CN105002302A - Primer and probe for detecting rift valley fever virus - Google Patents
Primer and probe for detecting rift valley fever virus Download PDFInfo
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- CN105002302A CN105002302A CN201510468141.0A CN201510468141A CN105002302A CN 105002302 A CN105002302 A CN 105002302A CN 201510468141 A CN201510468141 A CN 201510468141A CN 105002302 A CN105002302 A CN 105002302A
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Abstract
The invention aims at providing a primer and probe for detecting rift valley fever virus by applying an RPA technology. The sequence of a forward primer body is showed in SEQ ID NO:1, the sequence of a reverse primer body is showed in SEQ ID NO:2, and the sequence of a probe is showed in SEQ ID NO:3. According to the primer and probe for detecting the rift valley fever virus by applying the RPA technology, a great number of RPA primers and probes are designed according to a rift valley fever virus genomic sequence, and a primer and probe combination which can rapidly and effectively detect rift valley fever virus nucleic acid can be screened out from the great number of the RPA primers and probes. Rapid detection is conducted by utilizing the set of the primer and probe, an obvious amplification curve can be obtained by taking rift valley fever virus nucleic acid RNA in vitro transcription as a positive control, and other pathogenic nucleic acids such as foot and mouth disease virus, contagious pustular dermatitis virus, mycoplasma capricolum and bluetongue virus which serve as a template to conduct the reaction do not have the amplification curve.
Description
Technical field
The invention belongs to animal doctor's the pathogenic microorganism examination technical field, be specifically related to a kind of primer sets and the probe that detect Rift Valley fever virus.Namely recombinase polysaccharase isothermal amplification technique (RecombinasePolymerase Amplification, RPA) primer used by technology for detection Rift Valley fever virus and detection method is applied.
Background technology
Rift Valley fever (Rift valley fever, RVF) is the Amphixenosis causing ruminating animal and people to fall ill by Rift Valley fever virus (Rift valley fever virus, RVFV).Rift Valley fever virus can be propagated by mosquito or contact blood, body fluid, tissue or infected animal.This disease is comparatively serious on the impact of sheep, goat, ox, can cause the death of the miscarriage of pregnant animal and new livestock.Above the average age for marriage non-pregnant animal also comparatively susceptible, but clinical disease resistance is comparatively strong, the susceptibility of animal is relevant with the hereditary difference of itself, from beyond Africa and the animal of RVF Non-epidemic areas to this disease comparatively susceptible.People, to RVFV susceptible, by contact, processes infectious material or bites infection by mosquito medium, usually asymptomatic or relevant to main self-limited fever diseases after people infects.After the latent period of 2-5 days, patient may experience the disease of an influenza sample, with generalized fatigue, and low fever, headache, photophobia and arthralgia.Some patients develop into and faint.A large amount of Symptomatic patient will present hepatitis.Heating continues one week usually, and most of spontaneous remission, the patient being less than 5% will develop into syndrome as ephritis, hemorrhagic fever or encephalitis, and mortality ratio is up to 10-12%.
Rift Valley fever is mainly endemic conditions in West Africa and some other African country, periodic epidemic in livestock and crowd after heavy showers.Within 2000, virus is transmitted to the arabia, causes the epidemic situation of Saudi Arabia and Yemen to break out greatly, and this is also the place this disease first time being transmitted to beyond the African continent, and the possibility pointing out this disease to be diffused into other areas further increases further.Established several real-time fluorescence RT-PCR method to detect this disease at present, but these methods generally need high plant and instrument, and the needs of Fields detection cannot be met.For making diagnosis to Rift Valley fever fast, being badly in need of setting up a kind of use and easily reading high sensitivity on system-based and specific real-time detection method.
Summary of the invention
The object of this invention is to provide a kind of primer and the probe of applying RPA technology for detection Rift Valley fever virus, thus make up the deficiencies in the prior art.
Primer of the present invention and probe, the sequence of its forward primer is SEQ ID NO:1, and reverse primer sequences is for shown in SEQ ID NO:2, and probe sequence is for shown in SEQ ID NO:3.
The 27th kilobase marker BHQ1 quenching group in its middle probe RVFV-RPAp is modified, and the 28th bit base replaces with tetrahydrofuran (THF) (tetrahydrofuran), and the 29th bit base flag F AM luminophore is modified, and 3 ' end carries out phosphorylation modification.
Primer of the present invention and probe are for detecting Rift Valley fever virus;
Present invention also offers a kind of method being detected Rift Valley fever virus by recombinase polysaccharase isothermal amplification technique: the RNA in extraction measuring samples is as template, primer described in utilization and probe carry out fluorescence rapid detection, if there is obvious fluorescence curve to increase, then illustrate institute detect in sample contain Rift Valley fever virus nucleic acid.
The present invention devises a large amount of RPA primer and probe according to Rift Valley fever virus genome sequence, therefrom filters out primer and the probe combinations that effectively can detect Rift Valley fever virus nucleic acid fast.This cover primer and probe is utilized to carry out rapid detection, can obtain obvious amplification curve using the Rift Valley fever virus nucleic acid RNA of in-vitro transcription as positive control, other cause of disease nucleic acid such as foot and mouth disease virus, sheep infective pustule virus, mycoplasma capri and blue tongue virus are that template carries out reacting all do not have amplification curve.
Accompanying drawing explanation
Fig. 1 is Rift Valley fever virus specific detection, and wherein 1 is Rift Valley fever virus positive control, and 2-6 is respectively foot and mouth disease virus, sheep infective pustule virus, mycoplasma capri, blue tongue virus and Healthy Sheep lymphoglandula.
Fig. 2 is detection sensitivity test chart, and the RVFV RNA using in-vitro transcription is template, and copy number is from 10
6to 10
0low Positive rate successively.
Embodiment
The present invention, by after carrying out sequence alignment to RVFV strain, obtains the region that virus conservation is higher, then from this zone design primer and probe.If the primer used and probe detect specificity fluorescent curve, it is RVFV strain.
Detailed description below by embodiment illustrates the present invention further.For the method steps adopted in the specific embodiment of the invention, those of ordinary skill in the art can carry out selection and replace from prior art, and is not limited in concrete record of the present invention.
Embodiment 1:RPA detects the foundation of RVFV method
According to Rift Valley fever virus genome sequence in GenBank, design primer and probe.Primer length is about 30-35nt, owing to there is no the primer-design software for RPA at present, the a large amount of primer of design and synthesis in previous work process of the present invention, therefrom filter out a pair highly sensitive and primer that specificity is good, primer and probe sequence SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 (table 1).
Table 1: the primer and the probe that detect RVFV for RPA
Note: the K in RVFV-RPAf represents G or T, the Y in RVFV-RPAr represents T or C.The 27th kilobase marker BHQ1 quenching group in RVFV-RPAp is modified, and the 28th bit base replaces with tetrahydrofuran (THF) (tetrahydrofuran), and the 29th bit base flag F AM luminophore is modified, and 3 ' end carries out phosphorylation modification.
The sensitivity of primer of the present invention's design and specificity are detected, concrete steps and result as follows:
1. materials and methods
(1) the Rift Valley fever virus DNA of material synthetic, foot and mouth disease virus, blue tongue virus, sheep mycoplasma and sheep infective pustule virus, and Healthy Sheep node sample.Primer and probe are synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd, and other reagent are import packing or domestic analytical pure.
(2) cause of disease nucleic acid extraction gets 200 μ l cause of disease culture or the tissue homogenates after 2000g is centrifugal, and the High Pure PCR Template Preparation kit of use Roche company extracts the total nucleic acid in various material according to product description.
(4) in-vitro transcription is prepared the Rift Valley fever virus template target gene that the Rift Valley fever virus Sudan30-2010 strain (JQ820481) of synthetic increased and is connected on pGEM-T carrier, the linear plasmid of single endonuclease digestion, then in-vitro transcription is carried out according to the specification sheets of Riboprobe In vitro Transcription systems, product is after the sodium-acetate alcohol settling of DNase I digestion, 3M, be dissolved in the water without RNase, corresponding copy number is calculated, successively from 10 after measuring concentration
6copy/μ l is with 10 times of doubling dilutions to 10
0individual copy/μ l.-70 DEG C of preservations.
(3) RPA amplification uses TwistAmp exo (TwistDx, Cambridge, UK) test kit carries out RPA test in 50 μ l reaction systems, first in 1.5ml centrifuge tube, add rehydration damping fluid 29.5 μ l, Transcriptor ThermoScript II (Roche) 0.5 μ l, the each 2.1 μ l of primer of 10 μMs, the TwistAmp exo probe 0.6 μ l of 10 μMs, template ribonucleic acid and dH
2o 12.7 μ l, vortex mixing of short duration centrifugal after, aforementioned mixed solution is joined in the reaction tubes of RPA lyophozyme ball, add the magnesium acetate solution 2.5 μ l of 280mM, mixing, is placed in RPA augmentation detection instrument (Twista) or quantitative real time PCR Instrument by reaction tubes, 37 DEG C are reacted 15 minutes.With different types of cause of disease nucleic acid and Healthy Sheep total nucleic acid for template carries out specific detection, detect with the susceptibility that the dilution RNA standard of difference carries out reacting for template.
2. result
2.1 specific outcome
Rapid detection is carried out with the primer designed by the present invention and probe, with Rift Valley fever virus nucleic acid RNA for template all can obtain obvious amplification curve, other cause of disease nucleic acid as foot and mouth disease virus, sheep infective pustule virus, mycoplasma capri, blue tongue virus and Healthy Sheep lymphoglandula total nucleic acid be template carry out RPA reaction all there is no amplification curve (Fig. 1).
2.2 susceptibility results
Viral nucleic acid 10 is doubly diluted to multiple gradient, each gradient repeat 10 times detect, Using statistics computed in software go out template be low to moderate about 100 copy time have 95% recall rate, there is highly sensitive (Fig. 2).
The detection application of embodiment 2RPA detection technique in clinical sample
Use the method set up in embodiment 1, to 30 parts of sheep mouth and nose swabs of the three provinces censorships such as domestic Yunnan, Guangxi and Guangdong with organize pathological material of disease clinical sample to carry out sampling Detection during in July, 2014 to September.Whether commodity in use test kit (the High Pure PCR TemplatePreparation kit of Roche company) extracts the total nucleic acid template in sample, detect in these samples containing RVFV by the RPA method set up; The Real-timeRT-PCR method of simultaneously recommending with reference to OIE (OIE) detects.The detected result of result two kinds of detection methods is consistent, and a positive control has positive amplification curve, and does not all detect positive in clinical sample.
Claims (8)
1. primer and probe, is characterized in that, the sequence of the forward primer in described primer is SEQ IDNO:1, and reverse primer sequences is SEQ ID NO:2, and probe sequence is SEQ ID NO:3.
2. primer as claimed in claim 1 and probe, is characterized in that, the 27th base of described probe is labeled BHQ1 quenching group and modifies.
3. primer as claimed in claim 2 and probe, it is characterized in that, the 28th bit base of described probe replaces with tetrahydrofuran (THF).
4. primer as claimed in claim 3 and probe, is characterized in that, the 29th bit base flag F AM luminophore of described probe is modified.
5. primer as claimed in claim 4 and probe, it is characterized in that, 3 ' end of described probe carries out phosphorylation modification.
6. primer according to claim 1 and probe are for detecting Rift Valley fever virus.
7., for detecting a test kit for Rift Valley fever virus, it is characterized in that, described test kit includes primer according to claim 1 and probe.
8. detected a method for Rift Valley fever virus by recombinase polysaccharase isothermal amplification technique, it is characterized in that, the step of described method is as follows:
Extract RNA in measuring samples as template, utilize the primer described in claim 1 and probe to carry out fluorescence rapid detection, if there is obvious fluorescence curve increase, then illustrate detect in sample and contain Rift Valley fever virus nucleic acid.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510468141.0A CN105002302A (en) | 2015-07-31 | 2015-07-31 | Primer and probe for detecting rift valley fever virus |
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| CN201510468141.0A CN105002302A (en) | 2015-07-31 | 2015-07-31 | Primer and probe for detecting rift valley fever virus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105738624A (en) * | 2015-12-28 | 2016-07-06 | 中华人民共和国上海出入境检验检疫局 | Indirect immuno-fluorescence detection method of rift valley fever virus IgG antibody |
| CN108624720A (en) * | 2018-06-21 | 2018-10-09 | 浙江国际旅行卫生保健中心（浙江出入境检验检疫局口岸门诊部） | The primed probe group and kit of RAA Fluorometric assay Rift Valley fever virus |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103952494A (en) * | 2014-03-06 | 2014-07-30 | 中国动物卫生与流行病学中心 | Rift valley fever virus RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection method |
-
2015
- 2015-07-31 CN CN201510468141.0A patent/CN105002302A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103952494A (en) * | 2014-03-06 | 2014-07-30 | 中国动物卫生与流行病学中心 | Rift valley fever virus RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection method |
Non-Patent Citations (1)
| Title |
|---|
| MILENA EULER, ET AL: ""Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus"", 《JOURNAL OF CLINICAL VIROLOGY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105738624A (en) * | 2015-12-28 | 2016-07-06 | 中华人民共和国上海出入境检验检疫局 | Indirect immuno-fluorescence detection method of rift valley fever virus IgG antibody |
| CN105738624B (en) * | 2015-12-28 | 2018-07-10 | 中华人民共和国上海出入境检验检疫局 | A kind of indirect immunofluorescene assay method of Rift Valley fever virus IgG antibody |
| CN108624720A (en) * | 2018-06-21 | 2018-10-09 | 浙江国际旅行卫生保健中心（浙江出入境检验检疫局口岸门诊部） | The primed probe group and kit of RAA Fluorometric assay Rift Valley fever virus |
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