CN105112561B - CH/GDDG/2014 plants of whole genome sequences of PPR virus and its amplimer pair - Google Patents
CH/GDDG/2014 plants of whole genome sequences of PPR virus and its amplimer pair Download PDFInfo
- Publication number
- CN105112561B CN105112561B CN201510500411.1A CN201510500411A CN105112561B CN 105112561 B CN105112561 B CN 105112561B CN 201510500411 A CN201510500411 A CN 201510500411A CN 105112561 B CN105112561 B CN 105112561B
- Authority
- CN
- China
- Prior art keywords
- sequence
- ppr virus
- whole genome
- virus
- gddg
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
-
- 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
- C12Q1/70—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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses CH/GDDG/2014 plants of whole genome sequences of PPR virus and its amplimers pair.Virus genome sequence is divided into 11 segments by the present invention, expands corresponding cDNA with One step RT-PCR method, the PCR fragment of amplification is cloned on pGM-T carrier and carries out nucleic acid sequencing.The present invention can effectively amplify target fragment, provide whole genome sequence, be conducive to study science of heredity, molecular epidemiology and reverse genetics research of PPR virus etc..
Description
Technical field
The invention patent belongs to molecular biology field, is related to the measurement of 4 type whole genome sequence of peste des petits ruminants genotype
Method is specifically related to CH/GDDG/2014 plants of whole genome sequence measuring methods of PPR virus.
Background technique
Peste des petits ruminants is to seriously endanger the deadly infectious disease of the animal healths such as goat, sheep, wild ruminants, can be drawn
It plays various clinicals symptom, the World Organization for Animal Health such as pneumonia, diarrhea, stomatitis, the miscarriage of infection animal and is classified as A class animal
Epidemic disease.Peste des petits ruminants has 4 genotype, and there are biggish differences between the genome of different genotype or same gene type strain
It is different.2007 and 2013, two wave peste des petits ruminants epidemic situations, especially the second wave epidemic disease were broken out in the Tibet in China and Xinjiang
Feelings involve a provinces, cities and autonomous regions more than 20, have brought tremendous economic losses to China's sheep husbandry and wild ruminants.Study table
Bright 4 type PPR virus of novel gene is arch-criminal.Although farm, China has begun using peste des petits ruminants epidemic disease
Seedling (2 type of gene), but still have wild-type virus outbreak of epidemic.Though currently, there is the full base of 4 type PPR virus of novel gene
It because of a group sequence report, but does not disclose and shows how it expands acquisition, this is unfavorable for China and constructs novel peste des petits ruminants
Reverse genetics system, meanwhile, it further will affect us and understand the growth curve of the virus, virus titer and pathogenic etc. raw
Object feature.
Based on problem above, a kind of open full base of effective, simple, accurate 4 type PPR virus of novel gene
Because group sequencing methods are necessary.
Summary of the invention
The purpose of this patent is to carry out design of primers, sequence alignment with the methods of molecular biology, bioinformatics
And reaction system optimization, provide CH/GDDG/2014 plants of 4 type PPR virus of novel gene whole genome sequence and its
Amplimer pair.
PPR virus CH/GDDG/2014 plants of whole genome sequences provided by the invention, sequence such as SEQ ID
Shown in No.1.
The present invention also provides the primer for expanding CH/GDDG/2014 plants of whole genome sequences of PPR virus, packets
Include the primer as shown in No.2~23 SEQ ID.
The present invention also provides a kind of methods for expanding PPR virus CH/GDDG/2014 plants of full-length genome, including with
Lower step:
(1) PPR virus CH/GDDG/2014 plants of RNA is extracted；
(2) the primer pair PPR virus whole genome sequence shown in No.2~23 SEQ ID carries out segmentation one
Footwork RT-PCR amplification；
(3) recycling, connection, conversion, the identification of positive bacteria and the sequencing of target fragment；
(4) sequence measured is compared using bioinformatics softwares such as DNAStar, sequence assembly, it will
11 segments join end to end, and obtain CH/GDDG/2014 plants of whole genome sequences of PPR virus.
Wherein, the 50 μ L One step RT-PCR reaction systems include: 2 × Buffer, 25 μ L, every section of amplification upstream and downstream
Primer each 1 μ L of 1 μ L, Mix, 5 μ L of viral RNA, sterilize ddH2O 17mL；
Wherein, the One step RT-PCR response procedures include: 50 DEG C of reverse transcription 30min, 95 DEG C of initial denaturation 5min, and 35
A circulation (95 DEG C of denaturation 30s, 53 DEG C~60 DEG C annealing 30s, 72 DEG C of extension 50s~188s), 72 DEG C re-extend 10min.
Virus genome sequence is divided into 11 segments by the present invention, expands corresponding cDNA with One step RT-PCR method,
The PCR fragment of amplification is cloned on pGM-T carrier and carries out nucleic acid sequencing.The present invention can effectively amplify target fragment,
Whole genome sequence is provided, science of heredity, molecular epidemiology and the reverse genetics of studying PPR virus are conducive to
Research etc..
The present invention separates CH/GDDG/2014 plants of PPR virus identified and belongs to 4 type PPR virus of gene
Strain, the goat Sample preservation being related to is in Institute of Animal Health,Guangdong Academy Of Agricultural Sciences's animal epidemic diagnostic center, address:
The Wushan Road, Tianhe District, Guangzhou City street Bai Shigang No. 1 building of diagnostic center, preservation sample number into spectrum are GDDG.
Detailed description of the invention
Fig. 1 show the Ago-Gel electricity of amplification PPR virus CH/GDDG/2014 pnca gene segment 1~11
Swimming picture.M:DNA Marker 2000；1~11 indicates 11 DNA fragmentations of different sizes of amplification.
Fig. 2 show the Phylogenetic tree of each genotype of PPR virus.The strain of mark "●" is small to ruminate beast
CH/GDDG/2014 plants of epidemic disease poison；The strain for marking " * " is peste des petits ruminants vaccine strain.
Specific embodiment
The present invention will be further described combined with specific embodiments below, and operating procedure can further clarify.
Optimal enforcement example:
(1) viral RNA extracts
The goat lungs sample for being accredited as the PPR virus positive by RT-PCR is taken, ground, organize to be homogenized,
Be diluted at 1: 2, multigelation 3 times, carries out RNA extracting according to virus RNA extraction kit (Axygen company), finally, with
50~100 μ L RNase-free distilled waters carry out RNA elution, are stored in -20 DEG C of refrigerators；
(2) design of primers and synthesis
By the comparison of the multiple genotype whole genome sequences of PPR virus, using Primer Premier 5.0
Software chooses conservative sequence as primer, has overlap different in size between adjacent amplified fragments, convenient for the later period
Sequence assembly.It is related to 11 pairs of 22 primers (chemical synthesis of Suzhou Jin Weizhi Biotechnology Co., Ltd) altogether, by peste des petits ruminants
The whole genome sequence of virus is divided into 11 sections, and specific primer sequence and corresponding site are as shown in table 1." F " represents forward primer,
" R " represents reverse primer.Primer location refers to China/XJYL/2013 plants of (GenBank accession number: KM091959) genomes.
1 primer information of table
(3) the One step RT-PCR amplification of each genetic fragment
Full-length genome is divided into 11 segments to expand respectively, primer is as shown in No.2~23 SEQ ID.Paragraph 1 and the 11st
A segment is respectively the 5 ' ends and 3 ' ends of genome, and while carrying out PCR, we also use 5 ' ends and 3 ' ends
RACE processing, to obtain its most true 5 ' end and 3 ' terminal gene group sequences.One step RT-PCR amplification reaction system (50 μ
It L) include: 2 × Buffer, 25 μ L, every section of amplification upstream and downstream primer each 1 μ L of 1 μ L, Mix, 5 μ L of viral RNA, sterilize ddH2O
17mL。
PCR response procedures include: 50 DEG C of reverse transcription 30min, 95 DEG C of initial denaturation 5min, 35 circulations (95 DEG C of denaturation 30s,
53 DEG C~60 DEG C annealing 30s, 72 DEG C of extension 50s~188s), 72 DEG C re-extend 10min.Wherein, paragraph 1 annealing temperature is 60
DEG C, the 2nd section of annealing temperature is 53 DEG C, and the 4th section of annealing temperature is 57 DEG C, other segment annealing temperatures are 55 DEG C, and paragraph 1 extends
Temperature is 51s, and the 2nd section of elongating temperature is 66s, and the 3rd section of elongating temperature is 60s, and the 4th section of elongating temperature is 50s, the 5th section of extension
Temperature is 66s, and the 6th section of elongating temperature is 174s, and the 7th section and the 8th section of elongating temperature are 188s, the 9th section, the 10th section and the 11st section
Elongating temperature is 120s.
(4) recycling of target gene fragment
PCR plastic recovery kit is purchased from TIANGEN Biotech (Beijing) Co., Ltd., and specific recovery method is as follows:
1. column equilibration step: into adsorption column CB2, the equilibrium liquid BL of 500 μ l is added in (adsorption column is put into collecting pipe),
12,000rpm (~13,400 × g) are centrifuged 1min, outwell the waste liquid in collecting pipe, adsorption column CB2 is placed back in collecting pipe
In.2. estimates the volume of PCR reaction solution, the combination liquid PB of 5 times of volumes is added thereto, mixes well (without removing paraffin oil
Or mineral oil).3. previous step acquired solution is added in an adsorption column CB2 (adsorption column is put into collecting pipe), it is placed at room temperature for
2min, 12,000rpm (~13,400 × g) are centrifuged 30-60sec, outwell the waste liquid in collecting pipe, adsorption column CB2 is put into receipts
In collector.4. 600 μ l rinsing liquid PW, 12,000rpm (~13,400 × g) are added into adsorption column CB2 and are centrifuged 30-60sec by,
The waste liquid in collecting pipe is outwelled, adsorption column CB2 is put into collecting pipe.5. repetitive operation step is 4..6. puts adsorption column CB2
It recycles in collector, 12,000rpm (~13,400 × g) are centrifuged 2min, as far as possible removing rinsing liquid.Adsorption column CB2 is placed in room temperature
It places several minutes, thoroughly dries, to prevent remaining rinsing liquid from influencing the experiment of next step.7. adsorption column CB2 is put into one by
In a clean centrifuge tube, 30-50 μ l elution buffer EB is vacantly added dropwise to adsorbed film middle position, is placed at room temperature for 2min.12,
000rpm (~13,400 × g) is centrifuged 2min and collects DNA solution.
(5) target gene fragment is connected on pGM-T carrier:
The specific operation method is as follows: 3 μ l, 10 × T4DNA ligase of target DNA, 1 μ l is added in 0.2ml PCR pipe,
PGM-T cloning vector 1 μ l, ddH25 μ l of O, mixes gently, in 22 DEG C of connection 1-2h.After reaction, pipe is placed in cold on ice
But.
(6) conversion of connection product
It takes 5 μ L connection products to be added in 50-100 μ L TOP10 competent cell, flicks mixing, ice bath 30min；Then will
Centrifuge tube is placed in 42 DEG C of water-bath 90sec, is immediately placed in ice bath after taking-up pipe and places 2-3min, not shake centrifuge tube therebetween；
LB (being free of antibiotic) culture medium of 37 DEG C of 250-500 μ L preheatings, 150rpm, 37 DEG C of shaken cultivation 1h are added into centrifuge tube.
Purpose is to make relevant resistant maker gene expression on plasmid, and thallus is made to recover；Bacterium solution in centrifuge tube is mixed, 100 μ are drawn
L is added on the LB solid medium containing ampicillin, X-gal and IPTG, with sterile elbow glass rod with gentle that cell is equal
It is even spreadable.After planar surface is dry, it is inverted plate, 37 DEG C of culture 12-16h；Obtained white colony is inoculated into 4mL LB
In fluid nutrient medium, 37 DEG C of shaking table shaken cultivation 12-16h after saving strain, are extracted to plasmid.
(7) identification of positive plasmid
It takes a little plasmid to carry out PCR amplification, sees that (3) describe.
(8) sequencing
To positive plasmid is accredited as, Beijing Liuhe Huada Genomics Technology Co., Ltd, sequencing portion, Guangzhou is sent to be surveyed
Sequence.
(9) sequence assembly
Using the SeqMan program in DNAStar software, the sequencing result of 11 segments is subjected to sequence assembly, Bian Jiji
Correction.Meanwhile base check and correction is carried out in conjunction with sequencing result of the sequencing peak figure to different batches, finally obtain PPR virus
CH/GDDG/2014 plants of whole genome sequences (15,954bp), as shown in SEQ ID No.1.
Claims (1)
1. a kind of method for measuring PPR virus full-length genome, which comprises the following steps:
(1) RNA of PPR virus is extracted；
(2) 50 μ L PCR reaction systems are prepared, carry out segmentation RT-PCR with primer pair PPR virus whole genome sequence
Amplification；
(3) recycling, connection, conversion, the identification of positive bacteria and the sequencing of target fragment；
(4) sequence measured is compared using DNAStar bioinformatics software, sequence assembly, is obtained small anti-
Hay epizootic disease virus whole genome sequence；50 μ L RT-PCR reaction systems include: 2 times of concentration Buffer, 25 μ L, and every section expands
Downstream primer each 1 μ L of 1 μ L, Mix solution, 5 μ L of viral RNA, sterilize 17 μ L of distilled water；PCR response procedures include: 50 DEG C of reversions
Record 30min, 95 DEG C of initial denaturation 5min, 35 circulations, the circulation are as follows: 95 DEG C of denaturation 30s, 53 DEG C~60 DEG C annealing 30s, 72 DEG C
Extend 50s~188s, 72 DEG C of 10min；The PPR virus is CH/GDDG/2014 plants, the sequence such as SEQ ID
Shown in No.1；The primer sequence is as shown in No.2~23 SEQ ID；
The wherein corresponding relationship of primer and amplified fragments are as follows:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510500411.1A CN105112561B (en) | 2015-08-14 | 2015-08-14 | CH/GDDG/2014 plants of whole genome sequences of PPR virus and its amplimer pair |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510500411.1A CN105112561B (en) | 2015-08-14 | 2015-08-14 | CH/GDDG/2014 plants of whole genome sequences of PPR virus and its amplimer pair |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105112561A CN105112561A (en) | 2015-12-02 |
| CN105112561B true CN105112561B (en) | 2019-10-11 |
Family
ID=54660663
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510500411.1A Active CN105112561B (en) | 2015-08-14 | 2015-08-14 | CH/GDDG/2014 plants of whole genome sequences of PPR virus and its amplimer pair |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105112561B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192898B (en) * | 2018-01-12 | 2019-09-20 | 金宇保灵生物药品有限公司 | The whole genome sequence and its amplimer of Seneca Valley virus SVV/CH/ZZ/2016 |
| CN108085323B (en) * | 2018-01-12 | 2019-09-20 | 金宇保灵生物药品有限公司 | The whole genome sequence and its amplimer of Seneca Valley virus SVV/CH/NM/2016 |
-
2015
- 2015-08-14 CN CN201510500411.1A patent/CN105112561B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| 一步法RT-PCR扩增PRSV海南分离物全长基因组cDNA;庹德财;《热带作物学报》;20111231;第32卷(第7期);1347-1351 * |
| 中国小反刍兽疫病毒分离株China/Tib/07全基因组序列测定与分析;刘文华;《病毒学报》;20100731;第26卷(第4期);322-329 * |
| 小反刍兽疫病毒基因组全长cDNA的构建及其序列的测定;翟军军;《病毒学报》;20100731;第26卷(第4期);315-321 * |
| 牛病毒性腹泻病毒一步法RT-PCR检测方法的建立与应用;王春庆;《中国畜牧兽医》;20131231;第40卷(第1期);66-69 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105112561A (en) | 2015-12-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pyrc et al. | Identification of new human coronaviruses | |
| CN107299155A (en) | A kind of primer and probe of goose astrovirus real-time fluorescence quantitative PCR detection | |
| CN105018489A (en) | Kit for recognizing Brucella wild strain and vaccine strains A19 and S2 | |
| CN101624636B (en) | LAMP-LFD detection method of infectious spleen and kidney necrosis virus (ISKNV) | |
| CN105002173A (en) | Kit for identifying Brucella S2 vaccine strain and wild strain | |
| CN103382507A (en) | One-step dual RT-PCR detection kit of 1-type and 3-type duck hepatitis A virus, primer pairs and method thereof | |
| Wang et al. | Differentiation of canine distemper virus isolates in fur animals from various vaccine strains by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism according to phylogenetic relations in china | |
| CN105018628B (en) | Differentiate the kit of brucella A19 vaccine strains and street strain | |
| Raviv et al. | The Mycoplasma gallisepticum 16S–23S rRNA intergenic spacer region sequence as a novel tool for epizootiological studies | |
| Alvarado et al. | Molecular characterization of avian infectious bronchitis virus strains isolated in Colombia during 2003 | |
| Lee et al. | Development of PCR diagnosis system for plant quarantine seed-borne Wheat streak mosaic virus | |
| CN110669870A (en) | Real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer, probe and detection kit for serotype of Palima serogroup virus | |
| CN104109666A (en) | Chromosome specific marker of elytrigia elongata in wheat background and use thereof | |
| CN103088039B (en) | Amplification method of porcine epidemic diarrhea virus S-gene epitope | |
| CN105112561B (en) | CH/GDDG/2014 plants of whole genome sequences of PPR virus and its amplimer pair | |
| CN112746116B (en) | Litopenaeus vannamei female DNA label sequence, primer and application thereof in sex identification | |
| CN101875983A (en) | Method for rapidly detecting various viruses of melons | |
| CN107287352A (en) | The probe primer group and its method of duck enteritis virus and duck hepatitis virus quick detection | |
| CN113174446A (en) | One-step double RT-PCR detection method for bovine viral diarrhea virus typing | |
| CN102600466B (en) | Anti-influenza A virus and novel universal epitope vaccine and preparing method thereof | |
| CN103993102A (en) | Multiple fluorescent PCR method and kit for simultaneous detection of human adenovirus, human mycoplasma pneumonia and bocavirus | |
| CN104862423B (en) | The visualization genetic chip and kit of porcine pseudorabies virus, pig parvoviral and porcine circovirus 2 type | |
| CN107058630B (en) | Multiple RT-PCR detection method for 3 genotypes of bovine parainfluenza 3 virus | |
| CN110643740A (en) | Real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer, probe and detection kit for Palimam serogroup virus | |
| CN103667538B (en) | Gene chip, kit and method for detecting three viruses for immunosuppression disease of chickens |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |