CN105400904A - RPA kit used for detecting Ebola virus, and special-purpose primers, probes, and applications of RPA kit - Google Patents
RPA kit used for detecting Ebola virus, and special-purpose primers, probes, and applications of RPA kit Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
The invention provides a RPA (recombinase polymerase amplification) kit used for detecting Ebola virus, special-purpose primers and probes of the RPA kit, and applications of the RPA kit in detection of Ebola virus. The RPA kit, the special-purpose primers, and the probes are designed based on Ebola virus NP gene conserved sequence, and the Ebola virus NP gene conserved sequence possesses an oligonucleotide sequence represented by SEQ ID NO.1. It is shown by research results that the RPA kit can be used for rapid detecting of Ebola virus in samples with specificity and sensitivity.
Description
Technical field
The invention belongs to biological technical field, relate to the molecular biology of Ebola virus, particularly detecting the application in Ebola virus for detecting the RPA test kit of Ebola virus and primer special thereof and probe and its.
Background technology
Ebola virus can cause a kind of acute infectious disease propagated in the mankind and primate, is referred to as ebola hemorrhagic fever.It is 3-21 days that people infects the latent period after Ebola virus, and their early stage patient presents the symptom such as high heat, headache similar to general influenza, and state of an illness rapid progression worsens subsequently, shows as hemorrhage, multiple organ failure and shock-like syndrome.Ebola virus belongs to inovirus section, comprises 5 hypotypes, and wherein four kinds can cause human infection, and lethality rate can up to 90%.In addition, at present in the world also not for effective methods for the treatment of and the vaccine of Ebola.This makes Ebola virus be classified as Biosafety level Four pathogenic micro-organism and category-A biological threats pathogenic agent, and the World Health Organization has been classified as to one of the most serious transmissible disease of mankind's harm.2014, the Epidemic outbreak of disease that West Africa various countries Ebola virus infects, especially very serious with the state such as Sierra Leone, Liberia.Therefore, the prevention and control that the detection method setting up Ebola virus fast and effectively infects for Ebola virus have important practical significance.
At present, the laboratory diagnosis of Ebola virus mainly adopts the nucleic acid detection method of RT-PCR.For the diagnosis of Ebola virus, although RT-PCR method is a kind of sensitivity, special detection method, it is long that RT-PCR method detects required time, cannot use at epidemic situation scene.Breaking out of Ebola's epidemic situation mainly concentrates on disadvantaged country and the areas such as African, and lack sound health security system, even the most basic medical resource all cannot ensure.And RT-PCR testing process comprises complicated nucleic acid extraction step, and need to rely on special and expensive thermal cycler to complete amplification procedure, make it be difficult to operate outside the laboratory of equipment advanced person, and need the long period just can obtain a result, thus limit its application.Therefore, be badly in need of setting up a kind of Ebola virus in-situ check and test method easy again fast.
In recent years, some constant temperature nucleic acid amplification technology are rapidly developed, as depended on the amplification technique (NASBA) of nucleotide sequence, rolling circle amplification (RCA), loop-mediated isothermal amplification technique (LAMP) and recombinase polymeric enzymatic amplification technology (RPA) etc.Compared with traditional PCR technique, nucleic acid isothermal amplification can break away from traditional PCR instrument, obtains detected result accurately at short notice.But, determine suitable method for Ebola virus, also need technician to do a large amount of and deep research, there is no clearly referential thinking and result at present.
Summary of the invention
The invention provides the RPA test kit for detecting Ebola virus, its primer special and probe, to realize the rapid detection of Ebola virus.
Based on the com-parison and analysis to multiple constant temperature nucleic acid amplification technology, determine that the present invention detects Ebola virus based on RPA technology.Recombinase polymeric enzymatic amplification (RecombinasePolymeraseAmplification, RPA) be based on the polymerase-mediated amplification principle of recombinase, the enzyme reaction process of DNA replication dna in analogue body, rely on specific enzyme and protein combination (recombinase, single strand binding protein and archaeal dna polymerase) increases to DNA profiling, under the constant temperature of about 39 DEG C, in 20 minutes, amplified reaction can be completed.Very low to the requirement of hardware device, be particularly suitable for the fields such as in-vitro diagnosis, food safety, Biosafety.Owing to not needing temperature control device, RPA really can realize portable Rapid nucleic acid and detect.And RPA does not also need complicated sample preparation, is applicable to the detection on the spot cannot extracting nucleic acid.Compared with other nucleic acid isothermal amplification technology, RPA technology tool has the following advantages, and: RPA can exponential amplification, and can detect amplified production in real time by implementation sequence specific probe, and fluorescent signal can be read by portable scanning device; Oligonucleotide design is simple, and required target sequence conservative region is consistent with PCR primer and probe design; RPA reaction reagent can also dry powder particle form provide, and convenient preservation is transported and carry out Site Detection.
Therefore, the RPA primer for detecting Ebola virus that an object of the present invention is to provide, described primer designs, for the qualitative detection of Ebola virus according to Ebola virus NP gene conserved sequence; Described Ebola virus NP gene conserved sequence is the nucleotide fragments containing 185 bases, has the oligonucleotide sequence shown in SEQIDNO.:1.
Preferably, described primer comprises forward primer (EBO-RPA-F2) and reverse primer (EBO-RPA-R2) totally two, and its sequence is respectively as shown in SEQ ID NO.:2 and SEQIDNO.:3.
Second object of the present invention is to provide the probe for detecting Ebola virus, and this probe designs, for the qualitative detection of Ebola virus according to Ebola virus NP gene conserved sequence; Described Ebola virus NP gene conserved sequence is the nucleotide fragments containing 185 bases, has the oligonucleotide sequence shown in SEQIDNO.:1.
Preferably, the length of described probe is 46 ~ 52bp, and wherein 5 ' hold at least 30bp, 3 ' holds at least 15bp.
Preferably, the T that described probe is marked by 5 ' terminal sequence, fluorophor T, tetrahydrofuran (THF) (THF or be dSPACER), quenching group mark, 3 ' terminal sequence and 3 ' hold a few part of closure to form.
Preferably, described probe has the oligonucleotide sequence shown in SEQIDNO.:4, and within the probe the Nucleotide T of Nucleotide T and the 33rd of the 32nd is marked with fluorophor and quenching group respectively, there is an abasic site (THF) (probe sequence after modification is as the EBO-RPA-P of table 1) between two groups.After it is by exonuclease identification cutting, two groups are made to be separated and to produce fluorescent signal.
Any fluorophor, conventional is FAM (Fluoresceincarboxylic acid) or TAMRA (carboxyl tetramethylrhodamine); Quenching group is BHQ (Black hole quencher).The preferred FAM of conventional fluorescent group, the preferred BHQ1 of quenching group.
More preferably, described probe by probe 3 ' end add suitable modification group as: between C3-, arm, phosphate, vitamin H-TEG or amino are to prevent polymerase catalysed extension, make probe more stable.
3rd object of the present invention there is provided the RPA test kit for Ebola virus rapid detection, and described test kit comprises above-mentioned RPA primer and probe.
Conveniently detect, described test kit can also comprise the positive plasmid containing Ebola virus NP gene conserved sequence, and described positive plasmid is containing, for example the Ebola virus NP gene conserved sequence shown in SEQ ID NO:1.
Preferably, described test kit comprises the reagent of the following RPA reaction system for 50 μ L, and described reagent comprises: above-mentioned forward primer and each 20pmol of reverse primer, above-mentioned probe 5pmol.
Conveniently detect, described test kit also can comprise negative control, RPA detect required for enzyme, buffer solution system and/or magnesium ion, described negative control is not containing the RPA reaction system of Ebola virus NP gene conserved sequence.
Preferably, the RPA reaction system of 50 μ L is as follows: each 2 μ L of above-mentioned forward and reverse primer (10 μm of ol/L), above-mentioned probe 0.5 μ L (10 μm of ol/L), DNA profiling 1 μ L, deionized water 12.5 μ L, damping fluid 29.5 μ L, magnesium acetate solution 2.5 μ L, add the 0.2mL reaction tubes containing lyophozyme powder.Wherein from TwistAmpexoRT test kit, (exo represents ExonucleaseIII to lyophozyme powder.Lower same), provide with the reaction tubes form containing lyophozyme powder, damping fluid is also directly provided by Twist test kit; DNA profiling concentration is different because institute's test sample product are different.
Present invention also offers the application in Ebola virus detects of above-mentioned primer, probe or test kit.
Application of the present invention, comprises the steps:
(1) with testing sample genomic dna for template, under the guiding and described probe mark of described primer, carry out RPA reaction;
(2) reaction carries out result judgement after terminating: detect with RPA fluorescence detector, and the sample that curve rises is judged to be positive findings; The judgement that curve does not rise is negative findings.
Preferably, application of the present invention, comprises the steps:
(1) amplifing reagent prepares and application of sample: by each for forward and reverse primer 2 μ L (10 μm of ol/L), probe 0.5 μ L (10 μm of ol/L), sample 1 μ L, without DNase and RNase water 12.5 μ L and 29.5 μ L damping fluids in the TwistAmpRTexo reaction tubes of the 0.2mL containing lyophozyme powder.Then 2.5 μ L magnesium acetate solution are added in all reaction tubess, slightly put into RPA fluorescence detector ESEQantTubeScanner immediately after mixing and carry out Real-Time Monitoring.
(2) increase: 40 DEG C are increased 20 minutes, within each 20 seconds, detect a FAM channel fluorescence, at reaction 4-5 minute, reaction tubes is taken out fully mixing, put back to fluorescence detector continuation afterwards and detect.
(3) reaction carries out result judgement after terminating: the sample that curve rises is judged to be positive findings; The judgement that curve does not rise is negative findings.
The principle that the present invention detects Ebola virus adopts RPA technology, and to specific conservative's target sequence of Ebola virus, namely Ebola virus NP gene conserved sequence detects, and this sequence can be used as Ebola virus to one of marker gene.
This invention simplifies the trace routine of Ebola virus, detection can be completed in 20min, with Standard PCR detect need a few hours compared with highly shortened detection time.
Susceptibility of the present invention is high, high specificity.The present invention is combined with portable augmentation detection instrument, can be used for scene or the other detection of bed, has broad application prospects.
Below in conjunction with specific embodiment, the present invention is described in further details.
Accompanying drawing explanation
Fig. 1 shows different primers in the optimum combination of primers screening of RPA detection system and combines the fluorescence intensity produced in time.
Fig. 2 is the threshold time of different primers combination.
Fig. 3 is that the fluorescence intensity of different primers combination compares.
Fig. 4 A is the real-time amplification graphic representation of different copy number, and 1 ~ 8 is respectively: copy number is respectively 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10 and 0.
Fig. 4 B is the semi-logarithmic chart that RPA detects the susceptibility of Ebola virus.
Fig. 5 adds the serum of different volumes in detection system, to observe the restraining effect that serum detects RPA.5 ~ 7 curves marked in figure are by order from top to bottom; 1 ~ 7 respectively corresponding volume be: 0,2,4,6,8,10,12 μ L.
The RPA of Fig. 6 display optimization serum tolerance reacts amplification curve.A is the amplification curve of non-increase serum, and b is the amplification curve adding 5 μ L serum in 50 μ L systems.
Embodiment
Following embodiment is only not used in for illustration of the present invention and limits the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition as the people such as Sambrook compile the condition described in " Molecular Cloning: A Laboratory guide ", or according to manufacturer suggestion condition.
Unless there are specified otherwise, percentage concentration described in the present invention is mass/mass (W/W, unit g/100g) percentage concentration, mass/volume (W/V, unit g/100mL) percentage concentration or volume/volume (V/V, Unit/mL/100mL) percentage concentration.
The approach that obtains of the various biomaterials be described in embodiment is only to provide a kind of approach of testing acquisition to reach concrete disclosed object, should not become the restriction to biological material source of the present invention.In fact, the source of used biomaterial is widely, and any biomaterial that can obtain with moral ethics that keeps on the right side of the law can replace use according to the prompting in embodiment.
The primer is synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd, and probe is synthesized by precious biotechnology (Dalian) company limited, and the work of all sequences mensuration completes by Beijing Liuhe Huada Genomics Technology Co., Ltd.
Embodiment 1, Ebola virus RPA primer and probe design and screening
(1) design of primer and probe
Contriver, by literature search, analyzes and determines that the present invention uses the conserved sequence in Ebola virus NP gene to be target gene.Known target gene sequence is obtained, the oligonucleotide sequence namely shown in SEQIDNO.:1 from ncbi database.According to RPA primer and probe design principle, design 8 primers and 1 probe, as shown in table 1.
Table 1 primer and probe
(2) primer screening
Synthetic contains the positive plasmid of Ebola NP gene order, with this plasmid for template, primer and probe are carried out global combinatorial, carry out RPA detection respectively, with reaction accumulation fluorescence intensity and detection time for index, filter out the primed probe combination that susceptibility is the highest, the evaluation detected for follow-up RPA and application.
The RPA reaction system of 50 μ L is as follows: be 10 by each for forward and reverse primer 2 μ L (10 μm of ol/L), probe 0.5 μ L (10 μm of ol/L), concentration
3copy/microlitre DNA profiling 1 μ l, deionized water 12.5 μ L and 29.5 μ L damping fluids (Twist test kit directly provides) join in the TwistAmpexo reaction tubes of the 0.2mL containing lyophozyme powder (from TwistAmpexoRT test kit, providing with the reaction tubes form containing lyophozyme powder).Then 2.5 μ L magnesium acetate solution are added on the lid of all reaction tubess, put into RPA fluorescence detector after mixing immediately and carry out Real-Time Monitoring, 40 DEG C of reaction 20min.
16 pairs of combination of primers fluorescence detecting system data are shown in Fig. 1-Fig. 3.Fig. 1 (for enabling lines distinguish, point A, B width) fluorescence intensity for producing in time, X-coordinate is the reaction times, and ordinate zou is fluorescent signal value; Fig. 2 and Fig. 3 be different primers combination threshold time and fluorescence intensity compare, Fig. 2 X-coordinate is combination of primers, and ordinate zou is threshold time, and Fig. 3 X-coordinate is combination of primers, and ordinate zou is fluorescent signal value.Visible, primers F 2/R2 has the shortest threshold time and the strongest fluorescent signal value, and therefore the present invention finally chooses the short and combination of primers EBO-RPA-F2/R2 that fluorescent signal is strong of detection time.
The combination of primers that the present invention determines comprises: forward primer (EBO-RPA-F2), and its sequence is as shown in SEQ ID NO.:2; Reverse primer (EBO-RPA-R2), its sequence is as shown in SEQ ID NO.:3.
(3) determination of probe
The EBO-RPA-P probe that table 1 is listed is that the present invention is preferred, and the length of probe is 46 ~ 52bp, and wherein 5 ' hold at least 30bp, 3 ' holds at least 15bp.
T, 3 ' terminal sequence and a few part of 3 ' end closure that the T that probe is marked by 5 ' terminal sequence, fluorophor, tetrahydrofuran (THF) (THF or title dSPACER), quenching group mark form.
Probe EBO-RPA-P has the oligonucleotide sequence (49bp) shown in SEQIDNO.4, and within the probe the Nucleotide T of Nucleotide T and the 33rd of the 32nd is marked with fluorophor and quenching group respectively, there is an abasic site (THF) between two groups.After it is by exonuclease identification cutting, two groups are made to be separated and to produce fluorescent signal.
Any using fluorescence group all can be used for the present invention, and conventional is FAM (Fluoresceincarboxylic acid) or TAMRA (carboxyl tetramethylrhodamine); Quenching group is BHQ (Black hole quencher).The preferred FAM of conventional fluorescent group, the preferred BHQ1 of quenching group.
More preferably, described probe by probe 3 ' end add suitable modification group as: between C3-, arm, phosphate, vitamin H-TEG or amino are to prevent polymerase catalysed extension, make probe more stable.
The sensitivity assessment that embodiment 2:RPA detects
Positive plasmid is diluted to 10 by 10 multiple proportions leus
7to a series of different concns such as 10 copy/microlitres, respectively get 1 μ L and add RPA reaction system described in embodiment 1 respectively, the template of combination of primers EBO-RPA-F2/R2 to above-mentioned different copy number of screening is utilized to carry out RPA detection, every hole repeats 8 times, with molecule copy number and detection time for parameter, utilize the susceptibility that Prism computed in software RPA detects.
Result as shown in the figure.Fig. 4 A is the real-time amplification graphic representation of different copy number, and X-coordinate is the time, and ordinate zou is fluorescent signal value; Fig. 4 B is the semi-logarithmic chart that RPA detects the susceptibility of Ebola virus, and X-coordinate is copy number, and ordinate zou is threshold time.As seen from the figure, the detection of RPA method detection Ebola virus is minimum is limited to 10 copies/microlitre, and the time obtaining positive findings is 9 minutes.
The Evaluation on specificity that embodiment 3:RPA detects
Evaluation on specificity with influenza virus and adenovirus in contrast, to determine the specificity of RPA detection method of the present invention.
Extract the DNA of Influenza Virus RNA and adenovirus, carry out RPA detection respectively, adenovirus DNA carries out RPA detection according to the method in embodiment 2; Influenza Virus RNA adopts following 50 μ LRT-RPA systems to detect: by each for forward and reverse primer 2 μ L (10 μm of ol/L), probe 0.5 μ L (10 μm of ol/L), RNA template 1 μ L, RNA enzyme inhibitors 0.5 μ L, deionized water 12 μ L and 29.5 μ L damping fluids in the TwistAmpRTexo reaction tubes of the 0.2mL containing lyophozyme powder.Then 2.5 μ L magnesium acetate solution are added on the lid of all reaction tubess, put into RPA fluorescence detector after mixing immediately and carry out Real-Time Monitoring, 40 DEG C of reaction 20min.
All there is not amplification curve in result influenza virus and adenoviral nucleic acid sample, illustrates that RPA detection method of the present invention has very strong specificity to Ebola virus.
Embodiment 4: the RPA Evaluation of detection methods of simulation serum sample
Be template by Ebola's positive plasmid of synthesis, be that primer carries out pcr amplification with EBO-RPA-F4 (see table 1) and EBO-R4-T7:TAATACGACTCACTATAGGGAGGAGAAATTTGATCAG, with 1 μ L amplified production for template, carry out external reverse transcription and become RNA.
Mixing as simulating serum sample using 0 ~ 12 μ L normal human serum with 1 μ L Ebola virus RNA, detecting by the RT-RPA system in embodiment 3, assessment this method detects the susceptibility of analog sample.The amount of serum is added, the detection system of Optimized Simulated serum sample by detected result adjustment.
Fig. 5 and Fig. 6 shows serum detects Ebola virus impact on RPA.Fig. 5 is the serum (0,2,4,6,8,10,12 μ L) adding different volumes in detection system, and to observe the restraining effect that serum detects RPA, X-coordinate is the time, and ordinate zou is fluorescent signal value; The RPA reaction of Fig. 6 display optimization serum tolerance, a is the amplification curve of non-increase serum, and b is the amplification curve adding 5 μ L serum in 50 μ L systems, and X-coordinate is the time, and ordinate zou is fluorescent signal value.Result shows, and 50 μ LRT-RPA systems can tolerate the serum accounting for total system 10%, although the fluorescence intensity detected reduces, does not affect its detection sensitivity.
Adopt PCR method to detect Ebola virus, need complicated nucleic acid extraction step, otherwise PCR detection sensitivity can be affected.Therefore, the leaching process of RNA limits the widespread use of PCR detection method in Ebola's epidemic outbreak.By Ebola's serum sample directly or add after slightly carrying nucleic acid in this RPA reaction system optimized and detect, its detection sensitivity is unaffected, can improve detection efficiency and reduce costs.
Embodiment 5: the RPA of clinical Ebola's sample detects
With the RPA primer of above-mentioned screening and probe, establish RPA reaction system.
From serum and the swab sample extraction RNA of doubtful Ebola patient, utilize RPA to react and detect, and compare with quantitative PCR detection result, the Sensitivity and Specificity that comparative analysis RPA detects.Have chosen the clinical sample of 160 parts of doubtful Ebola patients, wherein quantitative PCR detection is 101 positive examples, negative 59 examples, RPA detected result is 94 examples of RPA test positive in 101 examples of the PCR positive, negative 7 examples, in 59 routine samples of quantitative PCR detection feminine gender, RPA is detected as 56 negative examples, 3 examples of test positive.With quantitative PCR result for reference, positive coincidence rate is 93.06%, and negative match-rate is 94.91%, and overall coincidence rate is 93.75%, and data are in table 2.
The RPA detected result of the clinical Ebola's sample of table 2
| Quantitative PCR is positive | Quantitative PCR is negative | Add up to | |
| RPA is positive | 94 | 3 | 97 |
| RPA is negative | 7 | 56 | 63 |
| Add up to | 101 | 59 | 160 |
Embodiment 6: the RPA test kit of Ebola virus rapid detection
The RPA test kit of Ebola virus rapid detection provided by the invention comprises above-mentioned RPA primer and probe, specifically comprises: forward primer (EBO-RPA-F2), and its sequence is as shown in SEQ ID NO.:2; Reverse primer (EBO-RPA-R2), its sequence is as shown in SEQ ID NO.:3; Probe EBO-RPA-P, its oligonucleotide sequence is as shown in SEQ ID NO.:4.
Conveniently detect, test kit can also comprise the positive plasmid containing Ebola virus NP gene conserved sequence, and described positive plasmid is containing, for example the Ebola virus NP gene conserved sequence shown in SEQ ID NO:1.
Preferably, test kit comprises the reagent of the following RPA reaction system for 50 μ L, and described reagent comprises: above-mentioned forward primer and each 20pmol of reverse primer, above-mentioned probe 5pmol.
Conveniently detect, described test kit also can comprise negative control, RPA detect required for enzyme, buffer solution system and/or magnesium ion, described negative control is not containing the RPA reaction system of Ebola virus NP gene conserved sequence.
Preferably, the RPA reaction system of 50 μ L is as follows: each 2 μ L of above-mentioned forward and reverse primer (10 μm of ol/L), above-mentioned probe 0.5 μ L (10 μm of ol/L), DNA profiling 1 μ l, deionized water 12.5 μ L, damping fluid 29.5 μ L, magnesium acetate solution 2.5 μ L, add in the 0.2ml reaction tubes containing lyophozyme powder.Wherein lyophozyme powder is from TwistAmpexoRT test kit, and provide with the reaction tubes form containing lyophozyme powder, damping fluid is also directly provided by Twist test kit; DNA profiling concentration is different because institute's test sample product are different.
Utilize described primer, probe or test kit can detect Ebola virus in sample (as serum sample, Nasopharyngeal swabs etc.).Detection can comprise the steps:
(1) with testing sample genomic dna for template, under the guiding and described probe mark of described primer, carry out RPA reaction;
(2) reaction carries out result judgement after terminating: detect with RPA fluorescence detector, and the sample that curve rises is judged to be positive findings; The judgement that curve does not rise is negative findings.
Concrete detection, can comprise the steps:
(1) amplifing reagent prepares and application of sample: by each for forward and reverse primer 2 μ L (10 μm of ol/L), probe 0.5 μ L (10 μm of ol/L), sample 1 μ L, without DNase and RNase water 12.5 μ L and 29.5 μ L damping fluids in the TwistAmpRTexo reaction tubes of the 0.2ml containing lyophozyme powder.Then 2.5 μ L magnesium acetate solution are added in all reaction tubess, slightly put into RPA fluorescence detector ESEQantTubeScanner immediately after mixing and carry out Real-Time Monitoring.
(2) increase: 40 DEG C are increased 20 minutes, within each 20 seconds, detect a FAM channel fluorescence, at reaction 4-5 minute, reaction tubes is taken out fully mixing, put back to fluorescence detector continuation afterwards and detect.
(3) reaction carries out result judgement after terminating: the sample that curve rises is judged to be positive findings; The judgement that curve does not rise is negative findings.
Claims (10)
1., for detecting the RPA primer of Ebola virus, according to the design of Ebola virus NP gene conserved sequence, described Ebola virus NP gene conserved sequence has the oligonucleotide sequence shown in SEQIDNO.1.
2. primer according to claim 1, is characterized in that: the forward primer in described primer has the oligonucleotide sequence shown in SEQIDNO.2, and reverse primer has the oligonucleotide sequence shown in SEQIDNO.3.
3., for detecting the probe of Ebola virus, according to the design of Ebola virus NP gene conserved sequence, described Ebola virus NP gene conserved sequence has the oligonucleotide sequence shown in SEQIDNO.1.
4. probe according to claim 3, is characterized in that: the length of described probe is 46 ~ 52bp, and wherein 5 ' hold at least 30bp, 3 ' holds at least 15bp.
5. probe according to claim 4, is characterized in that: T, 3 ' terminal sequence and a few part of 3 ' end closure that the T that described probe is marked by 5 ' terminal sequence, fluorophor, tetrahydrofuran (THF) (THF or title dSPACER), quenching group mark form.
6. probe according to claim 5, it is characterized in that: described probe has the oligonucleotide sequence shown in SEQIDNO.4, and within the probe the Nucleotide T of Nucleotide T and the 33rd of the 32nd is marked with fluorophor and quenching group respectively, there is an abasic site (THF) between two groups.
7. probe according to claim 4, is characterized in that: described reporter fluorescence group is FAM or TAMRA, and described quenching fluorescence group is BHQ.
8., for detecting the RPA detection kit of Ebola virus, comprise the primer of claim 1 or 2, and/or the arbitrary described probe of claim 3-7.
9. test kit according to claim 8, it is characterized in that: described test kit also comprises the positive plasmid containing Ebola virus NP gene conserved sequence, described positive plasmid is containing, for example the Ebola virus NP gene conserved sequence shown in SEQ ID NO:1.
10. test kit according to claim 9, is characterized in that: for the reagent of the RPA reaction system of 50 μ L in described test kit, described forward primer and each 20pmol of reverse primer, described probe 5pmol.
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| CN114606330A (en) * | 2022-04-20 | 2022-06-10 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894118A (en) * | 2015-06-01 | 2015-09-09 | 山东省农业科学院奶牛研究中心 | Primer, probe and kit for detecting bovine viral diarrhea virus |
| CN105018485A (en) * | 2015-07-31 | 2015-11-04 | 中国动物卫生与流行病学中心 | Primer and probe for detecting peste des petits ruminants virus by virtue of RPA (Recombinase Polymerase Amplification) technique |
-
2015
- 2015-12-18 CN CN201510956456.XA patent/CN105400904A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894118A (en) * | 2015-06-01 | 2015-09-09 | 山东省农业科学院奶牛研究中心 | Primer, probe and kit for detecting bovine viral diarrhea virus |
| CN105018485A (en) * | 2015-07-31 | 2015-11-04 | 中国动物卫生与流行病学中心 | Primer and probe for detecting peste des petits ruminants virus by virtue of RPA (Recombinase Polymerase Amplification) technique |
Non-Patent Citations (1)
| Title |
|---|
| MILENA EULER ET AL: "Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
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| CN106191298A (en) * | 2016-09-15 | 2016-12-07 | 宁波海洋研究院 | A kind of method detecting vibrio parahaemolyticus Vibrio parahaemolyticus |
| CN110042173A (en) * | 2019-02-28 | 2019-07-23 | 中国科学院广州生物医药与健康研究院 | Nano-gold biosensor and detection method a kind of while that detect four kinds of hemorrhagic fever viruses |
| CN110863058A (en) * | 2019-12-05 | 2020-03-06 | 河北省农林科学院植物保护研究所 | RPA primer for identifying potato rot stem nematode and application thereof |
| CN111705159A (en) * | 2020-07-28 | 2020-09-25 | 河北省农林科学院植物保护研究所 | Real-time fluorescent RPA detection primer for sweet potato black spot germs and application thereof |
| CN112322705A (en) * | 2020-12-02 | 2021-02-05 | 济南国益生物科技有限公司 | Isothermal amplification fluorescence RMA method for multiple nucleic acid detection |
| CN113373266A (en) * | 2021-06-22 | 2021-09-10 | 内蒙古农业大学 | Fluorescent RPA primer, kit and detection method for detecting sheep pulmonary adenomatosis virus |
| CN113373266B (en) * | 2021-06-22 | 2022-07-01 | 内蒙古农业大学 | Fluorescent RPA primer, kit and detection method for detecting sheep pulmonary adenomatosis virus |
| CN114410740A (en) * | 2021-12-31 | 2022-04-29 | 成都万众壹芯生物科技有限公司 | Nucleic acid amplification kit and preparation method and application thereof |
| CN114606330A (en) * | 2022-04-20 | 2022-06-10 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization |
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