Different (p is respectively 0.172 and 0.674, α=0.05)；In low concentration virus group 1 × 104PFU/100
During μ L, viral genome primer PVGPs of the present invention is Polio virus in 50 clones selected at random
Discrimination be 58%, the discrimination 8% than traditional random primer RP is significantly increased (X2=26.046,
P=0.000, α=0.05)；In least concentration virus group 1 × 103During PFU/100 μ L, primer of the present invention
PVGPs remains able to detect the Polio virus genome of 12%, and RP detected at all less than.Above
Result explanation relies on viral genome primer PVGPs of the present invention and possesses higher than relying on random primer RP
Sensitivity.
In order to reflection present invention expanding effect on all of reference virus genome, the present invention is also
Existing reference virus genome sequence is simulated amplification, i.e. with every viral genome as template,
Carry out forward and reverse identification with the PVGPs of the present invention, according to viral genome be combined with PVGPs result,
By every may amplification fragment according to different fragment length (1～99bp, 100～399bp,
400～699bp, 700～999bp, 1000～1299bp, 1300～1599bp, 1600～1899bp,
1900～2199bp and >=2200bp) adding up, wherein said viral genome is 4244, presses
Being divided into six classes according to different Virus Types, six viroid types are as follows: double-strand DNA (dsDNA)
viruses,single-strand DNA(ssDNA)viruses,double-strand RNA(dsRNA)viruses,
single-strand positive RNA(ssRNA(+))virus,single-strand negative
RNA(ssRNA(-))virus and retrovirus(RT)virus.The result of simulation amplification shows, Ji Husuo
Some viral genome can be identified amplification by PVGPs, except following 5 viral genomes, as
Mulard duck circovirus in ssDNA virus (accession:NC_005053, derives from duck,
Long 2kb), (accession:NC_013030 derives from sargassum to Circovirus-like genome SAR-A
Sea, long 1.74kb), Acartia tonsa copepod circovirus isolate 154_D11 (accession:
NC_020099, derives from Marine copepod, long 1.67kb), the Gremmeniella in dsRNA virus
Abietina RNA virus MS2 (accession:NC_006444, NC_006445 and NC_006446,
Derive from coniferous forest, long 1.19kb～1.78kb) and ssRNA (+) Saccharomyces 23S in virus
RNA narnavirus (GI:21557568 derives from yeast, long 2.89kb).Visible PVGPs can know
Not and expand known most viral genome and include from various animals, plant, bacterium and fungi
Virus Deng species.
The viral genome primer (PVGPs) of the present invention can be sensitiveer than traditional random primer RP
Nucleic acid sequence a small amount of in sample detected, this invention makes viral genome primer detection technique
The diagnosis of the especially unknown cause of disease of etiological diagnosis of clinical infection's case can extensively be applied.
The above is only the preferred embodiment of the present invention, it is noted that general for the art
For logical technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvement and profit
Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (6)
1. a viral genome primer, it is characterised in that include such as SEQ ID NO:1-SEQ ID NO:8
Eight shown nucleotide sequences.
2. the application in terms of detection viral genome of the viral genome primer described in claim 1.
3. a kit, it is characterised in that described kit includes the viral base described in claim 1
Because of group primer.
4. the application in terms of detection viral genome of the kit described in claim 3.
5. detect virus genomic method, its feature with the viral genome primer described in claim 1
It is, comprises the steps:
1) viral genome primer as claimed in claim 1 is synthesized；
2) to Sample pretreatment and digestion；
With centrifuge 5 minutes, the filter taking supernatant 0.22 μm filtered, and is adopted by the sample obtained
With the RNase A of the TURBO DNase and 50 μ g of the DNase I, 8U of 70U 37 DEG C of temperature
Lower digestion 90 minutes；
3) synthesis of the first chain；
Extract the nucleic acid compositions of the DNA virus/RNA virus comprised in sample, be dissolved into 20 μ L seedless
In the deionized water of enzyme, generate nucleic acid extraction liquid；The nucleic acid extraction liquid that simultaneously will extract, uses claim
Viral genome primer described in 1, carries out reverse transcription to the rna gene group of virus, generates DNA first
Chain；
4) synthesis and the PCR of the second chain reacts；
By step 3) obtain virus nucleic acid extraction liquid and the first chain solution, use such as claim 1 institute
The viral genome primer amplification stated obtains DNA the second chain；
The DNA double chain liquid that DNA the first chain and DNA the second chain form is stripped；
DNA double chain liquid SEQ ID NO:9 primer after extracting is carried out PCR reaction；
5) clone of PCR primer and sequencing；
PCR primer builds clone bank, and screening has the clone of insertion and checks order；
6) process of data and analysis；
Nucleotide sequence order-checking obtained is sheared, and removes the sequence of carrier part, and rejecting does not contains
The nucleotide sequence of SEQ ID NO:9 primer sequence, compares with database, finds the virus mated most
After kind/strain, it is judged that the viral species contained in this sample.
6. the virus genomic method of detection as claimed in claim 5, it is characterised in that described step
4) in, PCR reaction condition is as follows: 95 DEG C of 5min of 1 circulation, 95 DEG C of 15sec of 35 circulations,
50 DEG C of 15sec, 72 DEG C of 2min, 72 DEG C of 7min of 1 circulation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610347518.1A CN105838827A (en) | 2016-05-24 | 2016-05-24 | Virus genome primer and method for detecting virus genome through same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610347518.1A CN105838827A (en) | 2016-05-24 | 2016-05-24 | Virus genome primer and method for detecting virus genome through same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105838827A true CN105838827A (en) | 2016-08-10 |
Family
ID=56593197
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610347518.1A Pending CN105838827A (en) | 2016-05-24 | 2016-05-24 | Virus genome primer and method for detecting virus genome through same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105838827A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113265452A (en) * | 2021-05-14 | 2021-08-17 | 北京大学人民医院 | Bioinformatics pathogen detection method based on Nanopore metagenome RNA-seq |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651538A (en) * | 2015-03-11 | 2015-05-27 | 珠海国际旅行卫生保健中心 | Primer group and kit for simultaneously detecting four diarrhea viruses |
-
2016
- 2016-05-24 CN CN201610347518.1A patent/CN105838827A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651538A (en) * | 2015-03-11 | 2015-05-27 | 珠海国际旅行卫生保健中心 | Primer group and kit for simultaneously detecting four diarrhea viruses |
Non-Patent Citations (1)
| Title |
|---|
| JOHN D. NEILL ET AL.: "Simultaneous rapid sequencing of multiple RNA virus genomes", 《JOURNAL OF VIROLOGICAL METHODS》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113265452A (en) * | 2021-05-14 | 2021-08-17 | 北京大学人民医院 | Bioinformatics pathogen detection method based on Nanopore metagenome RNA-seq |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107988428B (en) | RAA constant temperature fluorescence detection method and reagent for Shrimp Iridovirus (SIV) | |
| CN101952461A (en) | Compositions, methods, and kits for detecting ribonucleic acid | |
| KR20180052317A (en) | Primer set for detection of MERS-coronavirus and uses thereof | |
| CN107475449A (en) | A kind of transcript profile sequence measurement spliced suitable for dwarf virus section and geminivirus infection coe virus genome | |
| CN110184387B (en) | RT-LAMP detection primer for detecting ANSVV, application thereof, detection reagent and detection method | |
| CN102676701A (en) | Universal RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection primer and detection method for avian pneumovirus | |
| CN105950785B (en) | Triple fluorescence RT-PCR detection kit, primer and probe for avian influenza virus, newcastle disease virus and infectious bronchitis virus | |
| CN111304365A (en) | Novel one-step loop-mediated isothermal detection reagent for gosling gout virus and application thereof | |
| CN113355459B (en) | Method and kit for detecting and screening N501Y mutation of new coronavirus | |
| Jamindar et al. | Evaluation of two approaches for assessing the genetic similarity of virioplankton populations as defined by genome size | |
| Li et al. | Morphology and molecular identification of the Eimeria spp. in domestic rabbits | |
| CN112011650B (en) | Chinese bee sacbrood virus RT-RPA detection primer, probe and kit | |
| CN111850153B (en) | Primer group for detecting prawn acute hepatopancreatic necrosis disease-vibrio parahaemolyticus and kit containing primer group | |
| CN110184386B (en) | RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection primer for detecting ANRSV (ANRSV), application thereof, detection reagent and detection method | |
| CN105838827A (en) | Virus genome primer and method for detecting virus genome through same | |
| CN107385108A (en) | A kind of detection kit and its detection method of new marmor upsilon coe virus in lotus rhizome | |
| CN107190103A (en) | Multiple PCR primer group, kit and the method for three kinds of fishes virus are detected simultaneously | |
| CN106086206A (en) | Green Wei Si Salmonella loop-mediated isothermal gene amplification fast detecting kit and detection method | |
| CN110592269A (en) | RAA constant-temperature fluorescence detection method and reagent for grass carp hemorrhagic disease type 2 virus (GCRV-2) | |
| CN110894550A (en) | RAA constant temperature fluorescence detection method and reagent for eel Herpes Virus (HVA) | |
| CN114231665A (en) | Phaseolus golden mosaic virus (RPA-LFD) detection kit and application thereof | |
| CN107988429B (en) | Reagent for detecting rabies virus and application thereof | |
| CN110894551A (en) | RAA constant-temperature fluorescence detection method and reagent for grass carp hemorrhagic disease type I virus (GCRV-I) | |
| CN104894125A (en) | RT-LAMP (reverse transcription loop-mediated isothermal amplification) reagent box detecting grapevine virus A and special primer thereof | |
| CN110656186B (en) | Kit and method for rapidly identifying walnut black spot aphids |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160810 |
|
| RJ01 | Rejection of invention patent application after publication |