CN102952892A - Loop-mediated isothermal amplification kit for detecting West Nile viruses, and its application - Google Patents
Loop-mediated isothermal amplification kit for detecting West Nile viruses, and its application Download PDFInfo
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Abstract
The invention discloses a loop-mediated isothermal amplification kit for detecting West Nile viruses, and its application. Special primers provided by the invention comprise a first primer, a second primer, a third primer and a fourth primer, and the nucleotide sequences of the first primer, the second primer, the third primer and the fourth primer are respectively represented by sequence 1, sequence 2, sequence 3 and sequence 4 in a sequence table. Experiments in the invention prove that the special primers are suitable for detecting the specificities of the West Nile viruses. The loop-mediated isothermal amplification kit has the advantages of performing of an amplification reaction at a constant temperature without special equipment, high specificity, rapidness and high efficiency to realize the completion of the amplification reaction within 35min, high sensitivity, and convenient and simple identification. The loop-mediated isothermal amplification kit is especially suitable for rapidly detecting clinic specimens and field onsite specimens.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of loop-mediated isothermal amplification kit and application thereof that detects west nile virus.
Background technology
The west Nile fever that West Nile virus infection causes is the zoonosis that the mosquito matchmaker propagates.Most of patients is inferior clinical symptom, and west Nile fever can appear in about 20% the infected behind 3-14d.Its illness may comprise fever, headache, physical distress, lymph gland swelling, and the eruption symptom is arranged sometimes.Illness continues an about week usually.West Nile virus infection can cause viral encephalitis, causes high fever, headache, mental derangement, faint, paralysis sometimes also can occur, can cause death accidentally.
West nile virus 1999 in the past mainly in Africa, the Middle East, West Asia and Europe is popular.Romania in 1996 break out Europe first west Nile fever be very popular.Popular 14 areas that concentrate on the basin, Danube, viral infection rate is about 12,14/,100,000.Russian south in 1999 breaks out also that west nile encephalitis is popular on a large scale, and 1000 cases are arranged approximately, and at least 40 people are dead.U.S.'s the first explosion west Nile fever was popular in 1999, and this is that west Nile fever is popular first in the western hemisphere.Since then, it is popular that the U.S. all breaks out west Nile fever/west nile encephalitis over the years, and west nile virus distributes and to spread to 2004 from 4 states in 1999 and be close on 50 states, and propagate into the Canadian middle and south and Caribbean.Even to this day, West Nile virus infection has become global serious public health health problem.
The main dependence of west nile encephalitis diagnosis detected virus and antibody thereof because virus load is low during viremia, and decubation virus be eliminated therefore isolated viral difficulty comparatively from blood or cerebrospinal fluid.New diagnostic techniques comprises with the ELISA method to be surveyed antigen or measures west nile virus nucleic acid with PCR and PCR in real time.Wherein PCR in real time is the most responsive in these diagnostic techniques methods, but still can not detect all west nile encephalitis patients.These traditional methods etc. often need to take a long time or need specific instrument, are difficult to satisfy the needs of epidemic outbreaks quick diagnosis.
Ring mediated isothermal amplification method (loop-mediated isothermal amplification, LAMP) method has the advantages that to get final product high speed, quick, high special, high-sensitive amplified target sequence under isothermal condition.When nucleic acid synthesized in a large number, dNTP can separate out a large amount of pyrophosphate ions, the Mg in pyrophosphate ion and the LAMP system
2+(or Mn
2+) combination, produce magnesium pyrophosphate or manganese pyrophosphate precipitation, thereby the turbidity of the liquid that induces reaction changes, and is highly susceptible to judging.In addition, utilize some fluorochromes, react such as SYBR green I dyestuff or with fluorexon indication LAMP.SYBRgreen I dyestuff can be combined with the double-stranded DNA product, and when having product to increase in a large number, reaction system presents significant fluorescent signal and changes.Fluorexon is in reaction when initial and Mn
2+In conjunction with, it is orange that reaction solution shows transparency, and when having nucleic acid product to synthesize in a large number, can generate the by product pyrophosphate, makes Mg
2+Dissociate out, be combined with fluorexon, finally make reaction solution show little turbid yellow-green colour.Because LAMP method test requirement condition is low, the result is easy to judge that the investigation of very suitable basic unit virus is carried out timely animal health prevention and control.
Summary of the invention
An object of the present invention is to provide a kind of primer special that detects west nile virus.
Primer special provided by the invention comprises primer 1, primer 2, primer 3 and primer 4, and the nucleotide sequence of described primer 1, described primer 2, described primer 3 and described primer 4 is respectively sequence 1, sequence 2, sequence 3 and the sequence 4 in the sequence table.
Described primer special also comprises primer 5, and the nucleotides sequence of described primer 5 is classified the sequence 5 of sequence table as.
Described primer special is comprised of described primer 1, described primer 2, described primer 3, described primer 4 and described primer 5;
Described west nile virus is specially west nile virus Chin-01 strain.
In the described primer special, 5 ' end and/or the 3 ' end of F3, B3, FIP, BIP, LB also can prolong according to target sequence; In the described primer special, F3, B3, FIP, BIP, LB also can carry out replacement and/or the replacement of base, keep the homology of 6 bases 100% of 3 ' end sequence to get final product.TTTT among FIP and the BIP (underscore person) is catenation sequence, and its length is generally 4, and number can corresponding shortening and prolongation.
Second purpose of the present invention provides a kind of ring mediated isothermal amplification (LAMP) reagent that detects west nile virus.
Reagent provided by the invention comprises RNA reversed transcriptive enzyme, Bst archaeal dna polymerase, 2 * ring mediated isothermal amplification damping fluid, described primer special.
Described amplifing reagent also comprises fluorexon;
Described amplifing reagent is comprised of RNA reversed transcriptive enzyme, Bst archaeal dna polymerase, 2 * ring mediated isothermal amplification damping fluid, described primer special and fluorexon;
Described primer 5 can be combined with loop-stem structure that to start strand displacement synthetic, improves amplification efficiency.The final product of amplification is the loop-stem structure with different numbers, the mixture of different lengths DNA, and its output can reach more than the 10 μ g, is at least 50 times of common PCR reaction DNA productive rate.
The concentration of described RNA reversed transcriptive enzyme in described ring mediated isothermal amplification reagent is 0.004U/ μ L;
Described RNA reversed transcriptive enzyme is specially the AMV reversed transcriptive enzyme;
The concentration of described Bst archaeal dna polymerase in described ring mediated isothermal amplification reagent is 0.32U/ μ L;
The concentration of described fluorexon in described ring mediated isothermal amplification reagent is 50 μ mol/L;
Described 2 * ring mediated isothermal amplification damping fluid is prepared as follows: with Repone K (KCl), sal epsom (MgSO
4), ammonium sulfate ((NH
4)
2SO
4), polysorbas20 (Tween-20), trimethyl-glycine (Betaine), Manganous chloride tetrahydrate (MnCl
2) and dNTPs (deoxynucleoside triphosphate mixture: dATP, dCTP, dGTP and dTTP) to be dissolved in concentration be that 40mmol/L, pH value are that 8.8 Tris hydrochloride damping fluid obtains;
In described 2 * ring mediated isothermal amplification damping fluid, the concentration of described Repone K is 20mmol/L, the concentration of described sal epsom is 16mmol/L, the concentration of described ammonium sulfate is 20mmol/L, the concentration of described polysorbas20 is 0.2% (quality percentage composition), the concentration of described trimethyl-glycine is 1.6mol/L, and the concentration of described Manganous chloride tetrahydrate is 1mmol/L, and the concentration of every kind of described deoxynucleoside triphosphate is 2.8mmol/L.
Described west nile virus is specially west nile virus Chin-01 strain.
The 3rd purpose of the present invention provides a kind of test kit that detects west nile virus.
Test kit provided by the invention comprises described ring mediated isothermal amplification reagent;
Described west nile virus is specially west nile virus Chin-01 strain.
The application in evaluation and/or assistant identification west nile virus of described primer special or described ring mediated isothermal amplification reagent or described test kit also is the scope of protection of the invention.
Described west nile virus is specially west nile virus Chin-01 strain.
The 4th purpose of the present invention provides the method for west nile virus in a kind of evaluation and/or the assistant identification testing sample.
Method provided by the invention comprises the steps:
With the described primer special in described primer special or described ring mediated isothermal amplification reagent or the described test kit testing sample is carried out ring mediated isothermal amplification, detection ring mediated isothermal amplification product;
In the described ring mediated isothermal amplification, take the RNA of testing sample as template;
Described loop-mediated isothermal amplification condition is: 60 ℃-65 ℃ are reacted 35-60min, then 75 ℃ of 2min termination reactions first.
Described loop-mediated isothermal amplification condition is: 63 ℃ are reacted 45min or 60min, then 75 ℃ of 2min termination reactions first;
Described sample to be tested is the brain of mouse.
Detected object of the present invention is not limited to the mouse brain tissue samples, also can relate to mosquito sample, animal or human serum, cerebrospinal fluid, cerebral tissue or organs and tissues etc. become celestial;
Described west nile virus is west nile virus Chin-01 strain.
The method of described detection ring mediated isothermal amplification product is following 1)-4) at least a:
1) observes described loop-mediated isothermal amplification product, if described product is yellow-green colour, and glassy yellow fluorescence is arranged under UV-light under natural light, then contain in the sample to be tested or the candidate is contained west nile virus; If described product is orange-yellow under natural light, and under UV-light without fluorescence, then do not contain in the sample to be tested or the candidate is not contained west nile virus;
2) detect described loop-mediated isothermal amplification product with real-time turbidimeter, if the specific amplified curve is arranged, then contain in the sample to be tested or the candidate is contained west nile virus; If without the specific amplified curve, then do not contain in the sample to be tested or the candidate is not contained west nile virus;
3) the described loop-mediated isothermal amplification product of electrophoresis detection obtains the amplified reaction product of gradient batten band, then contains in the sample to be tested or the candidate is contained west nile virus; If the loop-mediated isothermal amplification product is not gradient batten band, then do not contain in the sample to be tested or the candidate is not contained west nile virus;
The size of described gradient batten band is 200bp-2000bp, and the size of described gradient batten band mainly is distributed between 200bp-2000bp;
4) cut described loop-mediated isothermal amplification product with Sca I enzyme, the described enzyme of electrophoresis detection is cut product, is 244bp product and 164bp product if described enzyme is cut product, then contains in the sample to be tested or the candidate is contained west nile virus; If described enzyme is cut product for 244bp product and 164bp product, then do not contain in the sample to be tested or the candidate is not contained west nile virus.
Described detection ring mediated isothermal amplification product adopts the variation of visual inspection turbidity, visual inspection fluorescence dye to change or in real time turbidimeter detection, and is specific as follows:
1) visual inspection turbidity
If the visual inspection turbidity increases, then loop-mediated isothermal amplification is positive, shows to contain in the sample or the candidate is contained west nile virus; Otherwise negative.
Be specially nucleic acid when synthetic in a large number, dNTP can separate out a large amount of pyrophosphate ions, the Mg in pyrophosphate ion and the LAMP system
2+(or Mn
2+) combination, produce magnesium pyrophosphate or manganese pyrophosphate precipitation, thereby the turbidity of the liquid that induces reaction changes; Based on this principle, directly naked-eye observation change to have judged whether that specific amplification occurs and specific nucleic acid synthesizes in a large number according to the turbidity of reaction solution, thereby whether judge templet is target sequence.
2) the visual inspection fluorescence dye changes
When adopting the fluorexon fluorescence dye to react, if the LAMP reaction product is become yellow-green colour and shown fluorescence under UV-light by the orange of beginning under natural light, then the LAMP reacting positive shows that namely sample to be tested contains or the candidate is contained west nile virus.
Be specially: when application fluorescence dye colour-change is judged, can adopt SYBR green I dyestuff or fluorexon.SYBR green I dyestuff can be combined with double-stranded DNA, but when having a large amount of products to produce in the reaction, the reaction system color becomes yellow-green colour.When nucleic acid synthesizes in a large number, can generate the by product pyrophosphate, the existence of pyrophosphate ion is so that Mg
2+Dissociate out fluorexon and Mg
2+In conjunction with, reaction solution presents little turbid yellow-green colour.Based on this principle, can judge whether that according to the colour-change of reaction solution nucleic acid is synthetic in a large number, thereby whether judge templet is target sequence.Also can according to reaction principle, select other fluorescence dyes of this area to carry out the judgement of LAMP reaction.
3) turbidimeter detects in real time
Adopt the turbidity of real-time turbidimeter and the real-time detection reaction liquid of supporting program software thereof to change, if observe typical amplification curve on the turbidimeter in real time, then the LAMP reacting positive shows that namely sample to be tested contains or the candidate is contained west nile virus.
Can also detect with the following method:
4) electrophoresis:
The LAMP reaction product is cut with Sca I enzyme, obtain enzyme and cut product, agarose gel electrophoresis detects the LAMP reaction product and enzyme is cut product; (200～2000bp), and enzyme cuts the biobelt that product is 244bp and 164bp, then the LAMP reacting positive shows that namely sample to be tested contains or the candidate is contained west nile virus if the LAMP reaction product presents the scalariform banding pattern.
5) when adopting SYBR green I dyestuff, can judge by the fluorescence of PCR in real time instrument and the real-time detection reaction liquid of supporting program software thereof, when detecting fluorescent signal, show that the LAMP reaction is positive.
Of the present invention experimental results show that, primer special provided by the invention and method, be applicable to west nile virus (WNV) is carried out specific detection and quantitative analysis, its advantage is as follows: (1) only need in steady temperature with regard to the energy amplified reaction, not need specific installation; (2) specificity is high; (3) rapidly and efficiently, amplified reaction can be finished in 35 minutes; (4) highly sensitive; (5) evaluation is convenient and simple.The present invention is specially adapted to the rapid detection of clinical samples.
Description of drawings
Fig. 1 is natural light (A) and lower each sample LAMP result of ultraviolet lamp (B)
Fig. 2 is that LAMP reaction product and enzyme are cut the product agarose gel electrophoresis
Fig. 3 is real-time turbidimeter amplification curve
Fig. 4 is the real-time turbidimeter amplification curve that virus-specific detects
Fig. 5 is the real-time turbidimeter amplification curve of sample
Fig. 6 is that RT-PCR detects sample results
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Used inorganic chemical reagent and organic reagent all meet the requirement of molecular biology experiment.
Primer synthesizes AMV reversed transcriptive enzyme (Avian Myeloblastosis Virus Reverse Transcriptase) by Shanghai Ying Jun technology company: Pu Luomaige (Beijing) Bioisystech Co., Ltd (Promega), cat.No.M5101.Dezyribonucleoside phosphate mixt (dNTPs): Pu Luomaige (Beijing) Bioisystech Co., Ltd (Promega), cat.No.U1515.BstDNA polysaccharase: NEB (Beijing) company limited, cat.No.M0275.RNA extracts test kit: RNeasy Mini Kit, QIAgen company product, Cat No.74014.RNA enzyme inhibitors (Ribonuclease inhibitor): precious biological (TAKARA) company in Dalian product, cat.No.D2310.Tris hydrochloride (Tris-HCl) pH8.8:Sigma company, cat.No.T9443.Repone K (KCl): Sigma company, cat.No.P9514.Sal epsom (MgSO
4): Sigma company, cat.No.M3409, ammonium sulfate ((NH
4)
2SO
4): Sigma company, cat.No.A4418.Polysorbas20 (Tween20): Sigma company, cat.No.P9416.Trimethyl-glycine (Betaine): Sigma company, cat.No.B0300.Manganous chloride tetrahydrate (MnCl
2): Sigma company, cat.No.M3634.Fluorexon: Sigma company, cat.No.17783.DMEM substratum: Shanghai Ying Jun Bioisystech Co., Ltd, Cat No.C11885.The real-time turbidimeter of LA-320C, Japanese Rong Yan company product.% among the following embodiment if no special instructions, is the quality percentage composition.
West nile virus Chin-01 strain is documented in: Jiang Tao, and Deng Yongqiang, Fan Baochang, etc.West nile virus Chin-01 pnca gene group coding region sequence is measured and is analyzed.Institute of Military Medical Science Institute periodical.2003.27 (6): 401-403, the public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL, this institute guarantees, can meet under the relevant Biosafety management regulation with army of country, and provide through the backward unit that has qualification to be engaged in more than the Equations of The Second Kind viral pathogen of approval of relevant departments.
West nile virus is identified in embodiment 1, LAMP reaction
One, the preparation of the design of primer special and each primer special
As follows according to west nile virus virogene group sequences Design specific primer sequence, synthetic:
F3 (sequence 1, primer 1): CTGTGACAGCAGCTAGTGC;
B3 (sequence 2, primer 2): TTTTCACGCTCTGGTTGGTA;
FIP (sequence 3, primer 3): TCTTCATTCGTGTGCCCCCC
TTTTACGTGGACGCATCGGTAG;
BIP (sequence 4, primer 4): GACTCGAACTTCGCCCATTGGA
TTTTAATTGAGCGATCAGTCCGTT;
LB (sequence 5, primer 5): CTGAGGCACGAATCATGCTGGA;
Two, the preparation of Virus Sample
1, the preparation of Virus Sample
The virus liquid of west nile virus Chin-01 strain, by plaque experiment calculation plaque titre (plaque forming unit/milliliter, PFU/ml).Use the DMEM substratum that virus liquid is carried out gradient dilution, obtain the virus liquid (concentration is respectively 1000PFU/ml, 1000PFU/ml, 1OOPFU/ml, 10PFU/ml, 1PFU/ml, 0.1PFU/ml and 0.01PFU/ml) of 7 kinds of different concns.
2, the extraction of virus genome RNA (adopting RNA to extract test kit)
210 μ l Virus Samples are added in the 1.5ml centrifuge tube, add 700 μ l RLT solution and 7 μ l beta-mercaptoethanols, add 490 μ l dehydrated alcohols behind the vibration mixing, add at twice behind the thermal agitation in the silicagel column, carry out silicagel column and filter.
Silicagel column filters: with the silicagel column 10 behind the application of sample, and the centrifugal 15s of 000g; Add 700 μ l RW1 damping fluids, the centrifugal 15s of 10,000g; μ l RPE damping fluid is centrifugal washes silicagel column twice with 500; Silicagel column is gone in the new centrifuge tube the centrifugal 2min of 10,000g; Silicagel column is gone in the new centrifuge tube, add 50 μ l nuclease free water, room temperature leaves standstill 2min, and then 10, the centrifugal 2min of 000g collects elutriant; In elutriant, add 1 μ l RNA enzyme inhibitors, be RNA to be measured ,-80 ℃ of preservations.
7 kinds of viral RNAs obtaining as 7 samples, are used for following detection.
Sample 1: be the RNA that the Chin-01 virus liquid of 10000PFU/ml extracts from concentration.
Sample 2: be the RNA that the Chin-01 virus liquid of 1000PFU/ml extracts from concentration.
Sample 3: be the RNA that the Chin-01 virus liquid of 100PFU/ml extracts from concentration.
Sample 4: be the RNA that the Chin-01 virus liquid of 10PFU/ml extracts from concentration.
Sample 5: be the RNA that the Chin-01 virus liquid of 1PFU/ml extracts from concentration.
Sample 6: from concentration be 0.1PFU/ml the RNA that extracts of Chin-01 virus liquid.
Sample 7: be the RNA that the Chin-01 virus liquid of 0.01PFU/ml extracts from concentration.
Three, primer special is applied to the detection of west nile virus
The testing sample that the primer special first detecting step two that obtains with step 1 obtains carries out respectively the LAMP reaction in 7 0.2ml reaction tubess.
The concentration of LAMP reaction system 25 μ l:F3 and B3 is 5pmol/L, the concentration of BIP and FIP is 40pmol/L, the concentration of LB is 20pmol/L, AMV reversed transcriptive enzyme 0.004U/ μ L, Bst archaeal dna polymerase 0.32U/ μ L, fluorexon 50 μ mol/L, 2 * ring mediated isothermal amplification damping fluid of 12.5ul, 2.5 μ l RNA to be measured, the deionized water of nuclease free and DNAzyme supply 25 μ l; Above concentration is the final concentration in the system.
2 * ring mediated isothermal amplification damping fluid is prepared as follows: with Repone K (KCl), sal epsom (MgSO
4), ammonium sulfate ((NH
4)
2SO
4), polysorbas20 (Tween-20), trimethyl-glycine (Betaine), Manganous chloride tetrahydrate (MnCl
2) and deoxynucleoside triphosphate mixture (dNTPs:dATP, dCTP, dGTP and dTTP) to be dissolved in concentration be that 40mmol/L, pH value are that 8.8 Tris hydrochloride damping fluid obtains;
In described 2 * ring mediated isothermal amplification damping fluid, the concentration of described Repone K is 20mmol/L, the concentration of described sal epsom is 16mmol/L, the concentration of described ammonium sulfate is 20mmol/L, the concentration of described polysorbas20 is 0.2% (quality percentage composition), the concentration of described trimethyl-glycine is 1.6mol/L, and the concentration of described Manganous chloride tetrahydrate is 1mmol/L, and the concentration of every kind of described deoxynucleoside triphosphate is 2.8mmol/L.
The LAMP reaction system is at 63 ℃ of reaction 35min.Isothermal reaction can be undertaken by this area ordinary method, the real-time turbidimeter of LA320C for example, and regular-PCR instrument, controllable temperature water-bath etc.,
After the termination reaction, the photo of reaction tubes is seen Figure 1A under the natural light, and 1 to 7 is followed successively by sample 1 to sample 7, and the positive is yellow-green colour, and feminine gender is orange-yellow; Can find out that pipe 1 is to pipe 5 (being that sample 1 is to sample 5) positive (yellow-green colours), pipe 6 is to pipe 7 (being that sample 6 is to sample 7) negative (orange-yellow).
After the termination reaction under the ultra violet lamp photo of reaction tubes see Figure 1B, 1 to 7 is followed successively by sample 1 to sample 7, the positive is yellow-green fluorescence, and is negative without fluorescence; Can find out that pipe 1 is to pipe 5 (being that sample 1 is to sample 5) positive (yellow-green fluorescences), pipe 6 is to pipe 7 (being that sample 6 is to sample 7) negative (without fluorescence).
Adopt visual inspection, the sensitivity of detection method is 1PFU/ml under the natural light or under the UV-light.The concentration of viral RNA among the RNA to be measured=(1PFU/ml * 200 μ l)/50 μ l=0.004PFU/ μ l.The content of viral RNA in each LAMP reaction system=0.004PFU/ μ l * 2.5 μ l=0.01PFU.Be that sensitivity is the 0.01PFU/ reaction system.
Adopt under the ultra violet lamp and observe, the sensitivity of detection method is the 0.01PFU/ reaction system.
Four, primer special is applied to the detection (agarose electrophoresis observation) of west nile virus
The sample 1 that the professional primer detecting step two that obtains with step 1 obtains and sample 7 RNA to be measured.Can not add fluorexon in the LAMP reaction system, other is with the LAMP reaction system of step 3.
The LAMP reaction system is 63 ℃ of reactions 35min, then 75 ℃ of 2min termination reactions.
After the termination reaction, get product 5 μ l and carry out enzyme with Sca I restriction enzyme and cut.Get LAMP reaction product 0.5 μ l and enzyme and cut product 5 μ l and carry out agarose gel electrophoresis, sepharose concentration is 2.5%, and electrophoretic buffer is 1 * Tris-boric acid (TBE) damping fluid, and 100V electrophoresis 40 minutes is observed electrophoresis result under the ultraviolet source.
The results are shown in Figure 2, swimming lane 3 and swimming lane 4 are respectively LAMP reaction product and the enzyme thereof of sample 1 and cut product, positive, the band banding pattern (200bp-2000bp) of the visible typical gradient sample of LAMP product, visible 2 bands (244bp and 164bp) only after enzyme is cut; Swimming lane 1 and 2 is respectively LAMP reaction product and the enzyme thereof of sample 7 and cuts product, and is negative, without typical gradient belt transect type.
Five, primer special is applied to west nile virus and detects (in real time turbidimeter)
The LAMP reaction system is with the LAMP reaction system of step 3.The LAMP reaction system is placed the real-time turbidimeter of LA320C, 63 ℃ of reactions 35min, then 75 ℃ of 2min termination reactions.Can not add fluorexon in the reaction system.
The turbidimeter amplification curve is seen Fig. 3 (curve from top to bottom is followed successively by sample 1-7) in real time, and sample 1 to sample 5 has special amplification curve, and is positive, and sample 6 and sample 7 are negative.
The sensitivity that turbidimeter is observed is the 0.01PFU/ reaction system.
Six, the LAMP atopic detects
The sample 1 that the primer special detecting step two that obtains with step 1 obtains (west nile virus Chin-01 strain) RNA, with dengue type 2 virus 43 strains, the yellow fever virus 17D strain, tick-brone encephalitis virus (or claiming fores encephalitis virus) Senzhang strain, japanese encephalitis virus (or claiming epidemic encephalitis B virus) SA14-14-2 strain is contrast.After above-mentioned virus (being 100000PFU/ml) is extracted RNA, adopt primer of the present invention to carry out LAMP reaction (method is identical with step 4), the result is Fig. 4, only the reaction of west nile virus Chin-01 strain has amplification curve, the result is positive, other virus reactions show that all without obviously amplification curve is negative primer of the present invention all has good specificity.
The yellow fever virus 17D strain is documented in Co MD, Kilpatrick ED, Rothman AL.Dynamics of the CD8T-cell response following yellow fever virus 17D immunization.Immunology.2009.128 (1Suppl): e718-27; The nucleic acid database number of logging in X03700.
Tick-brone encephalitis virus Senzhang strain is documented in Ma Xinying, takes charge of bright silver, Gao Xuan, etc.China's Tick-borne encephalitis virus Senzhang strain Isolated coding region sequence is measured.China's virusology.2003.18 (4): 322-325; The nucleic acid database number of logging in AY182009.
Japanese encephalitis virus SA14-14-2 strain is documented in Li Yuhua, Li Hailing, and Wu Yonglin, etc.Epidemic encephalitis B virus living vaccine strain SA14-14-2 stable gene Journal of Sex Research.China's microbiology and Journal of Immunology.2003.23 (11): 858-861; The nucleic acid database number of logging in AF315119.
The above-mentioned viral public all can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.This institute guarantees, can meet the relevant Biosafety management regulation with army of country and provide to the unit that has qualification to be engaged in more than the Equations of The Second Kind viral pathogen after relevant departments' approval.
Above-mentioned virus all belongs to Flavivirus, with west nile virus be equal genus member.
China there is no the west Nile fever case at present.The present invention replaces with the mouse infection analog sample, and the virus infection method is documented in Deng Yongqiang, Jiang Tao, etc., Detection of West Nile virus using real-time PCR assay.China's microbiology and Journal of Immunology.2005.25(6)：519-522。Concrete grammar is: in three grades of laboratories of animal organism safety, get 4～6 all female BALB/c mouse in age (the SPF level are available from Military Medical Science Institute's Experimental Animal Center) 18, Transperitoneal injection west nile virus Chin-01 strain 100 μ l (10
5PFU/ml), after infecting, got the mouse brain tissue samples on the 6th day, be kept at-70 ℃ for subsequent use.
Employing is detected gathering from the mouse brain tissue samples of numbering the infection west nile virus Chin-01 strain of 1-18 respectively by the primer special that embodiment 1 step 1 obtains, with the sample 2 positive contrasts (+) of embodiment 1 step 2, with sample 7 negative contrasts (-).
After the infection west nile virus Chin-01 strain mouse brain tissue samples that will exsomatize respectively fully grinds, resuspended with the 1mlDMEM substratum, centrifugal 10 minutes of 5000g, get supernatant 210 μ l, extract the RNA (extracting method is with the step 2 of embodiment 1) of supernatant liquor, the ring isothermal amplification method is with the step 3 of embodiment 1.
Detected result is seen Fig. 5, and #1 to #18 is that mouse brain sample 1 is to sample 18.Can find out, #12, #18 and positive control have the specific amplified curve, and all the other samples and negative control are negative without the specific amplified curve.
The employing document (Deng Yongqiang, Jiang Tao, etc.Detection of West Nile virus using real-time PCR assay.China's microbiology and Journal of Immunology.2005.25 (6): 519-522) (upstream primer is the real-time RT-PCR method: TGATCCATGTAAGCCCTCAGAA, downstream primer is: ACATTGGGCTTTGAAGTTACAACA, probe is: 5 ' FAM-CGTCTCGGAAGGAGGACCCCA-3 ' BHQ1, annealing temperature is 60 ℃) #1 to #18 sample is detected, detected result is seen Fig. 6 (have three of obvious amplification curve and be followed successively by from top to bottom positive control, #18, #12), can find out, #12, #18 and positive control have the specific amplified curve, and be positive.All the other samples and negative control are without the specific amplified curve, and be negative.
Claims (10)
1. primer special that detects west nile virus, comprise primer 1, primer 2, primer 3 and primer 4, the nucleotide sequence of described primer 1, described primer 2, described primer 3 and described primer 4 is respectively sequence 1, sequence 2, sequence 3 and the sequence 4 in the sequence table.
2. primer special as claimed in claim 1, it is characterized in that: described primer special also comprises primer 5, the nucleotides sequence of described primer 5 is classified the sequence 5 of sequence table as.
3. primer special as claimed in claim 1 or 2, it is characterized in that: described primer special is comprised of described primer 1, described primer 2, described primer 3, described primer 4 and described primer 5;
Described west nile virus is specially west nile virus Chin-01 strain.
4. a ring mediated isothermal amplification reagent that detects west nile virus comprises RNA reversed transcriptive enzyme, Bst archaeal dna polymerase, 2 * ring mediated isothermal amplification damping fluid, the described primer special of claim 1-3.
5. ring mediated isothermal amplification reagent according to claim 4 is characterized in that:
Described amplifing reagent also comprises fluorexon;
Described amplifing reagent is comprised of RNA reversed transcriptive enzyme, Bst archaeal dna polymerase, 2 * ring mediated isothermal amplification damping fluid, the described primer special of claim 1-3 and fluorexon;
Primer 1 in the described primer special and the primer 2 final concentration in described ring mediated isothermal amplification reagent is 5pmol/L; Primer 3 in the described primer special and primer 4 final concentration in described ring mediated isothermal amplification reagent is 40pmol/L; The final concentration of primer 5 in the described primer special in described ring mediated isothermal amplification reagent is 20pmol/L;
The concentration of described RNA reversed transcriptive enzyme in described ring mediated isothermal amplification reagent is 0.004U/ μ L;
Described RNA reversed transcriptive enzyme is specially the AMV reversed transcriptive enzyme;
The concentration of described Bst archaeal dna polymerase in described ring mediated isothermal amplification reagent is 0.32U/ μ L;
The concentration of described fluorexon in described ring mediated isothermal amplification reagent is 50 μ mol/L;
Described 2 * ring mediated isothermal amplification damping fluid is prepared as follows: just to be dissolved in concentration be that 40mmol/L, pH value are that 8.8 Tris hydrochloride damping fluid obtains for Repone K, sal epsom, ammonium sulfate, polysorbas20, trimethyl-glycine, Manganous chloride tetrahydrate and dNTPs;
In described 2 * ring mediated isothermal amplification damping fluid, the concentration of described Repone K is 20mmol/L, the concentration of described sal epsom is 16mmol/L, the concentration of described ammonium sulfate is 20mmol/L, the concentration of described polysorbas20 is 0.2% (quality percentage composition), the concentration of described trimethyl-glycine is 1.6mol/L, and the concentration of described Manganous chloride tetrahydrate is 1mmol/L, and the concentration of every kind of described dNTP is 2.8mmol/L.
Described west nile virus is specially west nile virus Chin-01 strain.
6. a test kit that detects west nile virus comprises claim 4 or 5 described ring mediated isothermal amplification reagent;
Described west nile virus is specially west nile virus Chin-01 strain.
7. arbitrary described primer special or claim 4 or 5 described ring mediated isothermal amplification reagent or test kit claimed in claim 6 application in evaluation and/or assistant identification west nile virus among the claim 1-3;
Described west nile virus is specially west nile virus Chin-01 strain.
One kind identify and/or the assistant identification testing sample in the method for west nile virus, comprise the steps:
Testing sample is carried out ring mediated isothermal amplification, detection ring mediated isothermal amplification product with the described primer special in arbitrary described primer special or claim 4 among the claim 1-3 or 5 described ring mediated isothermal amplification reagent or the test kit claimed in claim 6;
In the described ring mediated isothermal amplification, take the RNA of testing sample as template;
Described loop-mediated isothermal amplification condition is: 60 ℃-65 ℃ are reacted 35-60min, then 75 ℃ of 2min termination reactions first.
9. method as claimed in claim 8 is characterized in that:
Described loop-mediated isothermal amplification condition is: 63 ℃ are reacted 35min, then 75 ℃ of 2min termination reactions first;
Described sample to be tested is mouse brain;
Described west nile virus is west nile virus Chin-01 strain.
10. method as claimed in claim 8 or 9 is characterized in that:
The method of described detection ring mediated isothermal amplification product is following 1)-4) at least a:
1) observes described loop-mediated isothermal amplification product, if described product is yellow-green colour, and glassy yellow fluorescence is arranged under UV-light under natural light, then contain in the sample to be tested or the candidate is contained west nile virus; If described product is orange-yellow under natural light, and under UV-light without fluorescence, then do not contain in the sample to be tested or the candidate is not contained west nile virus;
2) detect described loop-mediated isothermal amplification product with real-time turbidimeter, if the specific amplified curve is arranged, then contain in the sample to be tested or the candidate is contained west nile virus; If without the specific amplified curve, then do not contain in the sample to be tested or the candidate is not contained west nile virus;
3) the described loop-mediated isothermal amplification product of electrophoresis detection obtains the amplified reaction product of gradient batten band, then contains in the sample to be tested or the candidate is contained west nile virus; If the loop-mediated isothermal amplification product is not gradient batten band, then do not contain in the sample to be tested or the candidate is not contained west nile virus;
The size of described gradient batten band is 100bp-2000bp;
4) cut described loop-mediated isothermal amplification product with Sca I enzyme, the described enzyme of electrophoresis detection is cut product, if described enzyme is cut product is, then contains in the sample to be tested or the candidate is contained west nile virus; Be not the biobelt of 244bp and 164bp if described enzyme is cut product, then do not contain in the sample to be tested or the candidate is not contained west nile virus.
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| CN104152440A (en) * | 2013-05-15 | 2014-11-19 | 中国人民解放军军事医学科学院微生物流行病研究所 | Primer composition for identifying forest encephalitis virus and application of primer composition |
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| CN106884057A (en) * | 2017-03-22 | 2017-06-23 | 广州中医药大学 | A kind of visible detection method of zika virus |
| CN115981384A (en) * | 2023-02-07 | 2023-04-18 | 淮阴工学院 | Intelligent biomass ORC evaporation pressure control equipment |
| CN115981384B (en) * | 2023-02-07 | 2023-09-22 | 淮阴工学院 | Intelligent biomass ORC evaporation pressure control equipment |
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