CN106987656A - A kind of primer and detection kit for detecting bovine epizootic fever virus - Google Patents

A kind of primer and detection kit for detecting bovine epizootic fever virus Download PDF

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CN106987656A
CN106987656A CN201710246638.7A CN201710246638A CN106987656A CN 106987656 A CN106987656 A CN 106987656A CN 201710246638 A CN201710246638 A CN 201710246638A CN 106987656 A CN106987656 A CN 106987656A
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seq
fever virus
primer
epizootic fever
bovine epizootic
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殷宏
高闪电
独军政
田占成
郑福英
刘志杰
杨吉飞
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Lanzhou Veterinary Research Institute of CAAS
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • 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

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Abstract

The present invention discloses a kind of primer for being used to detect bovine epizootic fever virus, and the detectable detection bovine epizootic fever virus kit being made up of these primers.The a kind of of the present invention detects that bovine epizootic fever virus primer is：SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4.Include foregoing four primers in the kit of the detection bovine epizootic fever virus of the present invention.The present invention primer can with it has been reported that bovine epizootic fever virus G genes have it is preferable match, detection kit of the invention can be more beneficial for be used for detect bovine epizootic fever virus.

Description

A kind of primer and detection kit for detecting bovine epizootic fever virus
Technical field
The present invention relates to a kind of primer for being used to detect bovine epizootic fever virus, and the detectable inspection being made up of these primers Survey bovine epizootic fever virus kit.
Background technology
Bovine Ephemeral Fever (bovine ephemeral fever, BEF) is by bovine epizootic fever virus (bovine ephemeral Fever virus, BEFV) caused by ox, milk cow, a kind of acute untouchable infectious disease of beef cattle and buffalo.BEFV is by matchmaker Jie insect mosquito and Storehouse midge are propagated, and its prevalence has obvious seasonality, often occurs at the beginning of autumn late summer, there is certain periodicity. BEF propagates rapid, popular wide, in Egypt, Zambia, Kenya, Australia, India, Indonesia, Palestine, Japan and the multiple provinces of China repeatedly occur.The sick clinical symptoms include heating, anorexia, dropped tears and nasal mucus, muscle The symptoms such as stiff, the temporary cyllopodia of contraction, salivary secretion thing increase, depression, arthroncus, can then occur mucous membrane when serious Purulence, tremble, paralyse and dead.
BEFV is that Rhabdoviridae, ephemeral fever virus belong to member, only one of which serotype, but according to the difference of strain incite somebody to action It is divided into 4 different genotypes.Wherein I types include Japanese 1988 and the separation strains and China mainland of 1989 and Taiwan Separation strains in 1984, II types mainly include Japan 2001-2004 separation strains and China Taiwan 1996-2004 separation Strain, type III is Japan's separation strains of 1996, and IV types are made up of Australian separation strains.BEFV can be with animal movement and medium Migrate in great-jump-forward spreading and propagation, heavy economic losses is caused to cattle-raising, therefore set up quick, sensitive, accurate, easy disease Malicious diagnostic method, for suspected infection animal make a definite diagnosis and the malicious condition monitoring of band of susceptible medium is particularly important.At present, Niu Liu The laboratory diagnosis of row pyreticosis mainly has (1) virus purification, and such as suckling mouse intracranial inoculation, Vero, BHK-21 sensitive cells are passed on, And then carry out neutralization test identification with standard immunoassay serum；(2) Serologic detection, such as complement fixation test (CFT), immunofluorescent test, G1-EL1SA, blocking ELISA method and neutralization test；(3) molecular detection technology, such as conventional RT-PCR detection technique and LAMP Method etc..Virus purification is the reliable method for making a definite diagnosis BEFV infection, but this method depends on cell culture, is taken longer；Serum Detection method is learned to be applied to by detecting that antiviral antibody carries out epidemiology survey or the evaluation of immune effect of vaccine.Molecular Detection Technology is higher due to fast and convenient, sensitivity, is played an important role in BEFV detections.At present, molecular biology method Have been used to BEFV detection and gene sequencing (Kato et al., 2009；Zheng et al., 2012), especially fluorescence Quantitative RT-PCR more sensitive, special advantage, it is to avoid the cumbersome step such as electrophoresis, is more suitable for BEFV quick diagnosis. 2005, Israel researcher Stram etc. established the TaqMan-MGB fluorescence quantitative RT-RCRs for BEFV antigen genes G For detecting that BEFV is detected, local separation strains Yaqum-00 and Australia Reference strains BB7721 can be detected effectively, still Because there is larger variation in the G genes of different strains, it is difficult to ensure the absolute matches of probe and China's BEFV strains, China Application for a patent for invention 201210219927.5 discloses the TaqMan-MGB fluorescence quantitative RT-RCR sides for China's BEFV strains Method, introduces two degeneracy bases, patent application MGB on the basis of average probe is modified within the probe to solve matching Probe, its hybridization ability is stronger, more stable, but needs the company of specialty to prepare, and adds the cost of detection.
The content of the invention
The present invention, which provides one kind, can overcome prior art not enough, can with it has been reported that bovine epizootic fever virus G genes have Preferably match be used for detect bovine epizootic fever virus primer, and the detection kit being made up of these primers.
The a kind of of the present invention detects that bovine epizootic fever virus primer is：SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4。
Include SEQ ID No.1, SEQ ID No.2, SEQ in a kind of kit of detection bovine epizootic fever virus of the present invention ID No.3、SEQ ID No.4。
Use for convenience, the kit that can be used for detection ox fluidity fever virus of the invention also includes following composition：BEFV RNA standard items, negative quality control standard product, fluorescence quantitative RT-RCR reaction solution and fluorescent quantitation correcting fluid ROX Reference Dye II。
The present invention primer can with it has been reported that bovine epizootic fever virus G genes have it is preferable match, inspection of the invention Test agent box can be more beneficial for being used to detect bovine epizootic fever virus.
The present invention is actually that one kind is simultaneously interior using ox GAPDH genes using Bovine Ephemeral Fever G genes progress Viral diagnosis The real-time fluorescence relative RT-PCR method of ginseng.Real-Time Fluorescent Quantitative PCR Technique is public by U.S. Applied Biosystems Take charge of and released in 1996, the technology not only realizes the leap from qualitative to quantitative, and with specificity compared with Standard PCR By force, sensitiveness is high, reproducible, quantitative accurate, automaticity is high, it is totally-enclosed the advantages of, thus in gene expression research, turn The numerous areas such as gene studies, curative effect of medication examination, pathogen detection are by extensive use.The present invention is being analysed in depth both at home and abroad On the basis of bovine epizootic fever virus sequence, Select gene information the abundantest G genes are detection target, are devised specific Conservative primer, while being corrected to express stable GAPDH genes as internal reference to sample rna content, is prepared for Bovine Ephemeral Fever Virus real-time fluorescence relative RT-PCR kit.In the present invention, by the optimization of reaction condition, make real-time quantitative RT-PCR Kit has good sensitiveness, specificity, repeatability, and its sensitiveness is higher than conventional RT-PCR 100 times, can be used for ox stream The relative quantification detection of row fever virus, and the kit is by increasing degeneracy base, the primer and 4 genotype Niu Liuhang The G genes of fever virus can be matched preferably, reduce the possibility of missing inspection.
Brief description of the drawings
The matching of Fig. 1 bovine epizootic fever virus detection primers.
Fig. 2 real-time fluorescence quantitative RT-PCR kinetic curves.
Fig. 3 real-time fluorescence quantitative RT-PCR standard curves.
Fig. 4 real-time fluorescence quantitative RT-PCR sensitivity experiments.
Fig. 5 conventional RT-PCR detection method electrophoretograms.
Wherein M is DNA molecular amount standard DL2000；1st, 2,3,4,5,6,7,8,9,10 be respectively 1011-102Copy/μ L RNA standard items；10 be blank control.
Fig. 6 real-time fluorescence quantitative RT-PCR specific test curves.
Fig. 7 real-time fluorescence quantitative RT-PCR broad spectrum activity trial curves.
Replica test curve in Fig. 8 real-time fluorescence quantitative RT-PCRs batch.
Replica test curve between Fig. 9 real-time fluorescence quantitative RT-PCRs batch.
The relative level of BEFV nucleic acid in the 3rd day to the 7th day blood of Figure 10 artificial challenges ox.
Embodiment
The present invention detection bovine epizootic fever virus primer for SEQ ID No.1 5 '- TAGTCACMACVTATGCDATYTAC-3 ' (forward primer) ,-GTTTACTYCCACYYTTGATGCT-3 ' of SEQ ID No.2 5 ' (reverse primer), purpose fragment size is 201bp；SEQ ID No.3：(ox GAPDH is just by 5 '-GCTCTCTGCTCCTGCCCG-3 ' To primer) ,-CGACGATGTCCACTTTGCCA-3 ' of SEQ ID No.4 5 ' (ox GAPDH reverse primers), purpose fragment size For 159bp.Primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Also need in specific application：Fluorescence quantitative PCR reaction solution, standard positive RNA, negative quality control standard product.At this Fluorescence quantitative PCR reaction solution is by Premix ExTaq in embodimentTM(2 ×) 12.5 μ L, 10 μM of forward primer SEQ No.1 and 10 μM of reverse primer SEQ No.2 each 0.5 μ L, fluorescence correction liquid ROX Reference Dye II (50 ×) 0.5 μ L, DEPC The μ L of water 10.
Kit application example of the present invention
It is prepared by standard positive RNA：According to bovine epizootic fever virus G gene orders, design primer GF1 (forward primer)：5’- TGTGGAGAACTTCATCCTGTA-3 ' SEQ No.5, GR1 (reverse primer)：5’-AGAATCTATCATCACCGATTGG-3’ SEQ No.6, carry out RT-PCR amplifications and obtain JT02 plants of G genetic fragments of bovine epizootic fever virus, length is 1786bp, reclaims purpose Fragment, is attached with pGEM-Teasy (Promega companies), JM109 competent cells is converted, with carrier negative sense primer M13R 5 '-CAGGAAACAGCTATGAC-3 ' SEQ No.7 and GR1 primer carry out bacterium colony PCR identifications, alkaline lysis method of extracting positive restructuring Plasmid enzyme restriction is linearized with Not I restriction endonucleases after plasmid pGEM-BEFV-G, sequencing, TranscriptAid is usedTM T7 High Yield Transcription Kit carry out in-vitro transcription, and then utilize the mould in DNAse I digestion removal transcripts Plate DNA, after through phenol chloroform, prepare the G gene RNA consistent with bovine epizootic fever virus genomic, negative strand RNA directions Section, using NanoDrop2000 (Thermo Fisher Scientific) ultramicron spectrophotometry concentration, and converts Into copy/μ L, in this, as standard positive RNA.
Fluorescence quantitative RT-RCR reactivity：The RNA standard items of preparation are serially diluted to final concentration of 1.0 × 109-1.0 ×100Copy/μ L, carries out quantitative RT-PCR amplification, and reaction system is 25 μ L：Premix ExTaqTM(2 ×) 12.5 μ L, 10 μM Forward primer SEQ No1 and 10 μM of each 0.5 μ L of reverse primer SEQ No2, fluorescence correction liquid ROX Reference Dye The μ L of 0.5 μ L, DEPC water of II (50 ×), 10 μ L, RNA template 1.Reaction condition is 42 DEG C of 10min reverse transcriptions, 95 DEG C of pre-degeneration 30s, Then 95 DEG C of 5s, 60 DEG C of 30min, 40 circulations, finally increase a melting curve.Fluorescence is carried out at the end of each circulation Signal detects that each template concentrations do 3 repetitions in real time, the calculating coefficient of variation of averaging, and experiment is all provided with negative control.Built Vertical real-time quantitative RT-PCR kinetic curve and standard curve are referring to Fig. 2 and Fig. 3, template amount and the corresponding linear phase of Ct values Close, coefficient correlation is 0.999, and amplification efficiency is 92.6%, calibration curve equation is y=-3.513 × logX+40.66.
Fluorescence quantitative RT-RCR sensitiveness：It is 1.0 × 10 to take concentration9-1.0×100Copy/μ L RNA standard items carry out spirit Sensitivity is tested, and the minimum detectability for as a result showing this method is 101Copy/μ L (Fig. 4), and the detection of conventional RT-PCR is limited to 103 Copy/μ L (Fig. 5) is higher than conventional RT-PCR method sensitivity 100 times.
Fluorescence quantitative RT-RCR specificity：Niu Liuhang is have detected respectively using the real-time fluorescent quantitative RT-PCR method of foundation Fever virus, hydrophobin, vesicular stomatitis virus, blue tongue virus, bovine viral diarrhea virus, Japanese B encephalitis virus Etc. the nucleic acid of cause of disease, as a result only bovine epizootic fever virus is positive, and other cause of diseases are negative (Fig. 6).
Fluorescence quantitative RT-RCR broad spectrum activity：China ox is have detected respectively using the real-time fluorescent quantitative RT-PCR method of foundation Epizootic fever poison strain LS11 (1.0 × 108Copy/μ L), Henan02 (1.0 × 108Copy/μ L), JB76H (1.0 × 107Copy Shellfish/μ L), LYC11 (1.0 × 107Copy/μ L), JT02 (1.0 × 106Copy/μ L) G gene plasmid standards, as a result can examine Go out, and it is linear good, illustrate the broad spectrum activity (Fig. 7) of the present invention.
Fluorescence quantitative RT-RCR stability：Take concentration 1.0 × 106-1.0×101The RNA standard items of copy/concentration of μ L 6 Carry out stability analysis.Batch interior repeated experiment (Fig. 8) is first carried out, the standard items diluted to same batch, each concentration is repeated Detection 3 times, the analysis Ct value coefficient of variation is between 0.08-0.89%, less than 5%, referring to table 1.Then repeatability between criticizing is carried out real Test (Fig. 9), the standard items of 3 different batches dilution detected, through analysis Ct discriminations say coefficient 0.23-1.02% it Between, less than 10%, referring to table 2, thus illustrate that the present invention has preferable stability.
Table 1Repeatability in batchExperimental result
Standard concentration (copy/μL) 1.0×101 1.0×102 1.0×103 1.0×104 1.0×105 1.0×106
Ct1 37.60 35.29 31.13 27.76 24.55 20.86
Ct2 37.54 35.05 30.74 27.72 24.75 21.09
Ct3 37.71 35.42 30.60 27.76 24.32 21.02
C.V% 0.23 0.53 0.89 0.08 0.88 0.56
Repeated experiment result between 2 batches, table
Standard concentration (copy/μL) 1.0×101 1.0×102 1.0×103 1.0×104 1.0×105 1.0×106
Ct1 37.51 35.19 30.81 27.92 24.31 20.97
Ct2 37.62 34.84 30.93 27.68 24.51 21.09
Ct3 38.02 34.48 30.8 27.72 24.3 20.87
C.V% 0.71 1.02 0.23 0.46 0.49 0.53
Detect clinical sample detection：To BEFV JT02 strain artificial challenges oxen the 3rd day (PID3) to the 7th (PID7) day blood Viral nucleic acid in liquid carries out relative quantification detection.The μ L of anticoagulation 300 are taken, adds after 800 μ L Trizol acutely shake and adds 200 μ L chloroforms, are stored at room temperature 5min.4 DEG C, 12000rpm centrifugation 15min draw supernatant and add isometric isopropanol, mix And in being stored at room temperature 10min.4 DEG C, 12000rpm centrifugation 10min abandon supernatant, the ethanol lotions of 1mL 75% are added in precipitation, 4 DEG C afterwards, 12000rpm centrifugation 10min, drying at room temperature precipitation adds the dissolving of 30 μ L DEPC water.Respectively with SEQ No1 and SEQ No2, SEQ No3 and SEQ No4 carry out the reaction of 25 μ L systems, using the 2 of relative quantification- Δ Δ CtCarry out data analysis, meter Calculate the nucleic acid level difference of BEFV in different infection time bovine bloods.At the 3rd day to the 7th day of infection, have in bovine blood BEFV nucleic acid is detected, and the 4th day is top, is minimized (Figure 10) within the 7th day.
Above example is the specific embodiment of the present invention, but protection scope of the present invention is not limited to above-mentioned implementation Example.
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>A kind of primer and detection kit for detecting bovine epizootic fever virus
<160> 4
<210> 1
<211> 23
<212> DNA
<213>The forward primer of bovine epizootic fever virus
<400>
tagtcacmac vtatgcdaty tac 23
<210> 2
<211> 22
<212> DNA
<213>The reverse primer of bovine epizootic fever virus
<400>
gtttactycc acyyttgatg ct 22
<210> 3
<211> 18
<212> DNA
<213>Ox GAPDH forward primers
<400>
gctctctgct cctgcccg 18
<210> 4
<211> 20
<212> DNA
<213>Ox GAPDH reverse primers
<400>
cgacgatgtc cactttgcca 20
<210> 5
<211> 21
<212> DNA
<213>Standard positive RNA forward primers
<400>
tgtggagaac ttcatcctgt a 21
<210> 6
<211> 22
<212> DNA
<213>Standard positive RNA reverse primers
<400>
agaatctatc atcaccgatt gg 22
<210> 7
<211> 17
<212> DNA
<213>Carrier negative sense primer
<400>
caggaaacag ctatgac 17

Claims (3)

1. a kind of primer for detecting bovine epizootic fever virus, it is characterised in that described primer is：SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4。
2. a kind of kit for detecting bovine epizootic fever virus, it is characterised in that include in described kit SEQ ID No.1, SEQ ID No.2、SEQ ID No.3、SEQ ID No.4。
3. the kit of detection bovine epizootic fever virus according to claim 2, it is characterised in that kit also includes as follows Composition：BEFV RNA standard items, negative quality control standard product, fluorescence quantitative RT-RCR reaction solution and fluorescent quantitation correcting fluid ROX Reference Dye II。
CN201710246638.7A 2017-04-16 2017-04-16 A kind of primer and detection kit for detecting bovine epizootic fever virus Pending CN106987656A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108531659A (en) * 2018-05-31 2018-09-14 广西壮族自治区兽医研究所 A kind of RT-PCR primer and kit of quick detection bovine epizootic fever virus
CN114410841A (en) * 2022-01-14 2022-04-29 华南农业大学 Primer and probe for detecting bovine epidemic heat and bovine epidemic heat virus and application thereof

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CN102719566A (en) * 2012-06-27 2012-10-10 北京森康生物技术开发有限公司 Nucleotide sequences and kit for testing bovine ephemeral fever virus
CN103969435A (en) * 2014-06-04 2014-08-06 中国农业科学院兰州兽医研究所 BEFV (Bovine Ephemeral Fever Virus) indirect ELISA (Enzyme Linked Immunosorbent Assay) antibody detection kit and preparation method thereof

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CN102719566A (en) * 2012-06-27 2012-10-10 北京森康生物技术开发有限公司 Nucleotide sequences and kit for testing bovine ephemeral fever virus
CN103969435A (en) * 2014-06-04 2014-08-06 中国农业科学院兰州兽医研究所 BEFV (Bovine Ephemeral Fever Virus) indirect ELISA (Enzyme Linked Immunosorbent Assay) antibody detection kit and preparation method thereof

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李志等: "牛流行热研究进展", 《中国畜牧兽医》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108531659A (en) * 2018-05-31 2018-09-14 广西壮族自治区兽医研究所 A kind of RT-PCR primer and kit of quick detection bovine epizootic fever virus
CN114410841A (en) * 2022-01-14 2022-04-29 华南农业大学 Primer and probe for detecting bovine epidemic heat and bovine epidemic heat virus and application thereof

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