CN104328216A - Kit for rapid typing identification detection on Ebola viruses - Google Patents
Kit for rapid typing identification detection on Ebola viruses Download PDFInfo
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- CN104328216A CN104328216A CN201410530911.5A CN201410530911A CN104328216A CN 104328216 A CN104328216 A CN 104328216A CN 201410530911 A CN201410530911 A CN 201410530911A CN 104328216 A CN104328216 A CN 104328216A
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Abstract
The invention relates to a kit for rapid typing identification detection on Ebola viruses, and especially relates to utilization of a multiplex real-time fluorescent polymerase chain reaction technology to identify bundibugyo Ebola virus (BDBV), Zaire Ebola virus (EBOV), Sudan Ebola virus (SUDV) and Tai forest Ebola virus (TAFV). The kit mainly includes an RT-PCR reaction liquid, a primer probe mixed liquor and an RT-PCR reaction enzyme system, DEPC H2O, and a packing box for separation and concentration packing of reagent bottles or tubes. With the method for respective detection through various fluorescence channels and application of a one-step real-time fluorescence PCR reaction mode, the kit can accurately and quickly detect and identify four types of the Ebola viruses, and can be widely applied in Ebola virus disease early identification diagnosis, epidemic situation control, detection quarantine and multiple fields.
Description
Technical field
The present invention relates to a kind of test kit of Ebola virus type identification and detection, particularly relate to a kind of test kit utilizing multiple real time fluorescence polymerase chain reaction technology one tube reaction can identify Ebola virus four kinds of pathogenic types.This test kit mainly comprises RT-PCR reaction solution, primed probe mixed solution, RT-PCR reaction enzymes system, DEPC H
2o, and separate and concentrate the packing box packing these reagent bottles or pipe.Because namely this test kit can accurately distinguish the Ebola virus of 4 kinds of pathogenic types: Zaire type Ebola virus, Ben Dibujiao type Ebola virus, the Sudan type Ebola virus, Ta Yisen crop type Ebola virus, multiple fields such as the clinical early diagnosis of ebola disease viral disease, severe cases early warning, Check and Examination of Port quarantine, epidemic prevention and control, medium competition and scientific research can be widely used in.
Background technology
Ebola disease viral disease (EVD; Be called ebola disease toxicity hemorrhagic fever in the past) be serious, often fatal human diseases.Cd (front Zaire) Ebola's river valley is betided first in 1976, because it causes systemic bleeding, therefore called after ebola hemorrhagic fever.The World Health Organization (WHO) has been classified as one of the most serious transmissible disease of mankind's harm, and specifies to carry out in Biosafety 4 grades of laboratories about the research of its live virus.The pathogenic agent of EVD is Ebola virus, propagates mainly through body fluid, blood, secretory product.Ebola virus by interference the infected body coagulation homeostasis, cause vascular wall damage or function impaired, thrombocyte can not blood coagulation, and in 2-21 days, occur hemorrhagic shock or organ failure, mortality ratio is very high.
Ebola virus is under the jurisdiction of inovirus section (Filoviridae), and inovirus belongs to (Filovirus), the sub-thread minus-stranded rna virus that right and wrong are segmented.Ebola virus belongs to and comprises five kinds of different types: her forest Ebola virus (TAFV) of Ben Dibujiao Ebola virus (BDBV), Zaire Ebola virus (EBOV), Reston Ebola virus (RESTV), the Sudan Ebola virus (SUDV) and tower.Except Reston Ebola virus to people in except stealthy infection, all the other 4 hypotypes have lethality to people.Ben Dibujiao Ebola virus, Zaire Ebola virus and the Sudan Ebola virus are relevant to the large-scale epidemic situation of African ebola disease viral disease, and Reston Ebola virus and Ta Yi forest Ebola virus then have no truck with.
Ebola disease viral disease patient's sample has extreme organisms harm risk; Only just can detect under the biological protection condition of highest level.The test in laboratory acquisition that Ebola virus infects by some types is clarified a diagnosis, as: antibody capture enzyme linked immunosorbent assay (ELISA), antigen-detection test, serum neutralization test, conventional RT-PCR detection, Real time RT-PCR detection, submicroscopy and Viral isolation.But serological test, virus culture have that sensitivity is low, immunological cross-reaction and the shortcoming such as the cycle is long; And conventional RT-PCR easily produces pollution, cause false-positive generation.Multiple real time fluorescence round pcr is modern technique round pcr and multicolor fluorescence label probe combined, the method has quick, special, sensitive, level of automation high, in conjunction with pollution prevention technology process, be particularly suitable for demand that is extensive, quick diagnosis.This technology adopts multicolor fluorescence to mark many Species specific probes, coordinate multiple PCR technique, can increase to multiple virus in same PCR reaction tubes simultaneously, the growth signal of assorted fluorescence becomes geometric ratio relation with the increment of corresponding PCR primer, and the fluorescence detector that is automated is collected, and finally reaches the object detecting pathogen type by analysis of fluorescence growth curve.Due to, it is short that Fluorescence PCR assay has amplified fragments, primer, probe has the dual advantage such as special, therefore, multiple real time fluorescence round pcr is applied to the rapid detection of Ebola virus somatotype, can save time, reduce quantity of drawing materials, improve detection and identification efficiency, suspected case or suspicious medium whether Infective Ebola virus and simultaneously make a definite diagnosis infected type can be judged at short notice, be convenient to clinical prevention and control and the remedy measures of taking necessity in time, prevent these viruses from continuing to propagate, and improve diagnosis and treatment efficiency, for quick diagnosis, effective monitoring and immunotherapy targeted autoantibody provide support and foundation.
The present invention adopts the multiple real time fluorescence PCR method in real-time fluorescence PCR technology, four kinds of pathogenic types (Ben Dibujiao type, Zaire's type, the Sudan's type and the Ta Yisen crop type) nucleic acid of Ebola virus is increased, according to the amplification situation of four look fluorescence of mark, differentiate the pathogenic type of Ebola virus.Test kit of the present invention can apply to pattern detection and the laboratory diagnosis of ebola hemorrhagic fever or the doubtful ebola hemorrhagic fever patient caused by Ebola virus widely.
Summary of the invention
The object of the present invention is to provide a kind of identification reagent box utilizing multiple real time fluorescence polymerase chain reaction technology to carry out four kinds of pathogenic Ebola virus types, utilize this test kit accurately can distinguish Ben Dibujiao Ebola virus, Zaire Ebola virus, the Sudan Ebola virus and Ta Yi forest Ebola virus.
On the basis that the nucleotide sequence of each pathogenic type of known Ebola viruses all on GENBANK is compared, find the specific conservative district of each type nucleotide sequence respectively, and for conserved regions design target nucleotide primer and probe.These primed probe containing hot resistant DNA polymerase, the RT-PCR reaction enzymes system of reversed transcriptive enzyme, high quality deoxyribonucleoside triphosphate (dNTPs) and containing Mg
2+in RT-PCR reaction solution Deng composition, realized the cyclic amplification of external nucleic acid by fluorescent PCR instrument.
Test kit involved in the present invention mainly comprises: 1) RT-PCR reaction solution, primed probe mixed solution, RT-PCR reaction enzymes system, DEPC H
2o, negative quality control product, positive quality control product and 2) separate and concentrate the packing box packing these reagent bottles or pipe.
A preferred embodiment of the present invention is that primed probe mixed solution is by four kinds of specific forward and reverse primers of pathogenic Ebola virus, specific probe composition, is characterized in that the sequence of the specific forward of Ben Dibujiao Ebola virus and reverse primer is 5 '-TTGGAAAGTAATGCGGTAAAATA-3 ' (SEQ ID NO:1) and 5 '-GCAGCCAATGTTCTCTTGATGT-3 ' (SEQ ID NO:2) respectively; The sequence of the specific probe of Ben Dibujiao Ebola virus is 5 '-CTACTCCCTGCTGCCTCGAGTGGAAA-3 ' (SEQ ID NO:3), and the two ends of probe are combined with fluorescence generation group FAM and fluorescent quenching group B HQ1 respectively; The sequence of the specific forward of Zaire Ebola virus and reverse primer is 5 '-GATGCCAACGAYGCTGTGAT-3 ' (SEQ ID NO:4) and 5 '-TGCAAGAGGATGGAGACGAA-3 ' (SEQ ID NO:5) respectively, the sequence of the specific probe of Zaire Ebola virus is 5 '-TCAGTGGCTCAAGCTCGTTTTTCAGGT-3 ' (SEQID NO:6), and the two ends of probe are combined with fluorescence generation group HEX and fluorescent quenching group B HQ1 respectively; The sequence of the specific forward of the Sudan Ebola virus and reverse primer is 5 '-TGGTGTTGTTGACCCGTATG--3 ' (SEQID NO:7) and 5 '-CGTCGTCCAAATTGAAGAGAT-3 ' (SEQID NO:8) respectively, the sequence of the specific probe of the Sudan Ebola virus is 5 '-CCTGACTACGAGGATTCGGCTGAAG-3 ' (SEQ ID NO:9), and the two ends of probe are combined with fluorescence generation group Texas Red and fluorescent quenching group B HQ2 respectively; The sequence of the tower specific forward of her forest Ebola virus and reverse primer is 5 '-CAACAACAAACACAGTCTTACAGG-3 ' (SEQID NO:10) and 5 '-TTATCCCTGGCGTATTCTTGA-3 ' (SEQID NO:11) respectively, the sequence of the specific probe of her forest Ebola virus of tower is 5 '-CTCCACACAAAACAATGACAATCCTGC-3 ' (SEQ ID NO:12), and the two ends of probe are combined with fluorescence generation group Cy5 and fluorescent quenching group B HQ2 respectively.
Another preferred embodiment of the present invention is that in primed probe mixed solution, primer concentration is 0.2mmol/L, and concentration and probe concentration is 0.1mmol/L.
Another preferred embodiment of the present invention is that RT-PCR reaction solution is by Tris-HCl (50mmol/L, pH8.0), MgCl
2(8mmol/L), KCl (250mmol/L) composition.
Another preferred embodiment of the present invention is that RT-PCR reaction enzymes system is made up of hot start Taq polymerase, reversed transcriptive enzyme, dNTPs.Reversed transcriptive enzyme can select the conventional reversed transcriptive enzymes such as mMLV.Hot start Taq polymerase, reversed transcriptive enzyme, dNTPs all can adopt commercially available prod, and as the product of Qiagen company, wherein in every person-portion RT-PCR reaction enzymes system, the consumption of hot start Taq polymerase is 8U, and reversed transcriptive enzyme consumption is 3U, dNTPs consumption is 10mmol.
Another preferred embodiment of the present invention is the condition of pcr amplification: 50 DEG C 15 minutes, 95 DEG C 15 minutes; 95 DEG C 10 seconds, 58 DEG C 35 seconds, 40 circulations (collecting fluorescent signal when 58 DEG C).
A preferred embodiment of the present invention is that test kit provides quality control product, is respectively negative quality control product and positive quality control product.Negative quality control product is human normal plasma, and positive quality control product is the full-length RNA containing four kinds of pathogenic Ebola virus goal gene of synthetic, selects concentration to be 4.0 × 10
5the RNA of copies/mL, and mix with equal-volume ratio and be prepared into positive quality control product.The detection that Samples detection to be checked can carry out two special quality control product is simultaneously carried out in test kit, only when positive quality control product detects, FAM, HEX, Texas Red and CY5 channel fluorescence signal is all positive, negative quality control product detects four channel fluorescence signals when being all negative, and the detected result of sample to be checked just effectively.
The test kit of the present invention sample to be checked that increases is completed automatically by commercially available quantitative real time PCR Instrument, simple to operate, consuming time few, and decreases the generation of pollution to greatest extent.Detected result can be used for multiple area researches such as Ebola virus somatotype, adjuvant clinical diagnosis and routine monitoring.
The present invention compared with prior art advantage is: 1. detect Ebola virus four kinds of type nucleic acid amplification levels, the state of Ebola virus infection in sample can be reflected and be confirmed to be which kind of type Ebola virus infection, can be used for the monitoring of ebola disease viral disease and prevention and control and clinical diagnosis, contribute to the early diagnosis and therapy of ebola disease viral disease; 2. respectively for ebola disease viral disease 4 kinds pathogenic type viral nucleic acid specific sequence design specialized primer, probe, ensure specificity and the accuracy of detection, also there is higher flux, easy and simple to handle, the advantage that relatively reduces costs, be more suitable for most of patients and detect; 3. a sample one-time detection can differentiate the infection conditions of four kinds of types, and testing process only needs 1.5h, compares a sample and carries out 4 detections or only carry out Ebola virus Universal fluorescence method detecting, greatly reduce workload, improve detection efficiency; Relative to existing nucleic acid hybridization detection technique, detect required time and reduce by more than half; 4. reagent adopts the RNA that synthesizes of prosthesis as positive quality control product outward, namely can carry out Quality Control to reagent, have more validity and science than plasmid as Quality Control; 5. reagent adopts the large packaging setting of RT-PCR reaction solution, primed probe mixed solution, RT-PCR reaction enzymes system, is applicable to multiple fluorescence detection device, has suitability feature more widely.
Accompanying drawing explanation
Fig. 1 shows the reaction conditions of pcr amplification.
The amplification curve of the negative quality control product of Fig. 2 visualizingre agent box.There is no S type amplification curve in figure, illustrate that FAM, HEX, Texas Red and CY5 channel fluorescence signal is all negative.
The amplification curve of Fig. 3 visualizingre agent box positive quality control product.In figure, the amplification curve of FAM passage is S type, illustrates that the fluorescent signal of this passage is positive.
The amplification curve of Fig. 4 visualizingre agent box positive quality control product.In figure, the amplification curve of HEX passage is S type, illustrates that the fluorescent signal of this passage is positive.
The amplification curve of Fig. 5 visualizingre agent box positive quality control product.In figure, the amplification curve of Texas Red passage is S type, illustrates that the fluorescent signal of this passage is positive.
The amplification curve of Fig. 6 visualizingre agent box positive quality control product.In figure, the amplification curve of CY5 passage is S type, illustrates that the fluorescent signal of this passage is positive.
Fig. 7 shows the amplification curve of Ben Dibujiao Ebola virus standard substance.In figure, the amplification curve of FAM passage is S type, illustrates that test kit can detect Ben Dibujiao Ebola virus RNA.
Fig. 8 shows the amplification curve of Zaire Ebola virus standard substance.In figure, the amplification curve of HEX passage is S type, illustrates that test kit can detect Zaire Ebola virus RNA.
Fig. 9 shows the amplification curve of the Sudan's Ebola virus standard substance.In figure, Texas Red passage amplification curve is S type, illustrates that test kit can detect the Sudan Ebola virus RNA.
Figure 10 shows the amplification curve of her forest Ebola virus standard substance of tower.In figure, Cy5 passage amplification curve is S type, illustrates that test kit can detect her forest Ebola virus RNA of tower.
Figure 11 shows the amplification curve of dengue virus I type, dengue virus II type, dengue virus type III, dengue virus IV type, encephalitis b virus, heating companion thrombocytopenic syndromes bunyavirus, hantaan virus, Seroul virus sample.Without amplification curve in figure, illustrate in this sample not containing pathogenic Ebola virus nucleic acid.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.
Embodiment 1 Ebola virus somatotype differentiates detection kit and use thereof
1 preparation comprises the test kit of following moiety: primed probe mixed solution (25 μ l/ manage) 1 is managed, RT-PCR reaction solution (250 μ l/ manage), RT-PCR reaction enzymes system (75 μ l/ manage) 1 manages, positive quality control product (200 μ l/ manage) 1 is managed, negative quality control product (200 μ l/ manage) 1 pipe, DEPC H
2o (2000 μ l/ manage) 1 manages.
The preparation of 2 goal gene full-length RNAs
Synthesized and purifying 4 kinds of type total length goal gene RNA by authorized company, record concentration by UV detector, then calculate its RNA copy number, and by 4 kinds of RNA DEPC H
2o is diluted to 1.0 × 10 respectively
5copies/mL ~ 1.0 × 10
2the standard substance of copies/mL.
3 real-time fluorescence quantitative PCR amplification and detections
3.1 reagent prepare: primed probe mixed solution 1 μ l, RT-PCR reaction solution 5 μ l, RT-PCR reaction enzymes of getting respective amount is in proportion 3 μ l, DEPC H
2o11 μ l, fully for subsequent use after mixing.
3.2 application of samples: in PCR reaction tubes, add positive and negative quality control product, goal gene full-length RNA solution 5 μ l respectively, cover tightly pipe lid, put into instrument sample groove.
3.3 editors: (ABI Prism 7500 quantitative real time PCR Instrument)
Open Setup window, positive and negative quality control product and sample to be measured are set by correspondence order, and sample ID is set in Name hurdle.Choose and allly arrange sample well, double-click, select Add Detector, selecting Reporter to be FAM and Quencher is none, then to select Reporter to be HEX and Quencher be none; Selecting Reporter to be Texas Red and Quencher is again none; Then selecting Reporter to be Cy5 and Quencher is close window after none.(none) is selected in Passive Reference.Open instrument window and cycling condition be set: 50 DEG C 15 minutes, 95 DEG C 15 minutes; 94 DEG C 10 seconds, 58 DEG C 35 seconds, 40 circulations (see accompanying drawing 1).Allly be provided with rear preservation file, run.
3.4 interpretations of result:
Reaction terminates rear preservation and detects data file.Ampplot window is opened under Results.The object sample position of selection analysis.Change Baseline numerical value into start:3, stop:10, and open manual setting Threshold:1.5 ± 100000.Double-click numerical value on Rn coordinate and open Graph settings window, change Log in Post Run Settings into Linear, after OK, open Analysis preferences window, under Analysis menu, select Analyze automatic analysis result.
Embodiment 2 is applied Ebola virus somatotype and is differentiated detection kit detection specificity sample
Choose through virus culture process be accredited as respectively dengue virus I type, dengue virus II type, dengue virus type III, dengue virus IV type, Chikungunya virus, encephalitis b virus, heating companion thrombocytopenic syndromes bunyavirus, hantaan virus, the Seroul virus positive each 1 example of serum sample as specificity sample, nucleic acid extraction is carried out to all samples, pcr amplification and interpretation of result step are carried out with reference to embodiment 1, carry out the moon, the detection of positive quality control product simultaneously.
Detected result: the amplification curve of negative quality control product is not S-type (see accompanying drawing 2), the amplification curve of positive quality control product is that obvious S type curve is (see accompanying drawing 3,4,5,6), positive and negative quality control product all meets the Quality Control requirement of test kit, and therefore the detected result of sample to be checked is effective.Accompany thrombocytopenic syndromes bunyavirus, hantaan virus, Seroul virus without non-specific amplification (see accompanying drawing 11) by the detected result of sample to be checked known test kit to dengue virus I type, dengue virus II type, dengue virus type III, dengue virus IV type, Chikungunya virus, encephalitis b virus, heating.
In this test, detected result and the virus culture qualification result of 9 routine samples fit like a glove, and illustrate and utilize the detection of this test kit and discriminating Ebola virus type to be feasible.This test kit is easy and simple to handle, and detection time is short, and can realize high throughput testing, it is cheap in addition, is expected to be applied to the clinical detection of ebola disease viral disease, inspection and quarantine and epidemic monitoring.
Claims (8)
1. Ebola virus somatotype differentiates a detection kit, comprising: 1) RT-PCR reaction solution, primed probe mixed solution, RT-PCR reaction enzymes system, DEPC H
2o, negative quality control product, positive quality control product and 2) separate and concentrate the packing box packing these reagent bottles or pipe, wherein primed probe mixed solution is by the forward and reverse primer of Ebola virus four kinds of type specificities, and the probe composition of Ebola virus four kinds of type specificities, is characterized in that:
1) sequence of the specific forward of Ben Dibujiao Ebola virus and reverse primer is 5 '-TTGGAAAGTAATGCGGTAAAATA-3 ' and 5 '-GCAGCCAATGTTCTCTTGATGT-3 ' respectively, the sequence of Ben Dibujiao Ebola virus specific probe is 5 '-CTACTCCCTGCTGCCTCGAGTGGAAA-3 ', and the two ends of probe are combined with fluorescence generation group FAM and fluorescent quenching group B HQ1 respectively; This can detect Ben Dibujiao Ebola virus specifically to primed probe, to other type Ebola virus no cross reactions;
2) sequence of the specific forward of Zaire Ebola virus and reverse primer is 5 '-GATGCCAACGAYGCTGTGAT-3 ' and 5 '-TGCAAGAGGATGGAGACGAA-3 ' respectively, the sequence of Zaire Ebola virus specific probe is 5 '-TCAGTGGCTCAAGCTCGTTTTTCAGGT-3 ', and the two ends of probe are combined with fluorescence generation group HEX and fluorescent quenching group B HQ1 respectively; This can detect Zaire Ebola virus specifically to primed probe, to other type Ebola virus no cross reactions;
3) sequence of the specific forward of the Sudan Ebola virus and reverse primer is 5 '-TGGTGTTGTTGACCCGTATG-3 ' and 5 '-CGTCGTCCAAATTGAAGAGAT-3 ' respectively, the sequence of the Sudan's Ebola virus specific probe is 5 '-CCTGACTACGAGGATTCGGCTGAAG-3 ', and the two ends of probe are combined with fluorescence generation group Texas Red and fluorescent quenching group Eclipse respectively; This can detect the Sudan Ebola virus specifically to primed probe, to other type Ebola virus no cross reactions;
4) sequence of the tower specific forward of her forest Ebola virus and reverse primer is 5 '-CAACAACAAACACAGTCTTACAGG-3 ' and 5 '-TTATCCCTGGCGTATTCTTGA-3 ' respectively, her sequence of forest Ebola virus specific probe of tower is 5 '-CTCCACACAAAACAATGACAATCCTGC-3 ', and the two ends of probe are combined with fluorescence generation group Cy5 and fluorescent quenching group Eclipse respectively; This can detect her forest Ebola virus of tower, to other type Ebola virus no cross reactions specifically to primed probe.
2. test kit according to claim 1, be further characterized in that ' end can adopt arbitrary fluorescent quenching group mark in TAMRA, BHQ1, BHQ2, Eclipse, Dabcy 1 to probe 3 used, described 2) in, ' end can adopt arbitrary fluorophor mark in VIC, HEX, YELLOW to probe 5 used, described 3) probe used 5 in ' end can adopt arbitrary fluorophor mark in ROX, Texas Red, described 4) in probe 5 used ' end can adopt arbitrary fluorophor mark in CY5, CY5.5.
3. test kit according to claim 1, be further characterized in that in primed probe mixed solution, primer concentration is 0.2mmol/L, concentration and probe concentration is 0.1mmol/L.
4. test kit according to claim 1, is further characterized in that RT-PCR reaction solution is 50mmol/L Tris-HCl, 3 ~ 8mmol/L MgCl of 8.0 ~ 8.8 by pH value
2, 150 ~ 350mmol/L KCl forms.
5. test kit according to claim 1, wherein RT-PCR reaction enzymes system is made up of hot start Taq polymerase, reversed transcriptive enzyme, dNTPs, is characterised in that the consumption of hot start Taq polymerase in every person-portion RT-PCR reaction enzymes system is 5 ~ 10U; Reversed transcriptive enzyme consumption is 1.5 ~ 5U; DNTPs is 6 ~ 12mmol.
6. test kit according to claim 1, is further characterized in that the condition of pcr amplification is: 50 DEG C 15 minutes, 95 DEG C 15 minutes; 94 DEG C 15 seconds, 58 DEG C 35 seconds, 45 circulations.
7. test kit according to claim 1, additionally provides negative quality control product and positive quality control product, it is characterized in that negative quality control product is human normal plasma, and positive quality control product is the goal gene RNA containing Ebola virus four kinds of types.
8. test kit according to claim 7, positive quality control product is the RNA containing four kinds of type Ebola virus object total lengths prepared by gene chemical synthesis mode.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950775A (en) * | 2016-07-08 | 2016-09-21 | 中山大学达安基因股份有限公司 | Kit for detecting NPM (Nucleophosmin)1 gene mutation types |
| CN106188286A (en) * | 2016-07-21 | 2016-12-07 | 中国科学院武汉病毒研究所 | A kind of nano antibody neutralizing Ebola virus |
| CN106967846A (en) * | 2017-05-09 | 2017-07-21 | 广州和实生物技术有限公司 | A kind of fluorescence Constant Temperature Detection kit of quick detection Ebola virus |
| CN108165669A (en) * | 2018-02-24 | 2018-06-15 | 广东出入境检验检疫局检验检疫技术中心 | A kind of Ebola virus parting detection primer, probe and kit |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140532A (en) * | 2010-12-24 | 2011-08-03 | 中国检验检疫科学研究院 | Novel Ebola virus fluorescent quantitative PCR (Polymerase Chain Reaction) detection method and system |
| CN102719557A (en) * | 2011-12-13 | 2012-10-10 | 广东出入境检验检疫局检验检疫技术中心 | Multiplex fluorescent polymerase chain reaction (PCR) kit and primers for detecting Ebola viruses, Marburg viruses, Lassa viruses and Rift Valley fever viruses |
| CN103045755A (en) * | 2011-10-14 | 2013-04-17 | 中国农业科学院上海兽医研究所 | Fluorescent quantitative PCR (polymerase chain reaction) method, primer and kit for detecting EBOV (Ebola virus) |
| CN103045754A (en) * | 2011-10-14 | 2013-04-17 | 中国农业科学院上海兽医研究所 | One-step process real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method and kit for detecting Z/S subtype ebola viruses |
-
2014
- 2014-10-10 CN CN201410530911.5A patent/CN104328216A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140532A (en) * | 2010-12-24 | 2011-08-03 | 中国检验检疫科学研究院 | Novel Ebola virus fluorescent quantitative PCR (Polymerase Chain Reaction) detection method and system |
| CN103045755A (en) * | 2011-10-14 | 2013-04-17 | 中国农业科学院上海兽医研究所 | Fluorescent quantitative PCR (polymerase chain reaction) method, primer and kit for detecting EBOV (Ebola virus) |
| CN103045754A (en) * | 2011-10-14 | 2013-04-17 | 中国农业科学院上海兽医研究所 | One-step process real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method and kit for detecting Z/S subtype ebola viruses |
| CN102719557A (en) * | 2011-12-13 | 2012-10-10 | 广东出入境检验检疫局检验检疫技术中心 | Multiplex fluorescent polymerase chain reaction (PCR) kit and primers for detecting Ebola viruses, Marburg viruses, Lassa viruses and Rift Valley fever viruses |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950775A (en) * | 2016-07-08 | 2016-09-21 | 中山大学达安基因股份有限公司 | Kit for detecting NPM (Nucleophosmin)1 gene mutation types |
| CN105950775B (en) * | 2016-07-08 | 2019-06-11 | 中山大学达安基因股份有限公司 | A kind of kit detecting NPM1 gene mutation typing |
| CN106188286A (en) * | 2016-07-21 | 2016-12-07 | 中国科学院武汉病毒研究所 | A kind of nano antibody neutralizing Ebola virus |
| CN106188286B (en) * | 2016-07-21 | 2019-07-23 | 中国科学院武汉病毒研究所 | A kind of nano antibody neutralizing Ebola virus |
| CN106967846A (en) * | 2017-05-09 | 2017-07-21 | 广州和实生物技术有限公司 | A kind of fluorescence Constant Temperature Detection kit of quick detection Ebola virus |
| CN108165669A (en) * | 2018-02-24 | 2018-06-15 | 广东出入境检验检疫局检验检疫技术中心 | A kind of Ebola virus parting detection primer, probe and kit |
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Application publication date: 20150204 |