CN102433392A - Primers and probe for detecting fragment S of rift valley fever virus - Google Patents
Primers and probe for detecting fragment S of rift valley fever virus Download PDFInfo
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- CN102433392A CN102433392A CN201110415602XA CN201110415602A CN102433392A CN 102433392 A CN102433392 A CN 102433392A CN 201110415602X A CN201110415602X A CN 201110415602XA CN 201110415602 A CN201110415602 A CN 201110415602A CN 102433392 A CN102433392 A CN 102433392A
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Abstract
The invention provides primers and a probe for detecting the fragment S of a rift valley fever virus, wherein, a forward primer is 5'-GGATTACTTTCCTGTGATATCTGTTG-3'; a reverse primer is 5'-GTATCCTGGGAGGRCCATCWC-3', R refers to A or G and w refers to T or A); and a probe is 5'-F1-ACTCCACTGACACAACACGACGACCACT-Q1-3', F1 is a fluorescent reporter and Q1 is a fluorescent quencher. The invention also provides a real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection method for the fragment S of the rift valley fever virus. In the method, pseudovirus grains of the rift valley fever virus are taken as a template, and the primers and the probe are used to carry out real-time fluorescent RT-PCR, thus judging whether the detected virus is the rift valley fever virus according to the detected result. The probe provided by the invention has high sensitivity and good specificity, and has no cross reaction with all of Africa swine fever virus, irides virus and goatpox virus.
Description
Technical field
The present invention relates to the genetically engineered field, specifically, relate to a kind of be used to detect segmental primer of Rift Valley fever virus S and probe.
Background technology
Rift Valley fever (Rift valley fever; RVF) be a kind of serious Amphixenosis; It also is the endemy that is popular in Grand Canyon area, East Africa since ancient times; Mainly be in the middle of sheep, goat and ox, to propagate, often can cause the miscarriage storm (abortion storm) of pregnant domestic animal, children's sheep mortality ratio in age is up to more than 90%.This sick popular cycle is about 5-10.But this disease infected person, the patient shows influenza-like symptoms such as heating, headache and arthrodynia, and minority has the performance of hemorrhagic fever and encephalitis, and idol can cause the retinitis so that blind, and case fatality rate is about 1%, and mortality ratio can be up to about 50% when showing the blood-head symptom.RVF has obvious seasonal mainly through killing propagation, also can pass through contact infection.
Rift Valley fever virus (RVFV) is that phlebotomus fever virus belongs to (Phlebovirus), and Bunyaviridae (Bunyaviridae) member is a kind of virus of single serotype, has the representative configuration and the physicochemical property of cloth Buddhist nun virus.Virus has cyst membrane, and diameter 90～110nm is spherical in shape, and there is clearly gp projection on the surface, the long 10nm of protrusion diameter.Viral nucleic acid is positioned at the nucleocapsid of virus, is the sub-thread strand RNA, RNA can be divided into L (greatly), M (in), three sections of S (little).S sections wherein is the RNA of two meanings, promptly has the amphiorentation encoding function, coding N albumen and other a kind of Nonstructural Protein (NSs); M sections coding G1 and two kinds of outer membrane glycoproteins of G2 and two kinds of Nonstructural Proteins (NSm, size is respectively 14kD and 78kD), G1 and G2 gp can both stimulate body to produce antibody, but have only G2 can stimulate body to produce neutralizing antibody in vivo; L sections coding L albumen, L albumen has the function of virus transcription enzyme.Virus particle does not contain stromatin.Strain isolated and the testing laboratory that does not also find the at present RVFV specific antigens difference of strain that goes down to posterity, but proved each strain pathogenicly exist certain difference.
At present, the diagnostic method of Rift Valley fever virus mainly contains diagnostic techniquess such as virus separation, agarose immunodiffusion technique, immunofluorescence dyeing technology, serology, Protocols in Molecular Biology.Wherein viral separation method is the most efficient responsive method of RVF diagnosis, the classical way of always diagnosing as RVF.In recent years, along with the development of Protocols in Molecular Biology, the real-time fluorescence PCR technology accounts for its advantage quick, responsive, that specificity is good and has great advantage, and has caused numerous investigators' concern.
Fluorescent PCR (FQ-PCR) technology is a kind of new nucleic acid quantification technology that U.S. PE (Perkin Elmer) company develops nineteen ninety-five; This method is since producing; Constantly development is perfect, and is particularly along with being widely used of TaqMan probe, up to the present should technology very ripe; Have highly sensitive (higher more than 100 times), high specificity (on the basis of a pair of primer of regular-PCR than regular-PCR; The centre also is designed with probe, has only all pairings fully of primer and probe, just possibly obtain amplification; Can identify to the sequence difference of 2 Nucleotide few; Thereby guaranteed the specificity of reaction), advantage such as easy-to-operate (need not gel electrophoresis, avoided the harm of EB dyeing) to human body, be widely used in a plurality of fields such as research of pathogen detection, oncogene detection, immunoassay, genetic expression, sudden change and polymorphums thereof such as bacterium, virus at present.
Summary of the invention
The purpose of this invention is to provide and be used to detect segmental primer of Rift Valley fever virus S and probe.
Another object of the present invention provides a kind of fluorescence RT-PCR method that detects Rift Valley fever virus.
In order to realize the object of the invention; A kind of be used to detect segmental primer of Rift Valley fever virus S and probe of the present invention; Upstream primer RVFVU1:5 '-GGATTACTTTCCTGTGATATCTGTTG-3 '; Downstream primer RVFVL1:5 '-GTATCCTGGGAGGRCCATCWC-3 ' (R is A or G, and W is T or A), probe RVFVP1:5 '-F1-ACTCCACTGACACAACACGACGACCACT-Q1-3 '; Wherein, F1 is the report fluorophor, and Q1 is the cancellation fluorophor.Preferred F1 is FAM, and Q1 is ECLIPSE.Wherein F1 can also be fluorophors such as FAM, HEX, TET, and corresponding Q1 can also be quenching groups such as TAMARA, BHQ, MGB.
The present invention also provides the fluorescent PCR method that detects Rift Valley fever virus, and it is a template with sample RNA, carries out real-time fluorescence RT-PCR with above-mentioned primer and probe and detects, and judges in the sample whether carry Rift Valley fever virus according to amplification curve.
It is following that the reaction system of 25 μ L contains composition: 10 * PCR damping fluid (Mg
2+) 2.5 μ L; 2.5mM MgCL
25.0 μ L; 2.5mM dNTP 2.5 μ L; ThermoScript II AMV 2.5U; Rnase suppressor factor 20U; 10 μ M RVFVU1,0.5 μ L; 10 μ M RVFVL1,0.5 μ L, 10 μ MRVFVP1,0.5 μ L, rTaq polysaccharase 2.5U; RNA template 1.6 * 10
-6～1.6 * 10
-1Ng, surplus is a distilled water;
Response procedures is: 50 ℃ of reverse transcription 30min; 95 ℃ of preparatory sex change 5min; 95 ℃ of sex change 10s, 47 ℃ of annealing 20s, FAM passage fluorescent signal is gathered in 45 circulations during each loop ends.
After reaction finishes, judge yin and yang attribute according to the CT value that instrument provides automatically, when CT value＜38, be judged to be the positive, when CT value＞40, be judged to be feminine gender, when 38≤CT≤40, be judged to be suspiciously, suspicious specimen need be rechecked, and reinspection still is judged to the positive for suspicious person.
The present invention further is provided for detecting the segmental test kit of Rift Valley fever virus S, and it comprises aforesaid primer and probe.
The high specificity of primer of the present invention and probe, highly sensitive, with the Rift Valley fever virus S fragment real-time fluorescence RT-PCR method of its foundation, have reaction fast, the advantage of high, the high specificity of susceptibility, can realize the detection of Rift Valley fever virus.
Description of drawings
What Fig. 1 showed is the amplification curve diagram that Rift Valley fever virus S fragment real time fluorescent PCR method detects the gradient standard plasmid.
What Fig. 2 showed is Rift Valley fever virus S fragment real-time fluorescence PCR specific amplification graphic representation.
What Fig. 3 showed is that Rift Valley fever virus S fragment real-time fluorescence RT-PCR method detects Rift Valley fever virus appearance particulate amplification curve diagram.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment, the raw materials used commercial goods that is.
The preparation of embodiment 1 primer and probe
1.1 the design of primer and probe is with synthetic
Retrieval obtains the segmental conserved sequence of Rift Valley fever virus S in the U.S.'s state-run bioinformation center NCBI gene pool (GenBank), and accession number is (DQ380149).Use its gene order of MegAlign software analysis,, screen a pair of primer at the conservative region of sequence, and in the right amplification region of this primer, set a fluorescence TaqMan probe with BeaconDesigner 5.0 according to the principle of design of primer and probe.
Primer sequence is:
RVFVU1：5’-GGATTACTTTCCTGTGATATCTGTTG-3’
RVFVL1:5 '-GTATCCTGGGAGGRCCATCWC-3 ' (R is A or G, and W is T or A), amplified production length is 92bp;
The sequence of said probe (RVFVP1) is: 5 '-ACTCCACTGACACAACACGACGACCACT-3 ', and 5 ' end of this probe is with report optical dye FAM mark, and 3 ' end is with cancellation optical dye ECLIPSE mark.The design of this probe is directed to the high GC content district between institute's extension increasing sequence.
1.2 the preparation of primer and probe
After primer and probe were synthetic, the primer dilution was 10 μ mol/l, and probe dilution is 10 μ mol/l, and the upstream and downstream primer mixes according to 1: 1 ratio equal-volume, the probe lucifuge, and-20 ℃ of preservations are subsequent use after the packing.
The foundation of embodiment 2 fluorescence PCR detecting methods
1.1 the preparation of Rift Valley fever S fragment recombinant plasmid
Retrieval obtains the segmental conserved sequence of Rift Valley fever virus S in the U.S.'s state-run bioinformation center NCBI gene pool (GenBank); Introduce a restriction enzyme site (KpnI and HindIII) after the chemosynthesis respectively at two ends, be connected among the cloning vector pGEM-T transformed into escherichia coli competence BL21 then; Through blue hickie screening; The picking hickie shakes bacterium, and positive bacteria liquid extracts plasmid and recombinant plasmid (pGEM-T-rvfv-s) after checking order, and surveying concentration is 1.0 * 10
8Copy/μ L is prepared into the standard plasmid that contains Rift Valley fever virus S fragment conserved sequence.With Dilution damping fluid (TaKaRa) with this plasmid 10 times of doubling dilution to 1.0 * 10 successively
7, 1.0 * 10
6, 1.0 * 10
5, 1.0 * 10
4, 1.0 * 10
3, 1.0 * 10
2, 1.0 * 10
1Copy/μ L is prepared into the standard plasmid of a series of gradient concentrations.
1.2 the foundation of fluorescent PCR reaction system and reaction conditions
With 1.0 * 10
5The recombinant plasmid of copy/μ L is a template, adds the required reagent of amplified reaction and forms the reaction system shown in the table 1, and reaction is provided with negative control (replacing dna profiling to carry out with sterilized water) simultaneously, and reaction system is seen table 1.
The segmental fluorescent PCR reaction system of table 1 Rift Valley fever virus S
After reaction solution prepares, instantaneous centrifugal, sample hose is put into Real-time PCR appearance, adopt following condition to react: 95 ℃ of 5min; 95 ℃ of 10s, 47 ℃ of 20s, FAM passage fluorescent signal is gathered in 45 circulations during each loop ends.After amplified reaction finishes, according to the amplification curve result of determination.
1.3 the result judges
Adopting known 10 copy recombinant plasmids is that template is carried out fluorescent PCR detection discovery, and it is 37.62 that fluorescent signal arrives the cycle number (CT) that preset threshold experienced.When actual sample detects, consider of the influence of sample RNA purity to amplified reaction, the actual time of occurrence of amplification curve possibly have hysteresis slightly; For avoiding omission and guarantee the recall rate of minimum quantity,, confirm when CT value＜38, to be judged to be the positive at last through revision test repeatedly; When CT value＞40, be judged to be feminine gender; When 38≤CT≤40, be judged to be suspiciously, suspicious specimen need be rechecked, and rechecks and still is judged to the positive for suspicious person.
The sensitivity test of embodiment 3 Rift Valley fever virus S fragment real time fluorescent PCR methods
Standard plasmid with preparation among the embodiment 2 adopts the Rift Valley fever virus S fragment real-time fluorescence PCR reaction system of aforementioned foundation to make an experiment.Reaction finishes the back instrument and provides amplification curve diagram (Fig. 1) automatically.Fig. 1 has shown the amplification curve and the CT value of different copy plasmids, and wherein the detection CT value of 10 copy plasmids is 37.62, can judge that thus the susceptibility of this method can reach 10 copy DNAs.
The specificity test of embodiment 4 Rift Valley fever virus S fragment real time fluorescent PCR methods
1.1 the structure of African swine fever recombinant plasmid (pGEM-T-p72)
Retrieval obtains African swine fever p72 gene conservative sequence in the U.S.'s state-run bioinformation center NCBI gene pool (GenBank); Be connected into after the chemosynthesis among the cloning vector pGEM-T; Transformed into escherichia coli competence BL21; Through blue hickie screening, the picking hickie shakes bacterium, and positive bacteria liquid extracts plasmid and recombinant plasmid (pGEM-T-p72) after checking order.
1.2 specificity test
With 1.0 * 10
7Copy pGEM-T-rvfv-s DNA is template and African swine fever pGEM-T-p72 DNA, capripox virus DNA, and irido virus DNA and the DNA that extracts from the pathological material of disease pig tonsil, and establish a negative control and carry out the fluorescent PCR detection simultaneously.Reaction finishes the back instrument and provides amplification curve diagram (Fig. 2) automatically.As can be seen from Figure 2 have the amplification except that the pGEM-T-rvfv-s positive plasmid, all the other control samples and negative control be not amplification all, has shown that this method has good specificity.
The real-time fluorescence RT-PCR method of utilizing embodiment 5 detects Rift Valley fever virus appearance particle
1.1 the extraction of Rift Valley fever pseudovirion RNA
Moved with the moving plant quarantine of China Inst. of Quarantine Inspection Sciences that to examine the Rift Valley fever virus appearance particle that the chamber prepares in earlier stage be material; Adopt Trizol (Invitrogen; USA) reagent extracts according to process specifications that to survey concentration behind total RNA be 16ng/ μ L, and-86 ℃ of refrigerators are preserved subsequent use behind 10 times of doubling dilutions.
1.2 the foundation of fluorescent RT-PCR method for detecting
Rift Valley fever virus RNA with 10 times of doubling dilutions is a template, according to the reaction system among the embodiment 2, adds AMV enzyme 2.5U and Rnase suppressor factor 20U again and carries out the real-time fluorescence RT-PCR reaction, and reaction finishes instrument and provides amplification curve diagram (Fig. 3) automatically.As can be seen from the figure Rift Valley fever virus particulate LDL can reach 1.6 * 10
-6Ng/ μ L.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (5)
1. be used to detect segmental primer of Rift Valley fever virus S and probe, it is characterized in that,
Upstream primer RVFVU1:5 '-GGATTACTTTCCTGTGATATCTGTTG-3 ',
Downstream primer RVFVL1:5 '-GTATCCTGGGAGGRCCATCWC-3 ', wherein R is A or G, W is T or A.
2. be used to detect the segmental probe of Rift Valley fever virus S, it is characterized in that, probe is: 5 '-F1-ACTCCACTGACACAACACGACGACCACT-Q1-3 ', and wherein F1 is the report fluorophor, Q1 is the cancellation fluorophor.
3. detecting the fluorescence RT-PCR method of Rift Valley fever virus, it is characterized in that, is template with the RNA of Rift Valley fever virus, adopts described primer of claim 1 and the described probe of claim 2 to carry out fluorescence RT-PCR and reacts.
4. method according to claim 3 is characterized in that, it is following that the reaction system of 25 μ L contains composition: 10 * PCR damping fluid (Mg
2+) 2.5 μ L; 2.5mM MgCL
25.0 μ L; 2.5mM dNTP 2.5 μ L; ThermoScript II AMV 2.5U; Rnase suppressor factor 20U; 10 μ MRVFVU1,0.5 μ L; 10 μ M RVFVL1,0.5 μ L, 10 μ M RVFVP1,0.5 μ L, rTaq polysaccharase 2.5U; RNA template 1.6 * 10
-6～1.6 * 10
-1Ng, surplus is a distilled water;
Response procedures is: 50 ℃ of reverse transcription 30min; 95 ℃ of preparatory sex change 5min; 95 ℃ of sex change 10s, 47 ℃ of annealing 20s, 45 circulations.
5. be used to detect the segmental test kit of Rift Valley fever virus S, it comprises described primer of claim 1 and the described probe of claim 2.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624720A (en) * | 2018-06-21 | 2018-10-09 | 浙江国际旅行卫生保健中心（浙江出入境检验检疫局口岸门诊部） | The primed probe group and kit of RAA Fluorometric assay Rift Valley fever virus |
| CN111978400A (en) * | 2019-02-15 | 2020-11-24 | 中国科学院微生物研究所 | Rift valley fever virus humanized monoclonal antibody and application thereof |
| CN112921119A (en) * | 2021-02-24 | 2021-06-08 | 复旦大学 | Primer group, kit and method for loop-mediated nicking isothermal-CRISPR (clustered regularly interspaced short palindromic repeats) combined detection of rift valley fever virus |
| CN113308576A (en) * | 2021-06-10 | 2021-08-27 | 广州海关技术中心 | Kit for detecting rift valley fever virus based on digital PCR and detection method thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388584A (en) * | 2014-10-30 | 2015-03-04 | 薛芳 | Rift Valley fever virus nucleic acid molecule characteristic standard sample and preparation method thereof |
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| CN101487064A (en) * | 2009-02-24 | 2009-07-22 | 中国人民解放军军事医学科学院微生物流行病研究所 | Method and special reagent kit for detecting five zoonosis virus |
| CN102140528A (en) * | 2010-12-24 | 2011-08-03 | 中国检验检疫科学研究院 | New fluorescence quantitative polymerase chain reaction (PCR) detection method for rift valley fever virus and rift valley fever virus detection PCR system |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624720A (en) * | 2018-06-21 | 2018-10-09 | 浙江国际旅行卫生保健中心（浙江出入境检验检疫局口岸门诊部） | The primed probe group and kit of RAA Fluorometric assay Rift Valley fever virus |
| CN111978400A (en) * | 2019-02-15 | 2020-11-24 | 中国科学院微生物研究所 | Rift valley fever virus humanized monoclonal antibody and application thereof |
| CN111978400B (en) * | 2019-02-15 | 2022-04-01 | 中国科学院微生物研究所 | Rift valley fever virus humanized monoclonal antibody and application thereof |
| CN112921119A (en) * | 2021-02-24 | 2021-06-08 | 复旦大学 | Primer group, kit and method for loop-mediated nicking isothermal-CRISPR (clustered regularly interspaced short palindromic repeats) combined detection of rift valley fever virus |
| CN112921119B (en) * | 2021-02-24 | 2024-01-23 | 复旦大学 | Primer group, kit and method for loop-mediated nicking isothermal-CRISPR (clustered regularly interspaced short palace surface plasmon resonance) combined detection of rift valley fever virus |
| CN113308576A (en) * | 2021-06-10 | 2021-08-27 | 广州海关技术中心 | Kit for detecting rift valley fever virus based on digital PCR and detection method thereof |
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Inventor after: Wang Caixia Inventor after: Lin Xiangmei Inventor after: Wu Shaoqiang Inventor after: Zhang Yongning Inventor after: Deng Junhua Inventor before: Wang Caixia Inventor before: Lin Xiangmei Inventor before: Wu Shaoqiang Inventor before: Deng Junhua |
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