CN108624720A - The primed probe group and kit of RAA Fluorometric assay Rift Valley fever virus - Google Patents
The primed probe group and kit of RAA Fluorometric assay Rift Valley fever virus Download PDFInfo
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Abstract
The present invention relates to the primed probe group of RAA Fluorometric assay Rift Valley fever virus and kits, belong to technical field of molecular biological detection.The primed probe group of RAA Fluorometric assays Rift Valley fever virus provided by the invention includes sense primer, downstream primer and probe, the nucleotide sequence of the sense primer is as shown in SEQ ID NO.1, for the nucleotide sequence of the downstream primer as shown in SEQ ID NO.2, the probe is that the 28th bit base of the nucleotide sequence shown in SEQ ID NO.3 modifies fluorescent reporter group, the 29th bit base replaces with tetrahydrofuran residue and the substance of the 30th bit base modification quenching group.Primed probe group detection provided by the invention is quick, accurate, sensitive, easy to operate, and specificity can complete the detection to Rift Valley fever virus up to 100% in 15min.
Description
Technical field
The present invention relates to technical field of molecular biological detection, and in particular to RAA Fluorometric assay Rift Valley fever virus draws
Object probe groups and kit.
Background technology
Rift Valley fever (Rift Valley fever, RVF) be by Rift Valley fever virus (Rift Valley fever virus,
RVFV a kind of Amphixenosis caused by) is arbo heat generation, acute infectious disease, is mainly passed by mosquito matchmaker
It broadcasts, also can be sheep, goat and ox by aerosol and contact transmission, main host, epidemiological features are that animal pregnancy occurs
There is rapid miscarriage, the death rate of brood is high, while with the morbidity and death of crowd.The disease is popular in Africa and I
Primary Peninsula, it is popular often along with the loop cycle of especially big rainfall, it is less to be happened at semiarid zone, or relatively frequently occur
In the grassland ecological of larger rainfall, lesion characteristics are hepatic injury.WHO has been classified as one of biological warfare agent, world animal health
Tissue (OIE) is classified as A class epidemic diseases.
Rift Valley fever virus belongs to bunyaviridae phlebotomus fever virus category, there is cyst membrane, spherical in shape, a diameter of 90~100nm,
Surface has glycoprotein protrusion, nucleic acid to be located in the nucleocapsid of virus, is sub-thread strand RNA.Rift Valley fever virus is a kind of single
The virus of serotype, morphology typical and physicochemical property with cloth Buddhist nun's virus.It is in ball that virus, which has cyst membrane, 90~110nm of diameter,
There are clear glycoprotein protrusion, the long 10mm of protrusion diameter in shape, surface.Viral nucleic acid is located in the nucleocapsid of virus, negative for sub-thread
Chain RNA, RNA can be divided into L (big), M (in), three segments S (small).The segments S therein are the RNA of double meanings, that is, it is double fixed to have
To encoding function, coding N protein and another non-structural protein (Ns)；The segments M encode G1 and two kinds of outer membrane glycoproteins of G2 and
Two kinds of non-structural protein QNSm, size are respectively 14kD and 78kD), G1 and G2 glycoprotein can stimulate body to generate antibody, but
Only G2 can stimulate body to generate neutralizing antibody in vivo；The segments L encode L albumen, and L albumen has the function of virus transcription enzyme.
Virion is free of stromatin.The virus only has One serotype, does not find the separation strains and reality of Rift Valley fever virus also at present
Test the specific antigen difference of room pass on strain.This is viral to fatsolvent and thermo-responsive, and 56 DEG C can inactivate for 40 minutes.
In recent years, due to the intensification of international cooperation, there is frequent commercial intercourse between various countries, traveller's quantity cumulative year after year, these
All increase the possibility that Rift Valley fever is passed to China.Therefore, the method for quickly detecting of the virus is established in frontier port, and applied
In the monitoring of port Rift Valley fever virus, it is of great significance to preventing the incoming China of the disease.
The method of current diagnosis Rift Valley fever virus has isolation of virus, such as hemagglutination-inhibition test, virus of serodiagnosis method
Neutralization test and enzyme linked immunosorbent assay.But these methods or live virus or complicated for operation, time-consuming, expense need to be operated
Power or susceptibility are not high enough, cannot obtain diagnostic result in early days.In recent years, the molecular biology method to grow up is such as
RT-LAMP methods in PCR (RT-PCR) and isothermal nucleic acid have built up, and in Rift Valley fever
It has been well used in the detection of virus, has substantially reduced the time of detection.Using RT-PCR technology as example multiple RT- of representative
PCR, RT- nest-type PRC and real-time quantitative RT-PCR are with the specificity of its height and sensibility in Rift Valley fever virus context of detection
Achieve great breakthrough.But these methods are needed using complicated instrument and well-equipped laboratory.Equal isothermal nucleic acids skill
RT-LAMP methods in art overcome the shortcomings of RT-PCR technology needs expensive equipment and instrument, have easy to operate, as a result sentence
The advantages that disconnected simple.But RT-LAMP technologies require multipair primer and to the more demanding of target gene, false positive rate also compares
It is high.
Invention content
The purpose of the present invention is to provide the primed probe group of RAA Fluorometric assay Rift Valley fever virus and kits.This hair
The primed probe group of bright offer has the characteristics that high specificity, high sensitivity, reproducible, can be in 15min using the kit
Inside detect Rift Valley fever virus RNA.
The present invention provides the primed probe groups of RAA Fluorometric assay Rift Valley fever virus, including sense primer, downstream primer
And probe, the nucleotide sequence of the sense primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is such as
Shown in SEQ ID NO.2, the probe is that the 28th bit base of the nucleotide sequence shown in SEQ ID NO.3 modifies fluorescence report
Group, the 29th bit base replace with the substance of tetrahydrofuran residue and the 30th bit base modification fluorescent quenching group.
Preferably, the fluorescent reporter group includes FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5.
Preferably, the fluorescent quenching group includes TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.
The present invention also provides a kind of kits of RAA Fluorometric assays Rift Valley fever virus, and the kit includes above-mentioned
Primed probe group described in technical solution.
Preferably, the probe uses a concentration of 0.01~0.03mM.
Preferably, the use concentration of the sense primer and downstream primer independently is 0.02~0.05mM.
Preferably, the kit further includes the general reaction reagent of RAA basic fluorescences and reaction buffer.
Preferably, the reaction buffer includes：The Tris-HCl for the use of a concentration of 500mM pH value being 7.4 is buffered
The PEG 10000 that liquid, the MgAc of 240mM and mass fraction are 10%.
Preferably, the kit further includes：Negative quality-control product and positive quality control product；
The feminine gender quality-control product is ddH2O or purified water；
The positive quality control product is Rift Valley fever virus recombinant dna plasmid.
The present invention provides the primed probe groups of RAA Fluorometric assay Rift Valley fever virus.Primed probe provided by the invention
Group can realize the quick and precisely identification of Rift Valley fever virus, and easy to operate, detection time is short, specificity up to 100%.Experiment knot
Fruit shows that primed probe group provided by the invention only needs to can be completed in 15min, so that DNA is untwisted without passing through high temperature, only need to be
Isothermal duplication being carried out at 30~42 DEG C, detection can be completed, detection is quick, sensitive, specific good, non-false positive；Future is detected
Rift Valley fever virus propagation has very important significance, and has great application prospect.
Description of the drawings
Fig. 1 is the various concentration Plasmid DNA sensitivity technique result figure that the embodiment of the present invention 2 provides；
Fig. 2 is the Rift Valley fever virus Plasmid DNA repeatability testing result figure that the embodiment of the present invention 3 provides；
Fig. 3 is the specific detection result figure for the Rift Valley fever virus that the embodiment of the present invention 4 provides.
Specific implementation mode
The present invention provides the primed probe groups of RAA Fluorometric assay Rift Valley fever virus, including sense primer, downstream primer
And probe, the nucleotide sequence of the sense primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is such as
Shown in SEQ ID NO.2, the probe is that the 28th bit base of the nucleotide sequence shown in SEQ ID NO.3 modifies fluorescence report
Group, the 29th bit base replace with the substance of tetrahydrofuran residue and the 30th bit base modification fluorescent quenching group.
In the present invention, the nucleotide sequence SEQ ID NO.1 of the sense primer are 5 '-
CATTTTCATCATCATCCTCCKGGGCTTRTTG-3’；The nucleotide sequence SEQ ID NO.2 of the downstream primer are 5 '-
GARCTCYTAAAGCAGTATGGTGGGGCTGACT-3’；The present invention is to the source of the sense primer and downstream primer without spy
Different restriction, carried out using biotech firm well known to those skilled in the art it is artificial synthesized, as raw work bioengineering (on
Sea) limited liability company.
Probe of the present invention is that the 28th bit base of the nucleotide sequence shown in SEQ ID NO.3 modifies fluorescence report base
Group, the 29th bit base replace with the substance of tetrahydrofuran residue and the 30th bit base modification fluorescent quenching group.In the present invention,
The probe is before modification, i.e. nucleotides sequence is classified as 5 '-shown in SEQ ID NO.3
GGGAGAAGGATGCCAAGAAAATGATTGTTCTGGCTCTRACTCGTG-3’.In the present invention, the fluorescent reporter group
Including FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5, more preferably FAM or HEX；The quenching group include TAMRA,
Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL, more preferably BHQ1 or BHQ2；The present invention is to different fluorescent reporter genes
The not special restriction with the combination of quenching group, above-mentioned fluorescent reporter group and quencher, which are combined, can reach phase
Same effect, the present invention do not have the instrument of fluoroscopic examination special restriction, using well-known to those skilled in the art corresponding
Fluoroscopic examination instrument, such as when the fluorescent reporter group is FAM, and the fluorescent quenching group is BHQ1, using nothing
The RAA-F1620 fluorogene detectors of the strange day biology scientific instrument Co., Ltd production of tin are detected.
The sequence that probe is obtained after modification of the present invention is：
5’-GGGAGAAGGATGCCAAGAAAATGATTGT(BHQ1-dT)(THF)(FAM-dT)GGCTCTRACTCGTG-
3 ', wherein FAM is fluorescent reporter group, and BHQ1 is fluorescent quenching group, and THF is tetrahydrofuran residue.The present invention draws to described
The source of object and probe does not have special restriction, is synthesized using biotech firm well known to those skilled in the art.
The present invention also provides the kit of RAA Fluorometric assay Rift Valley fever virus, the kit includes above-mentioned technology
Primed probe group described in scheme.The present invention does not have special limit to storage concentration of the primer and probe in kit
It is fixed.In the present invention, the probe uses a concentration of 0.01~0.03mM, more preferably 0.02mM.In the present invention, described
The use concentration of sense primer and downstream primer independently is 0.02~0.05mM, independently more preferably is 0.03mM.
In the present invention, the kit further includes the general reaction reagent of RAA basic fluorescences and reaction buffer.In this hair
In bright, the general reaction reagent of RAA basic fluorescences is preferably the freeze-dried powder by frozen drying；In the implementation of the present invention
In example, the source of the general reaction reagent of RAA basic fluorescences can be Jiangsu Qi Tian genes bio tech ltd, commodity
Article No. is the commercial goods of F00001, and the reaction specification of the general reaction reagent of RAA basic fluorescences is 50 μ L, uses preceding application response
It is molten that buffer solution carries out weight.In the present invention, the general reaction reagent of RAA basic fluorescences also can be according to the Fluorometric assay sides RAA
The principle of method selects other can be as the product of the general reaction reagent of RAA basic fluorescences as replacement.The present invention is to described anti-
Storage concentration of the buffer solution in kit is answered not have special restriction；The reaction buffer includes：Use a concentration of 500mM
The PEG 10000 that the MgAc and mass fraction for the Tris-HCl buffer solutions, 240mM that pH value is 7.4 are 10%.In the present invention, institute
The concentration for stating Tris-HCl buffer solutions, MgAc and PEG 10000 is final concentration.The present invention to the Tris-HCl buffer solutions,
The source of MgAc and PEG 10000 are not particularly limited, using conventional commercial product.Described in the embodiment of the present invention
Tris-HCl's and PEG 10000 is purchased from Sigma-Aldrich, and MgAc is tried purchased from traditional Chinese medicines Shanghai.
In the present invention, the kit further includes：Negative quality-control product and positive quality control product；
The feminine gender quality-control product is ddH2O or purified water；
The positive quality control product contain be Rift Valley fever virus recombinant dna plasmid, the Rift Valley fever virus recombinant dna plasmid it is dense
Degree is 1.0 × 103~1.0 × 106copies/μL.In the present invention, the use concentration of the positive quality control product is more preferably 1.0
×104~1.0 × 105Copies/ μ L, most preferably 1.0 × 105copies/μL.Positive quality control product of the present invention is preferably logical
Recombinant plasmid switching Escherichia coli are crossed to cultivate and extract.Positive quality control product of the present invention can also act as the positive of sensitivity experiment
Standard items realize the detection of sensitivity, when the positive quality control product is used as positive criteria by adjusting the concentration of positive quality control product
When product, the concentration of the positive criteria product is preferably regulated as 1.0 × 100~1.0 × 1010Copies/ μ L are spare.
Kit of the present invention is for detecting Rift Valley fever virus, and in the present invention, the application method of the kit is excellent
Choosing includes the following steps：
(1) RNA of extraction sample to be tested (doubtful Rift Valley fever virus)；The method of extraction of the present invention preferably uses commercially available
The Viral nucleic acid extraction reagent of commercialization, by specification are operated, or using the grand nucleic acid automatic extracting instrument in Xi'an day, or west
Pacify the extraction that the grand mating nucleic acid extracting reagent in day carries out RNA；The RNA mentioned is saved backup at -20 DEG C；
(2) detecting instrument RAA-F1620 is powered on and is preheated, response parameter is configured, response parameter is set
39 DEG C are set to, the reaction time：20min.
(3) RNA for obtaining extraction carries out external reverse transcription；In the present invention, reverse transcriptase is preferably Promega M-
MLV (200U/ μ L) or Takara M-MLV (200U/ μ L), the amount that reverse transcriptase is added in external transcriptive process,reversed press different factories
The amount of recommending in the specification of family.Specifically, the present invention is directly added in each general reaction reagent of RAA basic fluorescences
0.2~0.6 μ L of reverse transcriptase, are preferably added to 0.4 μ L；It is directly realized by RT-RAA reactions；
(4) 1 μ L probes and 1 μ L primers are added in 42.6 μ L reaction buffers, 0.4 μ L reverse transcriptase adds after being sufficiently mixed
It is added in the general reaction reagent of RAA basic fluorescences and mixes；Obtain reaction mixture；
(5) sample to be tested obtained in step (1) described in 5 μ L is filled with the reaction mixture obtained in the step (4)
Point mixing, obtained reaction system, which carries out being put into detecting instrument RAA-F1620, is detected fluorescence signal；
(6) positive is determined as when by slope value K >=20 according to the positive determination method in RAA-F1620 detecting instruments.
It is determined as feminine gender when slope value K ＜ 20.
In the present invention, the sample to be tested is to be possible to the sample containing Rift Valley fever virus, and the present invention is to sample to be tested
Extracting method there is no special restriction, using Plasmid DNA or the general extraction methods of viral RNA.
In the present invention, the real-time RAA fluorescence methods reaction condition is preferably：30~42 DEG C of reaction temperature, reaction time 5
~20min, more preferable 39 DEG C for fluorescence real-time fluorescence, reaction time 20min.
When kit provided by the invention detection Rift Valley fever virus with other insect-borne infectious disease encephalitis, Dengue pyreticosis, yellow heat,
Marburg no cross reaction has high specificity, specific up to 100%.High sensitivity is reached for per reaction detection sensitivity
10Copies.And kit provided by the invention, large-scale instrument and equipment is not needed, is suitable for Site Detection and suitable for extensive
Screening.
With reference to specific embodiment to the primed probe group of RAA Fluorometric assays Rift Valley fever virus of the present invention and
Kit is further described in detail, and technical scheme of the present invention includes but not limited to following embodiment.
Embodiment 1
The present invention extracts Rift Valley fever virus sample rna and verifies, according to Gene Name Rift Valley fever virus (Rift
Valley fever virus, RVFV), corresponding complete genome sequence is found in genebank
(www.ncbi.nlm.nih.gov), homology analysis and b1ast sequence analyses are carried out with DNASTAR softwares, sifts out rift valley
Fever virus highly conserved sequence is as shown in SEQ ID NO.4：
GTTGATGAGAGCCTCCACAGTTGCTTTGCCTTCTTTCGACATTTTCATCATCATCCTCCGGGGCTTGTTGCCACGAG
TCAGAGCCAGAACAATCATTTTCTTGGCATCCTTCTCCCAGTCAGCCCCACCATACTGCTTTAAGAGTTCGATAACC
CTACGAGCATCAAACCCTTGATAAGCAAACTCTCGGACCCACT。
Target gene of the present invention according to highly conserved sequence as detection, synthesis Rift Valley fever virus recombinant dna plasmid is simultaneously
Design primer probe.
Entrust Sangon Biotech (Shanghai) Co., Ltd. according to the above sequence) limited liability company carry out synthesis Rift Valley fever virus recombination
DNA plasmid, plasmid size 197bp；
1 primed probe designs
It is finally determined according to the design that RAA technologies primer and probe design principle carry out by screening and evaluating：
Upstream primer sequence 5 '-CATTTTCATCATCATCCTCCKGGGCTTRTTG-3 '；
Downstream primer sequence 5 '-GARCTCYTAAAGCAGTATGGTGGGGCTGACT-3 '；
Unmodified probe is：
5’-GGGAGAAGGATGCCAAGAAAATGATTGTTCTGGCTCTRACTCGTG-3’。
2 selection fluorescent decoration groups and fluorescent quenching group
According to laboratory apparatus using the RAA-F1620 fluorescent bases of the strange day biology scientific instrument Co., Ltd production in Wuxi
Because of detector, the fluorescence detected is FAM fluorescence, therefore fluorescence modification group is selected as FAM, and fluorescent quenching group is selected as BHQ1.
The method of modifying of 3 probes includes：Fluorescent reporter group modification is in probe sequence from the ends 5' base number 28bp's
On position；Fluorescent quenching group modify on position of the probe sequence from the ends 5' base number 30bp, fluorescent reporter group be quenched
1 base C is spaced between group, which is replaced with tetrahydrofuran residue.It is by the probe of modification：5’-
GGGAGAAGGATGCCAAGAAAATGATTGT(BHQ1-dT)(THF)(FAM-dT)GGCTCTRACTCGTG-3’；
Wherein, FAM is fluorescent reporter group, and BHQ1 is fluorescent quenching group, and THF is tetrahydrofuran residue.
4 primed probes and plasmid entrust Sangon Biotech (Shanghai) Co., Ltd.) limited liability company synthesized.
It is prepared by 5 Rift Valley fever virus recombinant dna plasmid positive criteria products
Rift Valley fever virus recombinant dna plasmid is cultivated and is extracted by Escherichia coli of transferring, and uses ultramicron purple after obtaining plasmid
Outer spectrophotometer carries out concentration mensuration and carries out copy number calculating, and 1.0 × 10 are prepared by concentration gradient dilution0copies/μ
L~1.0 × 1010Copies/ μ L are spare.
6 composition kits
Kit of the present invention includes the general reaction reagent of RAA basic fluorescences, reaction buffer, positive quality control product, negative matter
Control product, primer and probe；
The concentration of sense primer and downstream primer stands alone as 0.04mmol/L.
A concentration of 0.03mmol/L of probe.
The general reaction reagent of RAA basic fluorescences is purchased from Jiangsu Qi Tian genes bio tech ltd, article No. F00001.
The reaction specification of the general reaction reagent of RAA basic fluorescences is 50 μ L, and it is molten to carry out weight using preceding application response buffer solution.
The reaction buffer of the molten general reaction reagent of RAA basic fluorescences of weight includes the component of following content：It is a concentration of
The PEG that Tris-HCl (PH7.4) buffer solution of 500mmol/L, the MgAc of a concentration of 240mmol/L and mass fraction are 10%
10000。
Positive quality control product：Containing Rift Valley fever virus recombinant dna plasmid, the concentration of the Rift Valley fever virus recombinant dna plasmid
It is 1 × 105Copies/μL。
Negative quality-control product：ddH2O。
Embodiment 2
Sensitivity experiment
Sense primer：
5’-CATTTTCATCATCATCCTCCKGGGCTTRTTG-3’；
Downstream primer：
5’-GARCTCYTAAAGCAGTATGGTGGGGCTGACT-3’。
Probe sequence is：
GGGAGAAGGATGCCAAGAAAATGATTGTTCTGGCTCTRACTCGTG
Fluorescent reporter group uses FAM, fluorescent quenching group to use BHQ1；
Probe after modification is：
GGGAGAAGGATGCCAAGAAAATGATTGT(BHQ1-dT)(THF)(FAM-dT)GGCTCTRACTCGTG；
Kit forms are as shown in table 1：
1 Kit components table of table
The working standard for the various concentration that Rift Valley fever virus recombinant dna plasmid positive criteria product prepares, respectively：
Working standard (positive quality control product) 1 contains 1.0 × 106Copies/ μ L Rift Valley fever virus recombinant dna plasmids.
Working standard (positive quality control product) 2 contains 1.0 × 105Copies/ μ L Rift Valley fever virus recombinant dna plasmids.
Working standard (positive quality control product) 3 contains 1.0 × 104Copies/ μ L Rift Valley fever virus recombinant dna plasmids.
Working standard (positive quality control product) 4 contains 1.0 × 103Copies/ μ L Rift Valley fever virus recombinant dna plasmids.
Working standard (positive quality control product) 5 contains 1.0 × 102Copies/ μ L Rift Valley fever virus recombinant dna plasmids.
Working standard (positive quality control product) 6 contains 1.0 × 101Copies/ μ L Rift Valley fever virus recombinant dna plasmids.
Implementation：
The preparation of 1 reaction buffer
301 μ L reaction buffers are drawn in reaction buffer pipe in kit, and preprepared 1.5mLPE pipes are added,
The mixture (a concentration of 0.02mmol/ of probe, a concentration of 0.05mmol/L of primer) for adding 16 μ L probes and primer, fills
Divide mixing, obtains the reaction buffer after mixing.
2RAA fluorescence basis reaction reagent weight is molten
Prepare 7 RAA fluorescence basis reaction reagents, the 45 μ L of reaction buffer of mixing are added separately in aspiration step 1
In the reaction reagent pipe of ready 7 RAA fluorescence basis, so that freeze-dried powder is fully dissolved and is mixed, become RAA reaction systems.
3, sample-adding reaction
5 μ L feminine genders quality-control products, 5 μ L works are separately added into the reaction reagent test tube of above 7 prepared RAA fluorescence basis
Make standard items 6,5 μ L working standards 5,5 μ L working standards 4,5 μ L working standards 3,5 μ L working standards 2,5 μ L work
Standard items 1 are template, and each reaction tube is fully mixed after having added sample, and each reaction tube total volume is 50 μ L.
Reaction tube is put into RAA-F1620 fluorescence detectors, sets reaction temperature as 39 DEG C, 20 minutes reaction time.
Testing result is as shown in Figure 1.As a result showing obviously there is amplification most fast 5 minutes, all standard work product have amplification in 15 minutes,
Sensitivity minimization can reach 1.0 × 101Copies/ μ L show that high sensitivity of the present invention, detection time are short.
Embodiment 3
Repeated experiment
Primed probe and positive quality control product sequence are same as Example 1.
Kit forms are as shown in table 2：
2 Kit components table of table
Reaction buffer is prepared：
173 μ L reaction buffers are drawn in reaction buffer pipe in kit, and preprepared 1.5mlPE pipes are added,
The mixture (a concentration of 0.02mmol/ of probe, a concentration of 0.04mmol/L of primer) for adding 8 μ L probes and primer, fills
Divide mixing, obtains the reaction buffer after mixing.
2RAA fluorescence basis reaction reagent weight is molten
Prepare 4 RAA fluorescence basis reaction reagents, the reaction buffer for drawing mixing in 45 μ L steps 1 every time adds respectively
Enter into ready 4 RAA fluorescence basis reaction reagent pipe, so that freeze-dried powder is fully dissolved and is mixed, become RAA reactants
System, and mark.
3, sample-adding reaction
Be separately added into the reaction reagent test tube of above 4 prepared RAA fluorescence basis 5 μ L feminine genders quality-control products, other 3
5 μ L positive quality control products are separately added into a reaction tube；Often plus what a sample just covers pipe lid.Each reaction tube after sample is added
It is fully mixed, each reaction tube total volume is 50 μ L.
4 reaction tubes of mixing are put into RAA-F1620 fluorescence detectors, set reaction temperature as 39 DEG C, when reaction
Between 20 minutes.Testing result is as shown in Figure 2.As a result show that amplified reaction is reproducible.
Embodiment 4
Specificity experiments
Primed probe and positive quality control product sequence are same as Example 1.
Kit forms are as shown in table 3：
3 Kit components table of table
Samples sources in specificity experiments：Encephalitis, Dengue pyreticosis, yellow heat, Marburg.
Wherein encephalitis, Dengue pyreticosis sample are provided by Zhejiang international travel health center, and grand full-automatic by Xi'an day
Instrument for extracting nucleic acid extracts viral RNA, and yellow fever virus RNA extracts gained, Marburg Plasmid DNA by Chinese disease from yellow hot vaccine
Prevention and control center virosis is provided.Rift Valley fever uses Plasmid DNA.
Implementation：
The preparation of 1 reaction buffer
Sample rna reaction buffer is prepared：127.8 μ L reaction buffers are drawn in reaction buffer pipe in kit to add
Enter preprepared 1.5mLPE pipe, adding the mixture of 6 μ L probes and primer, (a concentration of 0.03mmol/ of probe, draws
A concentration of 0.05mmol/L of object), 1.2 μ LPromega M-MLV (200U/ μ L) reverse transcriptase is added, mixes well, obtains mixed
Reaction buffer 1 after even.
DNA reaction buffers are prepared：130 μ L reaction buffers are drawn in reaction buffer pipe in kit to be added in advance
Ready 1.5mlPE pipe, add 6 μ L probes and primer mixture (a concentration of 0.03mmol/ of probe, primer it is dense
Degree is 0.05mmol/L), it mixes well, obtains the reaction buffer after mixing 2.
2RAA fluorescence basis reaction reagent weight is molten
Prepare 3 RAA fluorescence basis reaction reagents, the reaction buffer 2 for drawing mixing in 45 μ L steps 1 every time adds respectively
Enter into ready 3 RAA fluorescence basis reaction reagent pipe, so that freeze-dried powder is fully dissolved and is mixed, become RAA reactants
System, and mark.
Separately prepare 3 RAA fluorescence basis reaction reagents, the reaction buffer 1 for drawing mixing in 45 μ L steps 1 every time is distinguished
It is added in the reaction reagent pipe of ready 3 RAA fluorescence basis, so that freeze-dried powder is fully dissolved and is mixed, it is anti-to become RT-RAA
System is answered, and is marked.
3, sample-adding reaction
5 μ L feminine genders quality-control products, 5 μ L horses are separately added into the reaction reagent test tube of above 3 prepared RAA fluorescence basis
That fort Plasmid DNA, 5 μ L positive quality control products；Often plus what a sample just covers pipe lid.Each reaction tube is filled after having added sample
Divide and be mixed, each reaction tube total volume is 50 μ L.Divide in the reaction reagent test tube of above 3 prepared RT-RAA fluorescence basis
It is template that 5 μ L encephalitis B virus RNA, 5 μ L Dengue pyreticosis viral RNAs, 5 μ L yellow fever virus RNA are not added, and often plus what a sample just covers
Good pipe lid.Each reaction tube is fully mixed after having added sample, and each reaction tube total volume is 50 μ L.
6 reaction tubes of mixing are put into RAA-F1620 fluorescence detectors, set reaction temperature as 39 DEG C, when reaction
Between 20 minutes.Testing result is as shown in Figure 3.As a result display only has Rift Valley fever Plasmid DNA to have amplification, and for the positive, other samples are such as
Marburg Plasmid DNA, encephalitis B virus RNA, Dengue pyreticosis viral RNA, yellow fever virus RNA and negative quality-control product do not expand,
It is feminine gender.Show detection method high specificity provided by the invention.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Zhejiang International Travel Healthcare Center（Port clinic of Zhejiang Entry-Exit Inspection and Quarantine Bureau）
Jiangsu Qi Tian genes bio tech ltd
<120>The primed probe group and kit of RAA Fluorometric assay Rift Valley fever virus
<160> 4
<170> SIPOSequenceListing 1.0
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<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
garctcytaa agcagtatgg tggggctgac t 31
<210> 3
<211> 45
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gggagaagga tgccaagaaa atgattgttc tggctctrac tcgtg 45
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<213>Artificial sequence (Artificial Sequence)
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gggcttgttg ccacgagtca gagccagaac aatcattttc ttggcatcct tctcccagtc 120
agccccacca tactgcttta agagttcgat aaccctacga gcatcaaacc cttgataagc 180
aaactctcgg acccact 197
Claims (9)
- The primed probe group of 1.RAA Fluorometric assay Rift Valley fever virus, including sense primer, downstream primer and probe, feature It is, the nucleotide sequence of the sense primer is as shown in SEQ ID NO.1, the nucleotide sequence such as SEQ of the downstream primer Shown in ID NO.2, the probe is that the 28th bit base of the nucleotide sequence shown in SEQ ID NO.3 modifies fluorescence report base Group, the 29th bit base replace with the substance of tetrahydrofuran residue and the 30th bit base modification fluorescent quenching group.
- 2. primed probe group according to claim 1, which is characterized in that the fluorescent reporter group include FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5.
- 3. primed probe group according to claim 1, which is characterized in that the fluorescent quenching group include TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.
- The kit of 4.RAA Fluorometric assay Rift Valley fever virus, which is characterized in that the kit includes that claims 1 to 3 is appointed Primed probe group described in meaning one.
- 5. kit according to claim 4, which is characterized in that the probe uses a concentration of 0.01~0.03mM.
- 6. kit according to claim 4, which is characterized in that the use concentration of the sense primer and downstream primer is only It is on the spot 0.02~0.05mM.
- 7. according to the kit described in claim 4~6 any one, which is characterized in that further include that RAA basic fluorescences are general anti- Answer reagent and reaction buffer.
- 8. kit according to claim 7, which is characterized in that the reaction buffer includes：Use a concentration of 500mM The PEG 10000 that the MgAc and mass fraction for the Tris-HCl buffer solutions, 240mM that pH value is 7.4 are 10%.
- 9. kit according to claim 4, which is characterized in that further include：Negative quality-control product and positive quality control product；The feminine gender quality-control product is ddH2O or purified water；The positive quality control product is Rift Valley fever virus recombinant dna plasmid.
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| CN201810644415.0A CN108624720A (en) | 2018-06-21 | 2018-06-21 | The primed probe group and kit of RAA Fluorometric assay Rift Valley fever virus |
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| CN109593889A (en) * | 2018-12-29 | 2019-04-09 | 广州动佰生物科技有限公司 | Pig Delta coronavirus detection method based on real-time fluorescent reverse transcription recombinase-mediated chain replacement nucleic acid amplification technologies |
| CN110592285A (en) * | 2019-10-21 | 2019-12-20 | 中国动物卫生与流行病学中心 | RAA primer probe for detecting Nipah virus and detection method |
| CN112391493A (en) * | 2020-12-10 | 2021-02-23 | 浙江省检验检疫科学技术研究院 | RAA fluorescence detection method, primer probe and kit for citrus greening disease Asian species |
| CN112646911A (en) * | 2021-01-15 | 2021-04-13 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Primer probe composition and kit for real-time fluorescence RAA detection of DNA of circumsporozoon and application of primer probe composition and kit |
| CN114045362A (en) * | 2021-12-02 | 2022-02-15 | 四川农业大学 | Reagent and kit for detecting Japanese encephalitis virus based on RT-RAA fluorescence method |
| CN114959120A (en) * | 2022-06-30 | 2022-08-30 | 华南农业大学 | Primer probe set and kit for detecting chicken infectious anemia virus by RAA fluorescence method and application of primer probe set and kit |
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Cited By (7)
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| CN109593889A (en) * | 2018-12-29 | 2019-04-09 | 广州动佰生物科技有限公司 | Pig Delta coronavirus detection method based on real-time fluorescent reverse transcription recombinase-mediated chain replacement nucleic acid amplification technologies |
| CN110592285A (en) * | 2019-10-21 | 2019-12-20 | 中国动物卫生与流行病学中心 | RAA primer probe for detecting Nipah virus and detection method |
| CN112391493A (en) * | 2020-12-10 | 2021-02-23 | 浙江省检验检疫科学技术研究院 | RAA fluorescence detection method, primer probe and kit for citrus greening disease Asian species |
| CN112646911A (en) * | 2021-01-15 | 2021-04-13 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Primer probe composition and kit for real-time fluorescence RAA detection of DNA of circumsporozoon and application of primer probe composition and kit |
| CN114045362A (en) * | 2021-12-02 | 2022-02-15 | 四川农业大学 | Reagent and kit for detecting Japanese encephalitis virus based on RT-RAA fluorescence method |
| CN114045362B (en) * | 2021-12-02 | 2023-05-09 | 四川农业大学 | Reagent and kit for detecting Japanese encephalitis virus based on RT-RAA fluorescence method |
| CN114959120A (en) * | 2022-06-30 | 2022-08-30 | 华南农业大学 | Primer probe set and kit for detecting chicken infectious anemia virus by RAA fluorescence method and application of primer probe set and kit |
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