CN108796128A - A kind of GII.8 types norovirus genome amplification primer and amplification method - Google Patents
A kind of GII.8 types norovirus genome amplification primer and amplification method Download PDFInfo
- Publication number
- CN108796128A CN108796128A CN201810646112.2A CN201810646112A CN108796128A CN 108796128 A CN108796128 A CN 108796128A CN 201810646112 A CN201810646112 A CN 201810646112A CN 108796128 A CN108796128 A CN 108796128A
- Authority
- CN
- China
- Prior art keywords
- gii
- primer
- norovirus
- amplification
- types
- 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.)
- Granted
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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of amplimers and amplification method of GII.8 types norovirus genome.The present invention uses the segmentation amplification strategy of " 4+1+1 " for GII.8 type norovirus genome signatures, six groups of amplimers and two genome ends sequencing primers are devised in conservative region, RT-PCR amplifications and sequencing are carried out by template of target viral RNA, it is compared again by splicing, obtains GII.8 type norovirus genome sequences.The amplification method is applied to practical detection sample, successfully obtains the GII.8 type norovirus genome sequences in China source.The method of the present invention has many advantages, such as that easy to operate, the period is short, easy popularization, and can be widely applied to health care, inspection and quarantine etc. has norovirus detection demand and corresponding scientific research field.
Description
Technical field：
The invention belongs to biotechnologies, and in particular to a kind of GII.8 types norovirus genome amplification primer and expansion
Increasing method.
Background technology：
Norovirus is the important non-bacterial cause of disease of global acute gastroenteritis, can infect each age group crowd.Promise such as disease
Poison infection belongs to self-limited disease, in general 72 hours patient will self-healing, however for infant, old man and immune deficiency people
Group can be with the danger of dehydration property death.In addition, norovirus easily causes epidemic outbreak, in school, hospital, home for destitute, big
The semiclosed environment such as type steamer will infect wherein all Susceptible population in 24.It is reported that the virus causes about 2.3 hundred million every year
Person-time infection, need to spend multi-million dollar for associated treatment every year in developed country, and is particularly acute in developing country, until
Lead to the death of 200,000 5 years old or less children less.Norovirus causes great threat to public health security, but to current
Until, there are no effective antiviral drugs and treatment means.
As RNA virus, norovirus has abundant genetic diversity, according to the amino acid sequence of its capsid protein VP1
Row, the mankind can be infected by being divided into six genomes (Genogroup) GI-GVI, wherein GI, GII and GVI.Same gene
Norovirus in group can also be further separated into different genotype, such as GI includes at least nine kinds of genotype, and GII includes
22 kinds of genotype.GII.4 type norovirus is the Major Epidemic genotype in the whole world, and about 80% norovirus infection is by this
Caused by type strain.However, other genotype are also found during virus monitor, for example, GII.2 types, GII.3 types,
GII.6 types, GII.8 types etc., and also result in breaking out for epidemic situation.However these non-principal popular genotype still less focus on,
Therefore, the information collection work for reinforcing different genotype norovirus is of great significance.
GII.8 type norovirus is one of common non-principal popular genotype in epidemiological study, it is reported earliest
In Japan in 1967, hereafter it is found in multiple countries such as Holland, Turkey, South Korea, New Zealand, Brazil, Cameroon.Together
When, GII.8 types norovirus is often detected with low infection rate in China's clinical monitoring, mainly on Guangdong, Jiangsu and other places.This
Outside, GII.8 genotype is also found in oyster, earth's surface water monitoring.However, the genomic information that the genotype has been reported at present
Only one (AB039780 | U25), which is Japan to be found in 1997, and complete capsid protein VP1 sequences also only have
3.The accumulation of viral genetic information is to further investigate the important foundation of viral pathogenesis and evolutionary mechanism, therefore carry out viral gene
Group amplification is of great significance with sequencing approach.
As the RNA virus easily to make a variation, it is understanding norovirus to continue to monitor the prevalence of virus and variation situation
Important foundation.The genome of GII norovirus is about 7.5kb, including 3 open reading frame.It is directed in our previous works
" 4+1+1 " amplification strategy that GII.4 types, GII.17 types norovirus and GII.P12/GII.3 recombinant type norovirus are established
Have the characteristics that easy to operate, the period is short, at low cost and high sensitivity, it is therefore, tactful according to this novel amplification, we
A kind of genome amplification primer and amplification method for GII.8 type norovirus is had developed, to accumulate the promise of China GII.8 types such as
Viral genome resource provides a strong research tool.
Invention content：
The object of the present invention is to provide a kind of GII.8 types norovirus genome amplification primer and amplification methods.
Specifically, the amplimer of GII.8 types norovirus genome provided by the invention, including six pairs of amplimers and
The sequencing primer of two genome ends：
Primer pair 1：II-1F：5'-GTGAATGAAGATGGCGTCTAAC-3'；
II.8-1R：5'-CTGTAGGCATCCCAGTGATC-3'；
Primer pair 2：II.8-2F：5'-TCATCTTCGCCTGACATCGT-3'；
II.8-2R：5'-TCCTCTTCACAGAAGTTCTC-3'；
Primer pair 3：II.8-3F：5'-CACACTGCATTCTCCAGCAA-3'；
II.8-3R：5'-CAATCCTCCAAATGCCCGA-3'；
Primer pair 4：II.8-4F：5'-GAGGAAGCCAAGAAGACAGT-3'；
II-4R：5'-CCRCCNGCATRHCCRTTRTACAT-3'；
Primer pair 5：II-5F：5'-GGAGGGCGATCGCAATC-3'；
II-5R：5'-CCRGCRAAGAAAGCTCCAGCCAT-3'；
Primer pair 6：II.8-6F：5'-TCYAACAGTGGGGACCACC-3'；
II.8-6R：5'-TCACTAAGCCCGTGACTCC-3'；
Sequencing primer：II.8-seq1R：5'-AACTCACCATCCCACATCTC-3'；
II.8-seq6F：5'-GACAAGGAGATGTTGAATGC-3'；
R represents A/G, and H represents A/C/T, and N represents base A/C/T/G, Y and represents C/T.
The present invention also provides a kind of amplification methods of GII.8 types norovirus genome, specially：Drawn respectively with above-mentioned
Object to II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R,
Upstream and downstream primers of the II.8-6F/II.8-6R as amplimer carries out RT- by template of the RNA of GII.8 type norovirus
PCR amplification respectively obtains amplified production, then uses above each pair of amplimer and sequencing primer II.8-seq1R, II.8-
Seq6F carries out nucleic acid sequence sequencing to corresponding amplified production respectively, then is compared by splicing, and obtains GII.8 types promise such as disease
Virus gene group full length sequence.
Further, the RT-PCR, reaction system are：Contain 2 × one-step RT-PCR mixture, 10 μ
L, 10 μm of ol/L sense primers and each 0.8 μ L of 0.6 μ L, MLV/RNasin/HS-Taq enzyme mixation of 10 μm of ol/L downstream primers,
2 μ L of RNA templates, remaining complements to 20 μ L by distilled water；Reaction condition is：50 DEG C of reverse transcriptions 30min, 94 DEG C of pre-degeneration 3min,
Then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 75s, after totally 30 cycles, last 72 DEG C of extensions 7min.
Compared with prior art, the invention has the advantages that：
The present invention is directed to common GII.8 type norovirus genotype, using point of " 4+1+1 " of covering whole gene group
Section amplification strategy, by amplification, sequencing and the sequence assembly applied to practical detection sample, to obtain GII.8 types promise such as disease
Virus gene group sequence.There is behaviour using the method that primer pair GII.8 type norovirus provided by the invention is expanded and is sequenced
Make the features such as simple, the period is short, at low cost and high sensitivity, can be widely applied to health care, inspection and quarantine etc. has promise
Mechanism such as viral diagnosis demand and corresponding scientific research field.
Description of the drawings：
Fig. 1 is the design position of GII.8 type norovirus genome amplification strategy and corresponding primer.
Fig. 2 is the RT-PCR annealing temperatures optimization suitable for GII.8 type norovirus genome amplification primers, and electrophoresis is suitable
Sequence is M, 1-24, and wherein M is that DNA Ladder, 1-4,5-8,9-12,13-16,17-20,21-24 are respectively that genome amplification draws
Object II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R,
The electrophoresis result of II.8-6F/II.8-6R amplified productions under different annealing temperature (being followed successively by 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C).
Fig. 3 is the RT-PCR primer concentration optimization suitable for GII.8 type norovirus genome amplifications, figure A- figure F difference
For primer I I-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-
The optimum results of 5R, II.8-6F/II.8-6R, electrophoresis sequence are M, 1-9, and wherein M is DNA Ladder, and 1-3 is 0.2 μ L primers
It expands respectively and 10 times, electrophoresis result of 100 times of the RNA stostes dilution for template is diluted with RNA stostes, RNA stostes, 4-6 is 0.6 μ L
Primer expands respectively dilutes 10 times, electrophoresis result of 100 times of the RNA stostes dilution for template with RNA stostes, RNA stostes, and 7-9 is
1.0 μ L primers expand respectively dilutes 10 times, electrophoresis result of 100 times of the RNA stostes dilution for template with RNA stostes, RNA stostes.
Fig. 4 is the RT-PCR sensitivity evaluations of GII.8 type norovirus genome amplification primers, and electrophoresis sequence is M, 1-
35, wherein M are that DNA Ladder, 1-5,6-10,11-15,16-20,21-25,26-30,31-35 are respectively that genome amplification draws
Object II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R,
II.8-6F/II.8-6R and detection primer G2SKF/G2SKR (is followed successively by 10 in viral RNA difference dilution1-105Dilute again) under
The electrophoresis result of amplified production.
Fig. 5 is the expanding effect of GII.8 type norovirus genomes in actual sample, and electrophoresis sequence is M, 1-6, wherein M
It is the electrophoresis result that actual sample L601 carries out amplification gene 6 segments of group for DNA Ladder, 1-6.
Specific implementation mode：
The following examples are further illustrations of the invention, rather than limiting the invention.In the following example not
It is usually conventional means well-known to those skilled in the art to indicate specific experiment condition and method, used technological means.
The design of the amplification strategy and corresponding primer of embodiment 1GII.8 type norovirus genomes
GII.8 type norovirus Genome Size about 7.5kb, including three ORF, wherein ORF1 are about 5.1kb, ORF2 long
About 1.6kb, ORF3 are about 0.8kb.Based on generation Sang Ge deoxynucleotide PCR sequencing PCRs, each amplified fragments size model is set
It encloses for 1.3kb-1.6kb, wherein ORF1 points are 4 segments, and ORF2 and ORF3 are 1 segment.In addition, to obtain genome 5'
With the ends 3' complete sequence, respectively in genome both ends design amplification length in the segment of 100-800bp, corresponding primer names respectively
For II.8-Seq1R and II.8-Seq6F.Specific genome partition strategy and the visible Fig. 1 of corresponding primer location.
Under the above restrictive condition, using the corresponding primer of Oligo Software for Design, specific primer information is shown in Table 1.Wherein,
R in primer nucleotide sequences represents A/G, and H represents A/C/T, and N represents base A/C/T/G, Y and represents C/T.
Table 1：GII.8 types norovirus genome amplification primer and sequencing primer information
aThe GII.8 type norovirus of primer location reference represents sequence GenBank accession number as AB039778.
The RT-PCR annealing temperatures that embodiment 2 is suitable for GII.8 type norovirus genome amplification primers optimize
(1) Virus Sample processing and nucleic acid extraction：The pending sample collected by PBS solution (DEPC processing) dilution
(GII.8 type norovirus positive sample L601) vibrates mixing, 10min is centrifuged under 12000 × g to 10% (w/v) concentration,
140 μ L of supernatant are collected, and viral RNA totally 60 μ L in sample are extracted by RNA extracts kits.
(2) gene set of segmentation amplification：Divide 6 sections of amplifications, used primer pair as follows：II-1F/II.8-1R,
II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R, II.8-6F/II.8-6R, often
Secondary RT-PCR selecting responses pair of primers.Using the One step RT-PCR reaction system of 20 μ L, contain 2 × one-step RT-
PCR mixture10 μ L, 10 μm of ol/L sense primers and each 0.6 μ L, MLV/RNasin/HS-Taq enzyme of 10 μm of ol/L downstream primers
0.8 μ L of mixed liquor, 2 μ l of sample rna masterplate, remaining is by ddH2O is supplied.
Reaction condition is：50 DEG C of reverse transcriptions 30min, 94 DEG C of pre-degeneration 3min, then 94 DEG C of 30s, 45-60 DEG C of 30s, 72 DEG C
75s, after totally 30 cycles, last 72 DEG C of extensions 7min.
Annealing temperature is respectively selected as 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C.
(3) electrophoresis：5 μ L of amplified production are taken, carry out Ago-Gel nucleic acid electrophoresis, and observe and tie by gel imaging system
Fruit.By primer pair II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/
The sequence of II-5R, II.8-6F/II.8-6R, GII.8 type norovirus genome amplification bands be followed successively by 1629bp, 1469bp,
1509bp,1569bp,1671bp,902bp.Electrophoresis result such as Fig. 2, the results showed that, six pairs of genome amplification primers have wider
Annealing region, to reduce non-specific amplification in actual sample, 55 DEG C of final choice is using annealing temperature.
Embodiment 3 is suitable for the RT-PCR primer concentration optimization of GII.8 type norovirus genome amplifications
(1) Virus Sample processing and nucleic acid extraction：The pending sample collected by PBS solution (DEPC processing) dilution
(GII.8 type norovirus positive sample L601) vibrates mixing, 10min is centrifuged under 12000 × g to 10% (w/v) concentration,
140 μ L of supernatant are collected, viral RNA totally 60 μ L in sample are extracted by RNA extracts kits, and using nuclease free
ddH2O carries out the dilution processing of 10 × gradient, and choosing RNA stostes respectively, (concentration of stoste is 104RTPCRU), RNA stostes dilute
10 times dilute 100 times as amplification template with RNA stostes.It should be noted that the content of norovirus of the present invention uses
RTPCRU units define, and as use detection primer G2SKF/G2SKR, by 10 × gradient of RT-PCR method pair of standard dilute
The virus liquid released is detected, then it is a RTPCRU to be diluted to virus concentration when be able to detect.
(2) gene set of segmentation amplification：Divide 6 sections of amplifications, used primer pair as follows：II-1F/II.8-1R,
II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R, II.8-6F/II.8-6R, often
Secondary RT-PCR selecting responses pair of primers.Using the One step RT-PCR reaction system of 20 μ L, contain 2 × one-step RT-
PCR mixture10 μ L, 10 μm of ol/L sense primers and 10 μm of ol/L downstream primers press different condition and 0.2 μ L, 0.6 are added respectively
μ L, 1.0 μ L, MLV/RNasin/HS-Taq enzyme mixation, 0.8 μ L, different 2 μ l of dilution sample rna masterplate, remaining is by ddH2O
It supplies.
Reaction condition is：50 DEG C of reverse transcriptions 30min, 94 DEG C of pre-degeneration 3min, then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C
75s, after totally 30 cycles, last 72 DEG C of extensions 7min.
(3) electrophoresis：5 μ L of amplified production are taken, carry out Ago-Gel nucleic acid electrophoresis, and observe and tie by gel imaging system
Fruit.By primer pair II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/
The sequence of II-5R, II.8-6F/II.8-6R, GII.8 type norovirus genome amplification bands be followed successively by 1629bp, 1469bp,
1509bp,1569bp,1671bp,902bp.Electrophoresis result such as Fig. 3, the results showed that, different primers are to the requirement to primer concentration
It is inconsistent, but optimal conditions is or including 0.6 μ L, and therefore, 0.6 μ L of final choice are primer additive amount in amplification system.
The RT-PCR sensitivity evaluations of embodiment 4GII.8 type norovirus genome amplification primers
(1) Virus Sample processing and nucleic acid extraction：The pending sample collected by PBS solution (DEPC processing) dilution
(GII.8 type norovirus positive sample L601) vibrates mixing, 10min is centrifuged under 12000 × g to 10% (w/v) concentration,
140 μ L of supernatant are collected, and viral RNA totally 60 μ L in sample are extracted by RNA extracts kits, and using nuclease free
ddH2The appropriate dilution that O carries out 10 × gradient (is followed successively by 101-105Dilute again) processing.
(2) gene set of segmentation amplification：Divide 6 sections of amplifications, used primer pair as follows：II-1F/II.2-1R,
II.2-2F/II.2-2R, II.2-3F/II.2-3R, II.2-4F/II-4R, II-5F/II-5R, II.2-6F/II.2-6R, often
Secondary RT-PCR selecting responses pair of primers.Using the One step RT-PCR reaction system of 20 μ L, contain 2 × one-step RT-
PCR mixture10 μ L, 10 μm of ol/L sense primers and each 0.6 μ L, MLV/RNasin/HS-Taq enzyme of 10 μm of ol/L downstream primers
0.8 μ L of mixed liquor, 2 μ L of sample rna masterplate, remaining is by ddH2O is supplied.
Reaction condition is：50 DEG C of reverse transcriptions 30min, 94 DEG C of pre-degeneration 3min, then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C
75s, after totally 30 cycles, last 72 DEG C of extensions 7min.
With detection primer G2SKF/G2SKR (G2SKF：5'-CNTGGGAGGGCGATCGCAA-3'；G2SKR：5'-
CCRCCNGCATRHCCRTTRTACAT-3') it is control, is carried out according to above-mentioned system and reaction condition.
(3) electrophoresis：5 μ L of amplified production are taken, carry out Ago-Gel nucleic acid electrophoresis, and observe and tie by gel imaging system
The content of fruit, the norovirus is defined using the RTPCRU units of detection primer, as using common detection primer
G2SKF/G2SKR is detected the viral RNA for carrying out 10 × gradient dilution, then being diluted to virus concentration when be able to detect is
One RTPCRU.By primer pair II-1F/II.2-1R, II.2-2F/II.2-2R, II.2-3F/II.2-3R, II.2-4F/II-
The sequence of 4R, II-5F/II-5R, II.2-6F/II.2-6R, GII.8 type norovirus genome amplification bands are followed successively by
1629bp, 1469bp, 1509bp, 1569bp, 1671bp, 902bp, electrophoresis result such as Fig. 4, as a result show：Six fragment amplifications
The sensitivity of primer reaches 1RTPCRU, is compared with detection primer G2SKF/G2SKR, and all primers reach detection primer
Sensitivity.
The expanding effect of GII.8 type norovirus genomes in the practical detection sample of embodiment 5
(1) Virus Sample processing and nucleic acid extraction：GII.8 type norovirus positive sample L601 are taken, PBS solution is passed through
(DEPC processing) dilutes pending sample to 10% (w/v) concentration, vibrates mixing, centrifuges 10min under 12000 × g, in collection
140 μ L of clear liquid, and viral RNA totally 60 μ L in sample are extracted by RNA extracts kits.
(2) gene set of segmentation amplification：Divide 6 sections of amplifications, used primer pair as follows：II-1F/II.2-1R,
II.2-2F/II.2-2R, II.2-3F/II.2-3R, II.2-4F/II-4R, II-5F/II-5R, II.2-6F/II.2-6R, often
Secondary RT-PCR selecting responses pair of primers.Using the One step RT-PCR reaction system of 20 μ L, contain 2 × one-step RT-
PCR mixture10 μ L, 10 μm of ol/L sense primers and each 0.6 μ L, MLV/RNasin/HS-Taq enzyme of 10 μm of ol/L downstream primers
0.8 μ L of mixed liquor, 2 μ L of sample rna masterplate, remaining is by ddH2O is supplied.
Reaction condition is：50 DEG C of reverse transcriptions 30min, 94 DEG C of pre-degeneration 3min, then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C
75s, after totally 30 cycles, last 72 DEG C of extensions 7min.
(3) electrophoresis takes 5 μ L of amplified production, carries out Ago-Gel nucleic acid electrophoresis, and observe and tie by gel imaging system
Fruit.By primer pair II-1F/II.2-1R, II.2-2F/II.2-2R, II.2-3F/II.2-3R, II.2-4F/II-4R, II-5F/
The sequence of II-5R, II.2-6F/II.2-6R, GII.8 type norovirus genome amplification bands be followed successively by 1629bp, 1466bp,
In 1529bp, 1569bp, 1701bp, 927bp size environs, electrophoresis result such as Fig. 5, the results showed that, using above-mentioned amplification
Primer pair sample L601, which is expanded, to succeed.
(4) nucleic acid sequencing is compared with genome splicing：Above six pairs of amplimers and two sequencing primers are respectively adopted
II.8-seq1R, II.8-seq6F are sequenced and splice to the amplified production of the corresponding L601 samples amplified, finally obtain base
Because group sequence length be 7476bp, genome nucleotide sequence as shown in SEQ ID NO.1, by the genome sequence submit into
Row BLAST analyses, the results showed that, 90% homologous sequence only one is all higher than with L601 sample genome coverages and similarity
AB039780/U25/JP (coverage 99% and similarity 94%).
It the above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
The limitation of the present invention, protection scope of the present invention should be subject to claim limited range.For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
Protection scope of the present invention is also should be regarded as into retouching.
Sequence table
<110>Guangdong Microbes Inst（Microbiological analysis inspection center of Guangdong Province）
Huankai Microbes Tech Co., Ltd., Guangdong
<120>A kind of GII.8 types norovirus genome amplification primer and amplification method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7476
<212> DNA
<213>Norovirus GII.8 L601 (norovirus GII.8 L601)
<400> 1
tgtgaatgaa gatggcgtct aacgacgcta ccgctgccgc tggcaacttt aacattgcca 60
acaacagtga aaacactccc cctcccgtta aggaagaggc gagtgtccta tccaacgtaa 120
aagttggatt caagaaaata ttgggggctg tgcctaagca gcccaagcca ccagaagtga 180
aaaccaaaga tcaacccagg gtgctggtca atggtaaggc cctggaggtg ccgcccccac 240
caccaaatgg tgaagacgtg gtttactata acagcaagga tgacactgtg cacggcctac 300
caaatctaac aacagtcagc tgtgaaggcg aaaaccttcc gtacacagtt cctcctctca 360
gcgagagaga gcacagggca gcaccagaac cattacctgg caccatacta gagatgtggg 420
atggtgaatt ctaccattat gcgatatatg tctctggtgg taaggccata ggggtgcaca 480
aacccccagc tgcaatcagc cttgccacca tcgagcttac acctatatcg ctttactgga 540
ggcctgttta tgtccccaat tacttggtcc accctgacac actcaagagc ttagctggtg 600
aaaaatttcc atacaccgca tttagcaata attgttacaa tttctgttgc tgggttcttg 660
acttgaaaga ctcgtggctt aaccgcagat caataactag aacaaccggt ttcttcaagc 720
cttatcaacc ctggaacaag aaaccacttc cgacggtgga tgatggaaaa atcaagaaag 780
tcgccaatgc agtcctgtgt gcccttggtt cactattctc aaaacctatt aaggacttga 840
tcgggaagct gaaaccactg aatttcttga atctcctcgc ttcgtgcgac tggaccttct 900
ccggagtggt tgagaccgtc atactagctg ctgaactatt cggtgtgttt tggacccccc 960
ccgacgtgtc gaacttcatc gcgtcactca tcggggactt cgagttgcag ggtccagagg 1020
atttagctgt ggagttggtg ccgattatca tgggcggcat cgggatggtg ctgggattca 1080
ccgccgagaa gatcggccga atgctatcat cagctgcatc gacccttagg gcctgtaaag 1140
acctaggtaa ctacgccctt gacatactta aattggtcat gaagtggttc ttcccaaaga 1200
aggaggagaa ggcagagatg gagaccttga gggccataga ggatgctgtc ttagacatgg 1260
aagcgattgg aaataatcac ctcactgctc tgctgaaaga cagggacagc ctcctaacct 1320
acatgaagac tctagacctc gaagaggaaa aggcaagaaa gttatccaca aaatcatctt 1380
cccctgacat cgtgggtacc atcaatgcca tactagccag aattgccgcg gctaggtctc 1440
tcctacacag agccaaagaa gaaatgtaca gcagacctag gcctgtcgtg gtgatgatat 1500
caggaagacc aggggttggt aaaacccata tggctagaca cctcgccaag agtgtggcgg 1560
gcaccatgag cggtgaccaa agggtgggcc tcatcccccg taatggagtt gatcactggg 1620
atgcctacag gggggaacga gttgtcttgt gggacgacta tggcatgggt aacccagtga 1680
aggacgccct tgctctgcaa gaactagcag acacttgtcc agtcaccctc aattgtgaca 1740
ggatagagaa taagggaaag atgttcgata gcgaggtaat aattataaca accaatctgg 1800
ccaaccccgc cccgcttgat tatgtcaact ttgaggcatg ttcccgcaga attgacttcc 1860
tcgtctacgc agaggccccg accattgaga aggcgaagaa ggatttcccc gggcaaccag 1920
acatgtggaa ggagcacttc aaagcagact tctctcatct caaactcaca ctggctccgc 1980
aaggcggttt tgacaagaac ggcaacacgc cacatgggaa ggggactatg aagaccttaa 2040
cacagggttc cctcacagca agagtggctg ggcttgtgca cgaaaggaaa gacgagttcc 2100
agttacaggg tggggagttg caggtctaca actttgacac aaacaaggta tcggctttca 2160
ggaagcttgc tgcagacaac aactatggat ttctggagac aatgagagtc ggctcctcac 2220
tcaaggacgt caagacagtg gaagatctta aatgtgccct caggagtgtc agtttcaaag 2280
aatgtgagat aatctacagg aatgtcaagt accgcatatc atcagacggt tgcggtaagg 2340
tcagcataga gaaactggca gataacaact cccaaacaac cagagagatt atcacggccg 2400
tcactagact acaacaggct cgtgctaggt actacatcag ctgtttccaa gacctagtct 2460
acacactctt gcaggttgcc ggagcctcat ttgtcattaa tagaatagtt aagaagttca 2520
actgggaacg atgggtcaag gctccggaaa cagatgaacc cacaccaagt aatgacaagc 2580
ccgacactga agaggaatgg gagatcgccc ccaaagatgt ggactctgag ggtaaaaagg 2640
ggaagaacaa gaagggcagg ggacgtaagc acactgcatt ctccagcaaa gggcttagtg 2700
atgaggagta tgatgaattc aaaagactga gggaagagaa acagggccgc tacacaatag 2760
aggagtacct ccaggacaga gaccggtact atgaagagct ggcagtggcc cgggccactg 2820
aagagaactt ctgtgaagag gaagagataa agatcaggca aagaatattc cgccccacca 2880
aaaagcagag gaaggaggag cggggggttc tgggactcgt cacaggagca gacatcagga 2940
aacggagacc cgatgatttc caaccaaaag gaaaactgtg ggcagatgat tctaggagcg 3000
tcgactacaa tgagaagatc gagtttgaag ctcctcctag cgtctggtca agaatcgtcc 3060
cgcttgggac tggttgggga ttctgggtct catccaactt actaatcacc accacacatg 3120
tgctgcccaa aggcatcaaa gagctttttg gggtggacat caaacaagtc caagttcaca 3180
aatcaggtga attttgcaga ttcaggttcc caaaaccagt gaggccagat gtctctgggt 3240
tggtactaga agaaggggcc cccgaaggca cagtgtgctc tgttctcatc aagaggtcaa 3300
caggagagat gatccccctt gcagttagga tgggtaccca cgcctctatg aagatccagg 3360
ggcgcactgt tggcggccag atgggcatgc tgctcactgg agccaacgct aagaacatgg 3420
atttgggtac cggccccggg gattgtggtt gcccgtacat atacaagagg ggcaatgaca 3480
tcgtggtggc cggcgtccac acagcagcag cgcgtggggg caacacggtc atctgtgcaa 3540
cccaaggacc tgatggtgag gcagtgttgg aagggggaga ggaccacggt acttactgtg 3600
gagccccaat attagcccct ggtaaagcgc cgaagcttag cacaaagact aagttttggc 3660
gctcatcccc tgattccctt ccccctggga cgtatgaacc agcttacctt gggggtaagg 3720
atccccgtgt ggagaaaggg ccatcactcc aacaagtgat gcgagaccaa ctcaaaccct 3780
tcacagaacc caggggcaag ccaccgaggc cgtcagtgct ggaggaagcc aagaagacag 3840
tcatgaatgt actggagcag accatagaac cagctaagcc atggacctac tctcaagcct 3900
gtgcttcatt ggataaaaca acatccagtg gcagtcctca ccacgtcagg aagaatgaac 3960
actggaacgg ggagtctttc actgggcctc ttgcagatca agcctcaaaa gccaacctga 4020
tgtatgagga gggtaaacac atggacccta tgtacacagc agccctcaag gacgagctgg 4080
tgaaaactga caaaatctat aagaaaatca agaagaggtt gttgtggggg tctgatctgg 4140
ggacaatggt gaggtgtgct cgagcgtttg gaggattgct cgacagcatg aaagagagct 4200
gcatctcctt accctgtagg gttggcatga acataaatga agatggacca ctcatatttg 4260
aaaaacattc caaatacacc tatcactatg atgctgatta ctctaggtgg gactcaacac 4320
agcagagggc tgtcctgagt gcagccatgg aggtaatggt gaaattctct gctgagccgg 4380
aactggctca ggtggttgcc gaagacctcc ttgcacccag ccgtcttgac gtgggtgact 4440
tcgtcatctc cgttcaagag ggtcttccat cgggtgtccc ctgcacgtca cagtggaact 4500
caatcgctca ctggatcctg actcttagtg ctatgtctga ggtctctggt ctttcccctg 4560
aggttgtgca agccaactcc tgtttctcat tctatgggga tgatgagata gtcagcacag 4620
atataaacct agacccagaa aaactcacca ggaaactgag ggagtatggc ctcgtcccaa 4680
caaggccaga caaaactgag ggcccacttg tgatcactca ggatttgaat ggtctcacat 4740
tcttgaggcg aaccatagtg cgggaccccg caggttggtt tggaaaattg gaccgtgatt 4800
ccattctaag gcagttatac tggaccagag gacccaatca tgagaacccc tttgaaagca 4860
tgatccccca ctcccagaga gcaacccagt taatggccct tcttggggaa gcctcgttgc 4920
atggtcccca gttttacaag aaggtgagta aaatggtcat caatgagatc aagagtggtg 4980
gtctggagtt ttacgtgccc agacaggagg ccatgtttag atggatgaga ttttcagacc 5040
tcagcacgtg ggagggcgat cgcaatctgg ctcccgagaa tgtgaatgaa gatggcgtcg 5100
aatgacgcag ctccatcgaa tgatggcgcg gctggcctcg taccagagat caaccatgag 5160
gtcatggcca tagagcctgt tgcaggggcc tctttagcag cccctgtcgt aggacaactt 5220
aatataattg atccctggat tagaaataat tttgtacaag cccctgctgg agaattcact 5280
gtttcgccta gaaatgctcc aggtgaattt ttgttagatc tagagttagg tccagaactg 5340
aatccttatc ttgctcacct tgcacgcatg tataatgggc atgcaggtgg tatggaggtg 5400
cagatagtgc ttgctgggaa tgcgttcaca gcgggcaaaa tactgtttgc agtcatacca 5460
cctggattcc cctatgaaaa tttgtcacct gcacaattga ccatgtgccc gcatgtagtg 5520
gtggatgtga gacagcttga accaatcctg ctgcccatgc cagacatccg taatacattc 5580
ttccattaca atcagagcaa tggaccaaaa ctcagattag tagccatgct ttacacaccc 5640
ctaagggcta acaatgctgg tgatgatgtc ttcacagtct cctgcagggt tttgactcgt 5700
ccgtcaccag attttgaatt caattttctt gtccccccat ctgttgaatc taagactaaa 5760
gctttcactc ttccaattct taagatatct gagatgacaa attcaagatt tcccatacca 5820
gttgaccaga tgtatactag taggaatgaa aatatagttg ttcaaccaca gaatggcagg 5880
gtgacacttg atggagagtt acagggcaca accacacttc aaccggtgtc catttgtggc 5940
ttccgaggga cgctgcagac caggctggca gatcaaccaa attacacata ccaagtgcac 6000
cttgagaatt tggacggctc acccgtcgat ccgacagatg aagtcccagc ccccctgggg 6060
acgccagatt ttcaggccca gctatttgga gttgtcagtc aaaggagtag tgataatgcc 6120
accagggcac atgaggctag ggtcaacacc aacgatccta ccttcgcgcc ccagatcgcc 6180
caggtgcgtt tcaaatcacc atcccacgac ttctttgaca atgaacccat caagttcaca 6240
ccagttggaa ttagtgttga tagcgaaaac agctacaacc aatggctcct tcccaggtat 6300
ggcgggcact tgacaaacaa cacccacctg gccccctctg tgtccccaat gttccccggc 6360
gagcagattc tcttcttccg ctcgttcatg ccaggtgcca gtggtcatac tgatggtgct 6420
attgactgcc tcttaccaca agagtgggtt gcccacttct atcaggaggc cgcaactgcg 6480
caaacagatg tggctctgat tagatttgtg aaccccgaca caggcagagt gctatttgaa 6540
gggaagctac acaagcgggg ctttataacc atctccaaca gtggggacca ccccattgtt 6600
atgcctgcta atggatactt caggtttgag gcatgggtca accaatttta ctctctcgcc 6660
cccgtgggaa ctggaagtgg gcgcagaagg gttcaataat ggctggagct ttctttgcgg 6720
gtctggctgg tgatgtggtg acctctggca tcgggtcgct catcaatgca ggtgctaatg 6780
cagtgaacca aaaggttgaa tatgacttca ataggcaact tcaagaagct tcttttaaac 6840
atgacaagga gatgttgaat gctcaaatta atgccacaac caaattgcag agggacatga 6900
ttgccatcaa gcagggggtt ttgaccgctg gcggcttttc ccccactgat gctgcaagag 6960
gttccatcaa tgctccaacc accaaaattc ttgactggaa tggatctagg tactgggcgc 7020
caggatcaat gagaacaaca gcatactctg gcaatttcat atcacaggga gttaagcaac 7080
ctacacccat tgttagatct gtgaaacctc aagtacagca atctcctgcc ccttcttctg 7140
tttattctgt aggttctgtt aagtcacaat caacacaatc aactcaatta acagggtcag 7200
ggactgtatc aagggcaccc accccctcaa caaccacaac actgtccaga acaagtgagt 7260
gggtgaggag ccagaatgat aggctgagcc cctttatggg aggcgcactc cagacagctt 7320
tcatcacgcc cccatcgagt aaggcttcat cctcatcagg aacagtctca accgttccta 7380
gagaaatttt ggactcctgg actcctgcat ttaacacacg caggcagccg ctcttcgccc 7440
acatgcgtag gcgaggggag tcacgggctt agtgaa 7476
Claims (3)
1. a kind of GII.8 types norovirus genome amplification primer, which is characterized in that including six pairs of amplimers and two genes
The sequencing primer of group end：
Primer pair 1：
II-1F：5'-GTGAATGAAGATGGCGTCTAAC-3'；
II.8-1R：5'-CTGTAGGCATCCCAGTGATC-3'；
Primer pair 2：
II.8-2F：5'-TCATCTTCGCCTGACATCGT-3'；
II.8-2R：5'-TCCTCTTCACAGAAGTTCTC-3'；
Primer pair 3：
II.8-3F：5'-CACACTGCATTCTCCAGCAA-3'；
II.8-3R：5'-CAATCCTCCAAATGCCCGA-3'；
Primer pair 4：
II.8-4F：5'-GAGGAAGCCAAGAAGACAGT-3'；
II-4R：5'-CCRCCNGCATRHCCRTTRTACAT-3'；
Primer pair 5：
II-5F：5'-GGAGGGCGATCGCAATC-3'；
II-5R：5'-CCRGCRAAGAAAGCTCCAGCCAT-3'；
Primer pair 6：
II.8-6F：5'-TCYAACAGTGGGGACCACC-3'；
II.8-6R：5'-TCACTAAGCCCGTGACTCC-3'；
Sequencing primer：
II.8-seq1R：5'-AACTCACCATCCCACATCTC-3'；
II.8-seq6F：5'-GACAAGGAGATGTTGAATGC-3'；
R represents A/G, and H represents A/C/T, and N represents base A/C/T/G, Y and represents C/T.
2. a kind of GII.8 types norovirus genome amplification method, which is characterized in that respectively with primer described in claim 1
To II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R,
Upstream and downstream primers of the II.8-6F/II.8-6R as amplimer carries out RT- by template of the RNA of GII.8 type norovirus
PCR amplification respectively obtains amplified production, then uses above each pair of amplimer and sequencing primer II.8-seq1R, II.8-
Seq6F carries out nucleic acid sequence sequencing to corresponding amplified production respectively, then is compared by splicing, and obtains GII.8 types promise such as disease
Virus gene group full length sequence.
3. amplification method according to claim 2, which is characterized in that the RT-PCR, reaction system are：Contain 2
10 μ L of × one-step RT-PCR mixture, 10 μm of ol/L sense primers and 10 μm of ol/L downstream primers each 0.6 μ L, MLV/
0.8 μ L, RNA template of RNasin/HS-Taq enzyme mixations, 2 μ L, remaining complements to 20 μ L by distilled water；Reaction condition is：50℃
Reverse transcription 30min, 94 DEG C of pre-degeneration 3min, then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 75s, after totally 30 cycles, last 72 DEG C
Extend 7min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810646112.2A CN108796128B (en) | 2018-06-21 | 2018-06-21 | GII.8 type norovirus genome amplification primer and amplification method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810646112.2A CN108796128B (en) | 2018-06-21 | 2018-06-21 | GII.8 type norovirus genome amplification primer and amplification method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108796128A true CN108796128A (en) | 2018-11-13 |
| CN108796128B CN108796128B (en) | 2021-07-27 |
Family
ID=64084290
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810646112.2A Active CN108796128B (en) | 2018-06-21 | 2018-06-21 | GII.8 type norovirus genome amplification primer and amplification method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108796128B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725543A (en) * | 2021-03-04 | 2021-04-30 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Characteristic nucleic acid identification primer group, kit and detection method for human norovirus GII.4 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397105A (en) * | 2013-07-01 | 2013-11-20 | 上海海洋大学 | Kit for detecting GII type norovirus and applications thereof |
| CN105039330A (en) * | 2015-04-01 | 2015-11-11 | 广东省微生物研究所 | GII.4 type norovirus genome amplimer and amplification method |
| CN105176984A (en) * | 2015-09-23 | 2015-12-23 | 广东省微生物研究所 | GII.17 type norovirus genogroup amplification primer and amplification method |
| CN105925573A (en) * | 2016-06-30 | 2016-09-07 | 广东省微生物研究所 | GII.P12/GII.3 recombinant norovirus genome amplification primer and amplification method |
| CN106987655A (en) * | 2017-02-28 | 2017-07-28 | 北京出入境检验检疫局检验检疫技术中心 | A kind of norovirus source tracing method and its primer, kit, application |
-
2018
- 2018-06-21 CN CN201810646112.2A patent/CN108796128B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397105A (en) * | 2013-07-01 | 2013-11-20 | 上海海洋大学 | Kit for detecting GII type norovirus and applications thereof |
| CN105039330A (en) * | 2015-04-01 | 2015-11-11 | 广东省微生物研究所 | GII.4 type norovirus genome amplimer and amplification method |
| CN105176984A (en) * | 2015-09-23 | 2015-12-23 | 广东省微生物研究所 | GII.17 type norovirus genogroup amplification primer and amplification method |
| CN105925573A (en) * | 2016-06-30 | 2016-09-07 | 广东省微生物研究所 | GII.P12/GII.3 recombinant norovirus genome amplification primer and amplification method |
| CN106987655A (en) * | 2017-02-28 | 2017-07-28 | 北京出入境检验检疫局检验检疫技术中心 | A kind of norovirus source tracing method and its primer, kit, application |
Non-Patent Citations (2)
| Title |
|---|
| JULIANNE R. BROWN等: ""Norovirus Whole-Genome Sequencing by SureSelect Target Enrichment- a Robust and Sensitive Method"", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| KAZUHIKO KATAYAMA等: ""Phylogenetic analysis of the complete genome of 18 Norwalk-like viruses"", 《VIROLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725543A (en) * | 2021-03-04 | 2021-04-30 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Characteristic nucleic acid identification primer group, kit and detection method for human norovirus GII.4 |
| CN112725543B (en) * | 2021-03-04 | 2022-05-10 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Characteristic nucleic acid identification primer group, kit and detection method for human norovirus GII.4 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108796128B (en) | 2021-07-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Park et al. | Sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in Korea | |
| CN105176984B (en) | A kind of GII.17 types norovirus genome amplification primer and amplification method | |
| CN105039330B (en) | A kind of GII.4 types norovirus genome amplification primer and amplification method | |
| Short et al. | Temporal dynamics of natural communities of marine algal viruses and eukaryotes | |
| Jensen et al. | High prevalence of Aleutian mink disease virus in free-ranging mink on a remote Danish island | |
| KR101149422B1 (en) | Primers and its application in multiplex PCR to identify Rinderpest, Peste-des-petits-ruminants virus, Bluetongue virus and Rift Valley fever | |
| CN105925573A (en) | GII.P12/GII.3 recombinant norovirus genome amplification primer and amplification method | |
| Ryt-Hansen et al. | Outbreak tracking of Aleutian mink disease virus (AMDV) using partial NS1 gene sequencing | |
| JPH05508773A (en) | Nucleic acid sequences derived from the genome of Salmonella Typhi and their use for in vitro diagnosis of the presence of bacteria of the genus Salmonella, especially in food products | |
| Maganga et al. | Molecular characterization of complete genome of a canine distemper virus associated with fatal infection in dogs in Gabon, Central Africa | |
| Xue et al. | Isolation of feline panleukopenia virus from Yanji of China and molecular epidemiology from 2021 to 2022 | |
| Kreuze et al. | Challenge of virus disease threats to ensuring sustained uptake of vitamin-A-rich sweetpotato in Africa | |
| CN111187756A (en) | Areca-nut yellows-related virus and detection method thereof | |
| Varanda et al. | Multiplex RT-PCR for detection and identification of three necroviruses that infect olive trees | |
| Gambley et al. | Identification of viral and non-viral reverse transcribing elements in pineapple (Ananas comosus), including members of two new badnavirus species | |
| CN108796128A (en) | A kind of GII.8 types norovirus genome amplification primer and amplification method | |
| Koloniuk et al. | Molecular characterization and complete genome of a novel nepovirus from red clover | |
| Tian et al. | Host adaptation of soybean dwarf virus following serial passages on pea (Pisum sativum) and soybean (Glycine max) | |
| Shimizu et al. | A broad spectrum, one-step RT-PCR to detect Satsuma dwarf virus variants using universal primers targeting both segmented RNAs 1 and 2 | |
| Vargas-Asencio et al. | Limited Genetic Variability Among American Isolates of Grapevine virus E from Vitis spp. | |
| CN107604098A (en) | For detecting dove TTV EvaGreen real-time fluorescence quantitative PCRs primer and kit | |
| Nemchinov et al. | Identification of a novel isolate of alfalfa virus S from China suggests a possible role of seed contamination in the distribution of the virus | |
| CN108796129B (en) | GI.Pb/GI.6 recombinant norovirus genome amplification primer and amplification method | |
| Ashraf et al. | Development of differential RT-PCR assays and molecular characterization of the complete VP1 gene of five strains of very virulent infectious bursal disease virus | |
| CN106676117A (en) | Construction and authentication of newcastle disease virus overall-length cDNA clone |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |