CN102433392B - 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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- CN102433392B CN102433392B CN 201110415602 CN201110415602A CN102433392B CN 102433392 B CN102433392 B CN 102433392B CN 201110415602 CN201110415602 CN 201110415602 CN 201110415602 A CN201110415602 A CN 201110415602A CN 102433392 B CN102433392 B CN 102433392B
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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 primer for detection of fragment S of rift valley fever virus and probe.
Background technology
Rift Valley fever (Rift valley fever, RVF) be a kind of serious Amphixenosis, it is also the endemy that is popular in since ancient times Grand Canyon area, East Africa, mainly to propagate in the middle of sheep, goat and ox, 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.This disease can infect the people, and the patient shows the influenza-like symptoms such as heating, headache and arthrodynia, and minority has the performance of hemorrhagic fever and encephalitis, occasionally can cause the retinitis so that blind, case fatality rate approximately 1%, and when showing the blood-head symptom, mortality ratio can be up to 50% left and right.RVF has obvious seasonality 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 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 glycoprotein projection on the surface, the long 10nm of protrusion diameter.Viral nucleic acid be positioned at virus nucleocapsid, be 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, namely has the amphiorentation encoding function, coding N albumen and another 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 glycoprotein can stimulate body to produce antibody, but only have in vivo G2 can stimulate body to produce neutralizing antibody; L sections coding L albumen, L albumen has the function of virus transcription enzyme.Virus particle does not contain stromatin.Also do not find at present the strain isolated of RVFV and the testing laboratory specific antigens difference of strain that goes down to posterity, but proved the pathogenic different that exists of each strain.
At present, the diagnostic method of Rift Valley fever virus mainly contains the 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 advantage that the real-time fluorescence PCR technology is quick with it, responsive, specificity is good accounts for 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, development is perfect, being widely used along with the TaqMan probe particularly, up to the present this technology is very ripe, have highly sensitive (higher more than 100 times than regular-PCR), high specificity is (on the basis of regular-PCR pair of primers, the centre also is designed with probe, only have all pairings fully of primer and probe, just may be increased, can identify to the sequence difference of 2 Nucleotide few, thereby guaranteed the specificity of reaction), easy-to-operate (need not gel electrophoresis, avoided EB dyeing to the harm of human body) etc. advantage, be widely used at present bacterium, the pathogen detection such as virus, oncogene detects, immunoassay, genetic expression, a plurality of fields such as research of sudden change and polymorphism thereof.
Summary of the invention
The purpose of this invention is to provide primer and probe for detection of fragment S of rift valley fever virus.
Another object of the present invention is to provide 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 primer and probe for detection of fragment S of rift valley fever virus of the present invention, upstream primer RVFVU1:5 '-GGATTACTTTCCTGTGATATCTGTTG-3 ', (R is A or G to downstream primer RVFVL1:5 '-GTATCCTGGGAGGRCCATCWC-3 ', 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 the fluorophors such as FAM, HEX, TET, and corresponding Q1 can also be the quenching groups such as TAMARA, BHQ, MGB.
The present invention also provides the fluorescent PCR method that detects Rift Valley fever virus, and it carries out real-time fluorescence RT-PCR with above-mentioned primer and probe and detect take sample RNA as template, judges in sample whether carry Rift Valley fever virus according to amplification curve.
It is as follows 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 inhibitor 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 distilled water;
Response procedures is: 50 ℃ of reverse transcription 30min; 95 ℃ of denaturation 5min; 95 ℃ of sex change 10s, 47 ℃ of annealing 20s, 45 circulations gather FAM passage fluorescent signal during each loop ends.
After reaction finishes, judge yin and yang attribute according to the CT value that instrument provides automatically, be judged to be the positive when CT value＜38, be judged to be feminine gender when CT value＞40, be judged to be suspiciously when 38≤CT≤40, suspicious specimen need be rechecked, and reinspection still is judged to the positive for suspicious person.
The present invention further provides the test kit for detection of fragment S of rift valley fever virus, it comprises aforesaid primer and probe.
The high specificity of primer of the present invention and probe, highly sensitive with the fragment S of rift valley fever virus real-time fluorescence RT-PCR method of its foundation, has advantages of that reaction is quick, susceptibility is high, high specificity, can realize the detection of Rift Valley fever virus.
Description of drawings
What Fig. 1 showed is the amplification curve diagram that the fragment S of rift valley fever virus real time fluorescent PCR method detects the gradient standard plasmid.
What Fig. 2 showed is fragment S of rift valley fever virus real-time fluorescence PCR specific amplification graphic representation.
What Fig. 3 showed is the amplification curve diagram that fragment S of rift valley fever virus real-time fluorescence RT-PCR method detects Rift Valley fever virus sample particle.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, 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 conserved sequence of fragment S of rift valley fever virus in the state-run bioinformation of U.S. center NCBI gene pool (GenBank), and accession number is (DQ380149).Use its gene order of MegAlign software analysis, according to the principle of design of primer and probe, screen pair of primers with BeaconDesigner 5.0 at the conservative region of sequence, and set a fluorescence TaqMan probe in the amplification region of this primer pair.
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 described probe (RVFVP1) is: 5 '-ACTCCACTGACACAACACGACGACCACT-3 ', 5 ' end of this probe is with reporting fluorescence dye FAM mark, 3 ' end cancellation fluorescence 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 the ratio equal-volume of 1: 1, the probe lucifuge, and after packing ,-20 ℃ save backup.
The foundation of embodiment 2 fluorescence PCR detecting methods
1.1 the preparation of Rift Valley fever S fragment recombinant plasmid
Retrieval obtains the conserved sequence of fragment S of rift valley fever virus in the state-run bioinformation of U.S. center NCBI gene pool (GenBank), introduce respectively a restriction enzyme site (KpnI and HindIII) after chemosynthesis at two ends, then be connected in cloning vector pGEM-T, transform intestinal bacteria competence BL21, through blue hickie screening, the picking hickie shakes bacterium, and positive bacteria liquid extracts plasmid and recombinant plasmid (pGEM-T-rvfv-s) after order-checking, and surveying concentration is 1.0 * 10
8Copy/μ L is prepared into the standard plasmid that contains the fragment S of rift valley fever virus 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 Fluorescence PCR system and reaction conditions
With 1.0 * 10
5The recombinant plasmid of copy/μ L is template, adds the reaction system shown in the required reagent composition table 1 of amplified reaction, and reaction arranges negative control (replacing DNA profiling to carry out with sterilized water) simultaneously, and reaction system sees Table 1.
The Fluorescence PCR system of table 1 fragment S of rift valley fever virus
After reaction solution prepares, instantaneous centrifugal, sample hose is put into Real-time PCR instrument, adopt following condition to react: 95 ℃ of 5min; 95 ℃ of 10s, 47 ℃ of 20s, 45 circulations gather FAM passage fluorescent signal during each loop ends.After amplified reaction finishes, according to the amplification curve result of determination.
1.3 result is judged
Adopting 10 known copy recombinant plasmids is that template is carried out fluorescent PCR detection discovery, and the cycle number (CT) that the threshold value that fluorescent signal arrival is set experiences is 37.62.When actual sample detects, consider that sample RNA purity is on the impact of amplified reaction, the actual time of occurrence of amplification curve may slightly have hysteresis, for avoiding undetected and guarantee the recall rate of minimum quantity, through revision test repeatedly, determine at last to be judged to be the positive when CT value＜38, be judged to be feminine gender when CT value＞40, be judged to be suspiciously when 38≤CT≤40, suspicious specimen need be rechecked, and rechecks and still is judged to the positive for suspicious person.
The sensitivity test of embodiment 3 fragment S of rift valley fever virus real time fluorescent PCR methods
With the standard plasmid of preparation in embodiment 2, adopt the fragment S of rift valley fever virus real-time fluorescence PCR reaction system of aforementioned foundation to test.After reaction finishes, instrument provides amplification curve diagram (Fig. 1) automatically.Fig. 1 has shown 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 the method can reach 10 copy plasmid DNA.
The specific test of embodiment 4 fragment S of rift valley fever virus real time fluorescent PCR methods
1.1 the structure of African swine fever recombinant plasmid (pGEM-T-p72)
Retrieval obtains the conserved sequence of African swine fever p72 gene in the state-run bioinformation of U.S. center NCBI gene pool (GenBank), be connected into after chemosynthesis in cloning vector pGEM-T, transform intestinal bacteria competence BL21, through blue hickie screening, the picking hickie shakes bacterium, and positive bacteria liquid extracts plasmid and recombinant plasmid (pGEM-T-p72) after order-checking.
1.2 specific test
With 1.0 * 10
7Copy pGEM-T-rvfv-s plasmid DNA is template and African swine fever pGEM-T-p72 plasmid 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 simultaneously the fluorescent PCR detection.After reaction finishes, instrument provides amplification curve diagram (Fig. 2) automatically.As can be seen from Figure 2 have amplification except the pGEM-T-rvfv-s positive plasmid, all the other control samples and negative control be not amplification all, has shown that the method has good specificity.
The real-time fluorescence RT-PCR method of utilizing embodiment 5 detects Rift Valley fever virus sample particle
1.1 the extraction of Rift Valley fever pseudovirion RNA
Moved take the moving plant quarantine of China Inst. of Quarantine Inspection Sciences and examined Rift Valley fever virus sample particle that the chamber prepares in earlier stage as material, adopt Trizol (Invitrogen, USA) reagent, be 16ng/ μ L according to surveying concentration after the total RNA of process specifications extraction, after 10 times of doubling dilutions ,-86 ℃ of Refrigerator stores are standby.
1.2 the foundation of fluorescent RT-PCR method for detecting
Take the Rift Valley fever virus RNA of 10 times of doubling dilutions as template, according to the reaction system in embodiment 2, then add AMV enzyme 2.5U and Rnase inhibitor 20U to carry out the real-time fluorescence RT-PCR reaction, reaction finishes instrument and automatically provides amplification curve diagram (Fig. 3).As can be seen from the figure the lowest detectable limit of Rift Valley fever virus particle can reach 1.6 * 10
-6Ng/ μ L.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1. for detection of primer and the probe of fragment S of rift valley fever virus, 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;
Probe is:
5 '-F1-ACTCCACTGACACAACACGACGACCACT-Q1-3 ', wherein F1 is the report fluorophor, Q1 is the cancellation fluorophor.
2. detect the fluorescence RT-PCR method of Rift Valley fever virus, it is characterized in that, take the RNA of Rift Valley fever virus as template, adopts primer claimed in claim 1 and probe to carry out fluorescence RT-PCR and react.
3. method according to claim 2, is characterized in that, reaction system contains composition as follows: contain Mg in 25 μ L
2+10 * PCR damping fluid, 2.5 μ L; 2.5mM MgCL
25.0 μ L; 2.5mM dNTP 2.5 μ L; ThermoScript II AMV 2.5U; Rnase inhibitor 20U; 10 μ M RVFVU10.5 μ L; 10 μ M RVFVL10.5 μ L, 10 μ M RVFVP10.5 μ L, rTaq archaeal dna polymerase 2.5U; RNA template 1.6 * 1
-61.6 * 10
-1Ng, surplus is distilled water;
Response procedures is: 50 ℃ of reverse transcription 30min; 95 ℃ of denaturation 5min; 95 ℃ of sex change 10s, 47 ℃ of annealing 20s, 45 circulations.
4. for detection of the test kit of fragment S of rift valley fever virus, it comprises primer claimed in claim 1 and probe.
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Cited By (1)
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| 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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| 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 |
| CN114409769B (en) * | 2019-02-15 | 2023-05-09 | 中国科学院微生物研究所 | Setaria fever virus humanized monoclonal antibody and application thereof |
| 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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| 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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| 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 (1)
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| 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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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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Free format text: CORRECT: INVENTOR; FROM: WANG CAIXIA LIN XIANGMEI WU SHAOQIANG DENG JUNHUA TO: WANG CAIXIA LIN XIANGMEI WU SHAOQIANG ZHANG YONGNING DENG JUNHUA |
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