CN101967524A - Kit for detecting eastern equine encephalitis virus and west equine encephalitis virus by real-time fluorescence quantitative RT-PCR - Google Patents
Kit for detecting eastern equine encephalitis virus and west equine encephalitis virus by real-time fluorescence quantitative RT-PCR Download PDFInfo
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Abstract
The invention discloses a kit for detecting eastern equine encephalitis virus and west equine encephalitis virus by real-time fluorescence quantitative RT-PCR. The kit provided by the invention comprises a primer pair as shown in the following (1) and (2): (1) EEEV forward primer: TGTGCGTACCTCCTCATCGTT, EEEV reverse primer: GACTGGCGTGAATCTCTGCT; (2) WEEV forward primer: AGGGATACCCCCGAAGGTT, WEEV reverse primer: GTGAATAGCACACGGGTGGTT. The kit can be applied to detecting EEEV and WEEV and has good sensitivity and specificity; in addition, the lowest EEEV virus titer that the kit can detect is 0.2TCID50/ml and the WEEV virus titer that the kit can detect is 1TCID50/ml.
Description
Technical field
The present invention relates to the test kit that real-time fluorescence quantitative RT-PCR in the biological technical field detects Eastern equine encephalitis virus and Western equine encephalitis virus.
Background technology
Eastern equine encephalitis virus (eastern equine encephalitis virus, EEEV) and Western equine encephalitis virus (western equine encephalitis virus, WEEV) be two kinds of viruses that antigenicity is close in the Togaviridae alphavirus, mainly be popular in the area, America.They can infect horse all by killing propagation, the bird and the mankind, and can cause serious encephalitis symptom.The lethality rate that EEEV infects can reach 36%, is one of the strongest encephalitis of lethality rate.WEEV is lower to adult's toxicity, but childhood infection can produce serious encephalitis, mortality ratio about 10%.But more serious neural system sequela is arranged all.Although the Chinese domestic at present report that does not still have eastern equine encephalitis virus and western equine encephalitis virus to infect, but they are once the input propagation, will the serious harm people ' s health, therefore set up the detection method that sensitive can detect EEEV and WEEV simultaneously, significant for the propagation control of this disease.
Because WEEV and EEEV intensive are pathogenic, the research of more existing detection methods at present, the indirect hemagglutination that has that has been applied clinically suppresses (IHI), and the ELISA method detects patient's antiserum(antisera) and RT-PCR method etc.In addition, laboratory study is used also has common multiple RT-PCR and real-time quantitative RT-PCR etc., and common RT-PCR method need be carried out gel electrophoresis, tests consuming timely, and the result can't be quantitative.
Summary of the invention
The purpose of this invention is to provide a kind of test kit that detects equine encephalitis virus.
The test kit of detection equine encephalitis virus provided by the present invention comprises that the primer shown in following (1) and (2) is right:
(1) EEEV upstream primer: TGTGCGTACCTCCTCATCGTT,
EEEV downstream primer: GACTGGCGTGAATCTCTGCT;
(2) WEEV upstream primer: AGGGATACCCCCGAAGGTT;
WEEV downstream primer: GTGAATAGCACACGGGTGGTT.
Described test kit comprises probe shown in following (3) and (4):
(3) probe sequence be 5 '-AGCAGCCTACCTTTCCGACAATGGTTGTC-3 ', connect fluorophor HEX at 5 of sequence A GCAGCCTACCTTTCCGACAATGGTTGTC ' end, 3 ' end connects quencher TAMRA;
(4) probe sequence be 5 '-CTTTCGAATGTCACGTTCCCATGCG-3 ', connect fluorophor FAM at 5 of sequence C TTTCGAATGTCACGTTCCCATGCG ' end, 3 ' end connects quencher TAMRA.
Described equine encephalitis virus is Eastern equine encephalitis virus and/or Western equine encephalitis virus.
Another object of the present invention provides the real-time quantitative PCR reagent that detects equine encephalitis virus.
The real-time quantitative PCR reagent of detection equine encephalitis virus provided by the present invention is made up of, probe shown in following (3) and (4) and real time fluorescent quantitative reverse transcription PCR amplification buffer and enzyme liquid the primer shown in following (1) and (2);
(1) EEEV upstream primer: TGTGCGTACCTCCTCATCGTT,
EEEV downstream primer: GACTGGCGTGAATCTCTGCT;
(2) WEEV upstream primer: AGGGATACCCCCGAAGGTT,
WEEV downstream primer: GTGAATAGCACACGGGTGGTT;
(3) probe sequence be 5 '-AGCAGCCTACCTTTCCGACAATGGTTGTC-3 ', connect fluorophor HEX at 5 of sequence A GCAGCCTACCTTTCCGACAATGGTTGTC ' end, 3 ' end connects quencher TAMRA;
(4) probe sequence be 5 '-CTTTCGAATGTCACGTTCCCATGCG-3 ', connect fluorophor FAM at 5 of sequence C TTTCGAATGTCACGTTCCCATGCG ' end, 3 ' end connects quencher TAMRA;
(1) concentration of each primer of the centering of primer shown in real-time quantitative PCR reagent is 500nmol/L;
(2) concentration of each primer of the centering of primer shown in real-time quantitative PCR reagent is 250nmol/L;
Described real time fluorescent quantitative reverse transcription PCR amplification buffer and enzyme liquid are available from Qiagen company.
The concentration of probe in real-time quantitative PCR reagent is 250nmol/L shown in described (3);
The concentration of probe in real-time quantitative PCR reagent is 150nmol/L shown in described (4).
Described equine encephalitis virus is Eastern equine encephalitis virus and/or Western equine encephalitis virus.
Another purpose of the present invention provides the primer shown in following (1) and (2) to the application in the test kit that detects equine encephalitis virus in preparation:
(1) EEEV upstream primer: TGTGCGTACCTCCTCATCGTT,
EEEV downstream primer: GACTGGCGTGAATCTCTGCT;
(2) WEEV upstream primer: AGGGATACCCCCGAAGGTT,
WEEV downstream primer: GTGAATAGCACACGGGTGGTT.
Described equine encephalitis virus is Eastern equine encephalitis virus and/or Western equine encephalitis virus.
Another purpose of the present invention provides the primer shown in following (1) and (2) to detecting application in the test kit of equine encephalitis virus with the probe shown in following (3) and (4) in preparation:
(1) EEEV upstream primer: TGTGCGTACCTCCTCATCGTT,
EEEV downstream primer: GACTGGCGTGAATCTCTGCT;
(2) WEEV upstream primer: AGGGATACCCCCGAAGGTT,
WEEV downstream primer: GTGAATAGCACACGGGTGGTT;
(3) probe sequence be 5 '-AGCAGCCTACCTTTCCGACAATGGTTGTC-3 ', connect fluorophor HEX at 5 of sequence A GCAGCCTACCTTTCCGACAATGGTTGTC ' end, 3 ' end connects quencher TAMRA;
(4) probe sequence be 5 '-CTTTCGAATGTCACGTTCCCATGCG-3 ', connect fluorophor FAM at 5 of sequence C TTTCGAATGTCACGTTCCCATGCG ' end, 3 ' end connects quencher TAMRA.
Described equine encephalitis virus is Eastern equine encephalitis virus and/or Western equine encephalitis virus.
This research and design the test kit that detects of the real-time quantitative PCR of WEEV and EEEV, the RNA that utilizes vitro culture virus is as template, this test kit can be used for detecting EEEV and WEEV, has susceptibility and specificity preferably, and the minimum virus titer that can detect EEEV is 0.2TCID
50/ ml, can detect the WEEV virus titer is 1TCID
50/ ml.
Description of drawings
Fig. 1 is the typical curve to the susceptibility detection of WEEV.
Fig. 2 is the typical curve to the susceptibility detection of EEEV.
Fig. 3 is the susceptibility detected result to WEEV.
Fig. 4 is the susceptibility detected result to EEEV.
Fig. 5 is the detected result of EEEV simulated samples.
Fig. 6 is the detection of WEEV simulated samples.
The real-time quantitative RT-PCR amplification of the negative contrast of Fig. 7.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
One, the design of primer and probe
Utilize SDS2.0 software to design.The real-time quantitative of Western equine encephalitis virus (WEEV) detects primer and probe is selected from virus structural protein E3 zone, the real-time quantitative of Eastern equine encephalitis virus (EEEV) detects primer and probe is selected viral Nonstructural Protein NSP3 zone, by the sequence alignment analysis, with other viral sequence homologies less than 50%.Detection primer and the probe sequence of WEEV and EEEV see Table 1.
Detection primer and the probe sequence of table 1WEEV and EEEV
Two, the foundation of reaction system and optimization
1, the preparation of sample
With Western equine encephalitis virus WEEV strain McMillan (McMillan virus strain, GenBank accession number: GQ287640.1, the public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL, the non-patent literature of putting down in writing this material is: Nagata LP, Hu WG, Parker M, Chan D, Rayner GA etal., Infectivity variation and genetic diversity among stains of western equine encephalitisvirus, J.Gen.Virol.2006,87,2353-2361) with Eastern equine encephalitis virus EEEV strain SSP.NorthAmerican Variant (SSP.North American Variant virus strain, GenBank accession number: X63135, the public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL, the non-patent literature of putting down in writing this material is: Volchkov.V E, Volchkova.V.A.and Netesov SV, Complete nucleotidesequence of the Eastern equine encephalitis virus genome, Mol.Gen.Mikrobiol.Virusol.1991,5,8-15) inoculate the BHK-21 cell respectively (available from ATCC, CAT.CCL 10), obvious pathology appears in cell after 2-3 days, the collecting cell culture supernatant, and the virus titer in the mensuration culture supernatant, EEEV:2 * 10
7TCID50/ml, WEEV:10
7TCwID50/ml; TCID (tissue culture infective dose).
(public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL to cultivate datum hole Kenya virus simultaneously, the non-patent literature of putting down in writing this material is: Shi Huafang, Zhang Hailin, from stepping on cloud, Li Zhaoxiang etc., Yunnan is separated to datum hole Kenya virus first in patient's body, China Amphixenosis magazine, first phase nineteen ninety), (public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL encephalitis b virus Japanese B encephalitis virus (JEV), the non-patent literature of putting down in writing this material is: Yang Yinhui, Zhu Xiaoguang, Zhang Yongguo, Kang Xiao equality.Utilize examination and the evaluation of biochip technology to a strain unknown virus.China's laboratory medicine magazine, 2007,30 (12): 1360-1363), (public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL fores encephalitis virus, the non-patent literature of putting down in writing this material is: Hu Yuyang, Yang Yinhui, Liu Hong, Kang Xiaoping, Zhu Xiaoguang, take charge of bright silver, Zhu Qingyu, the foundation of fores encephalitis virus (TBEV) real-time quantitative TaqMan PCR detection method, PLA's medical journal 2006,31,745-748), (public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL dengue virus 2 types, the non-patent literature of putting down in writing this material is: Murthy HM, Clum S, Padmanabhan R.Dengue virus NS3 serine protease.Crystal structure and insightsinto interaction of the active site with substrates by molecular modeling and structuralanalysis ofmutational effects.J Biol Chem, 1999,274 (9): 5573-5580.), (public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL St. Louis encephalitis virus, the non-patent literature of putting down in writing this material is: Phillpott RJ, Venugopal K, Brooks T.Immunization with DNA polynucleotidesprotects mice against lethal challenge with St.Louis encephalitis virus.Arch.Virol.1996,141:743-749), (public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL the sindbis virus, the non-patent literature of putting down in writing this material is: Xiong Hongyan, Liu Yujing etc., short wave ultraviolet and cross-linking starch iodine are to the research of sindbis virus's deactivation in the blood plasma, China's Disinfection magazine, the fourth phase in 1998) as negative control.Extract the RNA that WEEV (McMillan virus strain .) and EEEV (SSP.North American Variant virus strain) cultivate virus, the template of reacting as real-time quantitative RT-PCR behind 10 times of serial dilutions.Extract datum hole Kenya virus, encephalitis b virus, fores encephalitis virus, dengue virus 2 types, St. Louis encephalitis virus, sindbis virus RNA negative control simultaneously as this reaction system.Virus culture and RNA extract and all carry out in BSL-3 level biocontainment laboratory.
2, virus infected cell carries out the virulence titration
The culture supernatant of WEEV and EEEV is carried out 10 times of serial dilution (10-10 respectively
8, totally 8 concentration, 10 times of serial dilutions), the viral supernatant of each weaker concn infects the BHK-21 cell (available from ATCC, CAT.CCL 10) that is incubated in the 96 porocyte plates respectively, each cell hole inoculation 100ul, each concentration repeats 4 holes.Establish the normal cell contrast of uninfecting virus simultaneously.Observe the cell growing state under the different concns every day after the inoculation, observed continuously 7.If virus successfully infects culturing cell and increases clay, then can make cytopathy and dead.Can cause in 4 multiple holes the virus quantity that occurs tangible pathology death in 2 cell holes and be defined as 1TCID
50, determine virus titer in the virus culture supernatant stoste by the cytopathy situation in each micropore of observing.
The result shows that in the above-mentioned virus culture supernatant stoste: the virus titer of EEEV is: 2 * 10
7TCID
50/ ml, the virus titer of WEEV is: 10
7TCID
50/ ml.
3, the optimization of reaction system
In order to improve the sensitivity of detection, primer and the concentration and probe concentration in the reaction system optimized in this research, determined The optimum reaction conditions.Primer concentration 125nmol/L, the 250nmol/L, 375nmol/L, 500nmol/L, 625nmol/L, the 750nmol/L that get eastern equine encephalitis virus EEEV at first respectively detect, and the concentration of probe and other reacted constituents remains unchanged.RNA template with serial dilution detects, choosing the minimum primer concentration of the detected rna content limit is suitable detectable level, and then the detectable level of probe is optimized, concentration and probe concentration 100nmol/L, the 150nmol/L, 200nmol/L, 250nmol/L, the 300nmol/L that choose eastern equine encephalitis virus detect, and choosing the minimum concentration and probe concentration of the detected rna content limit is suitable detectable level.Taking identical method that primer and the concentration and probe concentration of WEEV are groped then, be suitable detectable level with minimum primer and the concentration and probe concentration of the detected rna content limit of energy equally.
4, the foundation of peak optimization reaction system
Contain real time fluorescent quantitative reverse transcription PCR amplification reaction solution, upstream primer, downstream primer, fluorescent probe and template in the reaction system, wherein, upstream primer sequence and downstream primer sequence are respectively:
EEEV upstream primer: TGTGCGTACCTCCTCATCGTT (sequence 1 in the sequence table) and
EEEV downstream primer: GACTGGCGTGAATCTCTGCT (sequence 2 in the sequence table);
WEEV upstream primer: AGGGATACCCCCGAAGGTT (sequence 3 in the sequence table) and
WEEV downstream primer: GTGAATAGCACACGGGTGGTT (sequence 4 in the sequence table);
The fluorescent probe sequence is:
EEEV fluorescent probe: HEX-AGCAGCCTACCTTTCCGACAATGGTTGTC-TAMRA (probe sequence is a sequence 5 in the sequence table),
WEEV fluorescent probe: FAM-CTTTCGAATGTCACGTTCCCATGCG-TAMRA (probe sequence is a sequence 6 in the sequence table).
Described real time fluorescent quantitative reverse transcription PCR amplification reaction solution is by real time fluorescent quantitative reverse transcription PCR amplification buffer (Qiagen company product, Quanti-tect Probe RT-PCR kit, cat.204443) and enzyme be mixed solution (Qiagen company product, Quanti-tect Probe RT-PCR kit cat.204443) forms.
The reaction system of being set up is as follows:
Real time fluorescent quantitative reverse transcription PCR amplification reaction solution damping fluid 10 μ L
Enzyme is mixed solution 0.2 μ L
EEEV upstream primer 10mM 1 μ L (final concentration 500nmol/L)
EEEV downstream primer 10mM 1 μ L (final concentration 500nmol/L)
WEEV upstream primer 10mM 0.5 μ L (final concentration 250nmol/L)
WEEV downstream primer 10mM 0.5 μ L (final concentration 250nmol/L)
EEEV fluorescent probe 10mM 0.5 μ L (final concentration 250nmol/L)
WEEV fluorescent probe 10mM 0.3 μ L (final concentration 150nmol/L)
Moisturizing to 20 μ L
Three, real-time fluorescence quantitative RT-PCR detects susceptibility and the specificity of WEEV and EEEV
Extract in the experiment two RNA of WEEV and EEEV virus in the virus culture supernatant stoste respectively, the pairing virus titer of the RNA of the WEEV that is extracted is 10
7TCID
50/ ml, 10 times of serial dilutions, totally 8 extent of dilution, pairing virus titer is 10
6TCID
50/ ml-10
-1TCID
50/ ml, the virus titer value of each extent of dilution correspondence specifically sees Table 2, respectively the template of reacting as real-time quantitative PCR; The pairing virus titer of the RNA of EEEV is 2 * 10
7TCID
50/ ml, 10 times of serial dilutions, totally 8 extent of dilution, pairing virus titer is 2 * 10
6TCID
50/ ml-2 * 10
-1TCID
50/ ml, the virus titer value of each extent of dilution correspondence specifically sees Table 2, respectively the template of reacting as real-time quantitative RT-PCR.Extract datum hole Kenya virus, encephalitis b virus, fores encephalitis virus, dengue virus, St. Louis encephalitis virus, sindbis virus RNA simultaneously as the negative control of setting up this reaction system.Virus culture and RNA extract and all carry out in BSL-3 level biocontainment laboratory.
Respectively with the RNA of the WEEV of above-mentioned serial dilution and EEEV as template, carry out the real-time quantitative RT-PCR reaction, determine the susceptibility of this detection method, get 6 negative controls simultaneously: the RNA of datum hole Kenya virus, encephalitis b virus, fores encephalitis virus, dengue virus, St. Louis encephalitis virus and sindbis virus's encephalitis correlated virus verifies the specificity of this detection method as template.
The instrument that uses is Roche LightCycler2.0.Reaction system is as shown in step 2.
Amplification condition is: 50 ℃ of reverse transcription 30min, 95 ℃ are heated 10min with the deactivation ThermoScript II then, and then carry out amplified reaction: 95 ℃ of 15s, 55 ℃ of 1min, totally 45 circulations.In the reaction process, carry out fluoroscopic examination in 55 ℃ of annealing processes, the 530nm wavelength detects the amplification of WEEV, and the 560nm wavelength detects the amplification of EEEV.
Detected result is as follows:
(1) negative control result: the detected result of negative control is seen Fig. 7, and A figure is the amplification under the 560nm wavelength among Fig. 7, has reacted the specific amplification of EEEV primer and probe; B figure is the amplification under the 530nm wavelength, has reacted the specific amplification of WEEV primer and probe.Every shown fluorescence intensity of amplification curve all fails to occur along with the increase of cycle number exponential growth among A figure and the B figure, amplification show these 6 negative control templates all fail the amplification, show that the primer of this amplification system and design and probe have specificity preferably.
(2) WEEV sensitivity result: the WEEV test result of samples under the different extension rates is shown in Fig. 3 and table 2.Amplification with CP value＜38 is judged as the positive.The cycle number that the CP value is experienced when arriving the fluorescence thresholding of setting for the fluorescent signal in each reaction tubes.Fluorescence thresholding is 10 times of PCR 3-15 circulation fluorescent signal standard deviation.
With each dilution logarithmic value is X-coordinate, is ordinate zou with its corresponding CP value, the drawing standard curve, and the result is as shown in Figure 1.
In double reaction system, can detected WEEV minimum virus titer be 1TCID
50/ ml, the signal that each reaction system occurred is the signal of single passage, illustrates that this detection architecture no cross reaction occurs.
3 repetitions are established in experiment, and the result is all consistent.
(3) EEEV sensitivity result:
EEEV test result of samples under the different extension rates is shown in Fig. 4 and table 2.Amplification with CP value＜38 is judged as the positive.
With each dilution logarithmic value is X-coordinate, is ordinate zou with its corresponding CP value, the drawing standard curve, and the result is as shown in Figure 2.
In double reaction system, can detected EEEV minimum virus titer be 0.2TCID
50/ ml, the signal that each reaction system occurred is the signal of single passage, illustrates that this detection architecture no cross reaction occurs.
3 repetitions are established in experiment, and the result is all consistent.
Table 2 real-time quantitative RT-PCR detects
| Virus | Virus titer | |
| WEEV | ||
| 10 6TCID 50/ml | 15.25 | |
| 10 5TCID 50/ml | 18.29 |
| 10 4TCID 50/ml | 22.02 | |
| 10 3TCID 50/ml | 25.49 | |
| 10 2TCID 50/ml | 29.28 | |
| 10 1TCID 50/ml | 32.79 | |
| 10 0TCID 50/ml | 36.03 | |
| 10
-1TCID
50/ | ||
| EEEV | ||
| 2×10 6TCID 50/ml | 11.37 | |
| 2×10 5TCID 50/ml | 16.25 | |
| 2×10 4TCID 50/ml | 18.54 | |
| 2×10 3TCID 50/ml | 22.03 | |
| 2×10 2TCID 50/ml | 27.35 | |
| 2×10 1TCID 50/ml | 30.18 | |
| 2×10 0TCID 50/ml | 34.79 | |
| 2×10 -1TCID 50/ml | 37.59 |
Utilize the reaction system of this test kit, can detected minimum virus titer be: the minimum virus titer of EEEV is 0.2TCID
50/ ml, the minimum virus titer of WEEV is 1TCID
50/ ml, the signal that each reaction system occurred is the signal of single passage, illustrates that this detection architecture no cross reaction occurs.
Four, real-time fluorescence quantitative RT-PCR detect simultaneously WEEV and EEEV susceptibility and specificity
After testing the viral RNA mixing of three WEEV that extract and EEEV, carry out 10 times of serial dilutions, carry out real-time quantitative RT-PCR, amplification system and reaction conditions are identical with experiment three.
Under two kinds of simultaneous situations of viral nucleic acid, the minimum virus titer that can detect is that the minimum virus titer of EEEV is 2TCID
50/ ml, the minimum virus titer of WEEV is 1TCID
50/ ml.
Five, the detection of simulated samples
With the EEEV that extracts and WEEV viral RNA solution respectively with an amount of murine brain RNA solution mix back (viral RNA: mouse cerebral RNA volume ratio is 1: 10) (in the viral solution concentration of two kinds of viral RNAs and with murine brain RNA solution in the concentration of RNA identical), get the 2ul mixture as template, carry out real-time quantitative RT-PCR and detect, amplification system and reaction conditions are identical with experiment three.Detected result is distinguished as shown in Figure 5 and Figure 6, and as seen from the figure, amplification curve is exponential growth, and detected result is positive.(amplification with CP value＜38 is judged as the positive).The real-time quantitative RT-PCR detection method that is used for EEEV and WEEV detection that this institute sets up is described, can be used for pathogen detection and epidemiological surveillance that EEEV and WEEV infect.
Claims (10)
1. detect the test kit of equine encephalitis virus, comprise that the primer shown in following (1) and (2) is right:
(1) EEEV upstream primer: TGTGCGTACCTCCTCATCGTT,
EEEV downstream primer: GACTGGCGTGAATCTCTGCT;
(2) WEEV upstream primer: AGGGATACCCCCGAAGGTT,
WEEV downstream primer: GTGAATAGCACACGGGTGGTT.
2. test kit according to claim 1 is characterized in that: described test kit comprises the probe shown in following (3) and (4):
(3) probe sequence be 5 '-AGCAGCCTACCTTTCCGACAATGGTTGTC-3 ', connect fluorophor HEX at 5 of sequence A GCAGCCTACCTTTCCGACAATGGTTGTC ' end, 3 ' end connects quencher TAMRA;
(4) probe sequence be 5 '-CTTTCGAATGTCACGTTCCCATGCG-3 ', connect fluorophor FAM at 5 of sequence C TTTCGAATGTCACGTTCCCATGCG ' end, 3 ' end connects quencher TAMRA.
3. test kit according to claim 1 and 2 is characterized in that: described equine encephalitis virus is Eastern equine encephalitis virus and/or Western equine encephalitis virus.
4. detect the real-time quantitative PCR reagent of equine encephalitis virus,, probe shown in following (3) and (4) and real time fluorescent quantitative reverse transcription PCR amplification buffer and enzyme liquid are formed by the primer shown in following (1) and (2);
(1) EEEV upstream primer: TGTGCGTACCTCCTCATCGTT,
EEEV downstream primer: GACTGGCGTGAATCTCTGCT;
(2) WEEV upstream primer: AGGGATACCCCCGAAGGTT,
WEEV downstream primer: GTGAATAGCACACGGGTGGTT;
(3) probe sequence be 5 '-AGCAGCCTACCTTTCCGACAATGGTTGTC-3 ', connect fluorophor HEX at 5 of sequence A GCAGCCTACCTTTCCGACAATGGTTGTC ' end, 3 ' end connects quencher TAMRA;
(4) probe sequence be 5 '-CTTTCGAATGTCACGTTCCCATGCG-3 ', connect fluorophor FAM at 5 of sequence C TTTCGAATGTCACGTTCCCATGCG ' end, 3 ' end connects quencher TAMRA;
(1) concentration of each primer of the centering of primer shown in real-time quantitative PCR reagent is 500nmol/L;
(2) concentration of each primer of the centering of primer shown in real-time quantitative PCR reagent is 250nmol/L.
5. real-time quantitative PCR reagent according to claim 4 is characterized in that:
The concentration of probe in real-time quantitative PCR reagent is 250nmol/L shown in described (3);
The concentration of probe in real-time quantitative PCR reagent is 150nmol/L shown in described (4).
6. according to claim 4 or 5 described real-time quantitative PCR reagent, it is characterized in that: described equine encephalitis virus is Eastern equine encephalitis virus and/or Western equine encephalitis virus.
7. the primer shown in following (1) and (2) is to the application in the test kit that detects equine encephalitis virus in preparation:
(1) EEEV upstream primer: TGTGCGTACCTCCTCATCGTT,
EEEV downstream primer: GACTGGCGTGAATCTCTGCT;
(2) WEEV upstream primer: AGGGATACCCCCGAAGGTT,
WEEV downstream primer: GTGAATAGCACACGGGTGGTT.
8. application according to claim 7 is characterized in that: described equine encephalitis virus is Eastern equine encephalitis virus and/or Western equine encephalitis virus.
9. the primer shown in following (1) and (2) is to detecting application in the test kit of equine encephalitis virus with the probe shown in following (3) and (4) in preparation:
(1) EEEV upstream primer: TGTGCGTACCTCCTCATCGTT,
EEEV downstream primer: GACTGGCGTGAATCTCTGCT;
(2) WEEV upstream primer: AGGGATACCCCCGAAGGTT,
WEEV downstream primer: GTGAATAGCACACGGGTGGTT;
(3) probe sequence be 5 '-AGCAGCCTACCTTTCCGACAATGGTTGTC-3 ', connect fluorophor HEX at 5 of sequence A GCAGCCTACCTTTCCGACAATGGTTGTC ' end, 3 ' end connects quencher TAMRA;
(4) probe sequence be 5 '-CTTTCGAATGTCACGTTCCCATGCG-3 ', connect fluorophor FAM at 5 of sequence C TTTCGAATGTCACGTTCCCATGCG ' end, 3 ' end connects quencher TAMRA.
10. application according to claim 9 is characterized in that: described equine encephalitis virus is Eastern equine encephalitis virus and/or Western equine encephalitis virus.
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| CN102796827A (en) * | 2011-05-24 | 2012-11-28 | 上海透景生命科技有限公司 | Method and kit for detecting multiple encephalitis related viruses |
| CN102808044A (en) * | 2012-09-12 | 2012-12-05 | 北京出入境检验检疫局检验检疫技术中心 | Nucleotide sequence and kit for detecting western equine encephalomyelitis virus |
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| CN102796827A (en) * | 2011-05-24 | 2012-11-28 | 上海透景生命科技有限公司 | Method and kit for detecting multiple encephalitis related viruses |
| CN102796827B (en) * | 2011-05-24 | 2016-11-09 | 上海透景生命科技股份有限公司 | A kind of method detecting multiple encephalitis correlated virus and test kit |
| CN102808044A (en) * | 2012-09-12 | 2012-12-05 | 北京出入境检验检疫局检验检疫技术中心 | Nucleotide sequence and kit for detecting western equine encephalomyelitis virus |
| CN102808043A (en) * | 2012-09-12 | 2012-12-05 | 北京出入境检验检疫局检验检疫技术中心 | Nucleotide sequence and kit for detecting eastern equine encephalomyelitis virus |
| CN102808043B (en) * | 2012-09-12 | 2013-12-11 | 北京出入境检验检疫局检验检疫技术中心 | Nucleotide sequence and kit for detecting eastern equine encephalomyelitis virus |
| CN102808044B (en) * | 2012-09-12 | 2013-12-11 | 北京出入境检验检疫局检验检疫技术中心 | Nucleotide sequence and kit for detecting western equine encephalomyelitis virus |
| CN104928401A (en) * | 2015-06-09 | 2015-09-23 | 山东出入境检验检疫局检验检疫技术中心 | Nucleotide sequence and kit for detecting eastern equine encephalomyelitis viruses |
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