CN103436635B - Primers for detecting plum pox viruses as well as kit and detection method - Google Patents

Abstract

The invention discloses primers for detecting plum pox viruses as well as a kit and a detection method, wherein the primers comprise the forward direction outer primer F3: 5'-GATGATGGATGGGGAAACA-3', the reverse direction outer primer B3: 5'-AGGCTGTAGTCTGTCAGG-3', the forward direction inner primer FIP: 5'-GCCATAATTTGTCTAAAAGTGGGTT-CAAGTGGAGTATCCAATAAAGC-3', and the reverse direction inner primer BIP: 5'-ACATTTCAGTAACGTGGCTGAA-GCTGAATCCCATACCTTGG-3'; the kit comprises the primers and the detection method adopts the primers; with adoption of the primers and the detection method, the plum pox virus detection is high in speed, simple and convenient, efficient, high in accuracy, strong in specificity and high in sensitivity.

Description

For detecting the primer of plumpox virus, test kit and detection method

Technical field

The present invention relates to a kind of primer, test kit and detection method for detecting plumpox virus, belonging to biological technical field.

Background technology

Plumpox virus (Plum pox virus, PPV) be one of dangerous maximum virus of tone fruit trees, fruit tree is once infect by it, immature fruit then can be caused to come off in a large number, output is seriously reduced, and fruit quality is significantly declined, and this virus disseminating speed is fast, namely may gives bringing on a disaster property of the fruit growing development loss of whole country at short notice.This virus is found in Bulgaria for 1915 first, then spread all over rapidly European most countries and Mediterranean Sea, in non-, India and Chile, current America & Canada also has its report occurred.Due to this viral hazardness and serious, therefore it has been listed in A2 class quarantine pathogen by European plant protection tissue, other country also shows the attention of high degree to it, numerous and confused strengthen dynamics of its research and to formulate a series of strict detection and elimination measure, takes precautions against in possible trouble.Current China is not found the formal report about this virus temporarily; because current China exchanges more and more frequent with other countries' germ plasm resource; China is entered along with the introduction of the reproductive materials such as nursery stock in order to prevent this virus; to protect health and the Sustainable development of China's agricultural, the inspection and quarantine to this virus must be strengthened!

At present, China's entry and exit plant virus detects the main technique methods adopted is that Enzyme-linked Immunosorbent Assay (ELISA) detects and inverse transcription polymerase chain reaction (RT-PCR) detects.To there is detection sensitivity in traditional elisa technique low in actual application, detects length consuming time, easily occur false positive results; Although RT-PCR technology is highly sensitive, there is RNA extracting loaded down with trivial details and easily degrade, PCR reaction easily by the interference of polyphenol, polysaccharide material in host, is difficult to quick, easy, the efficient detection realizing viral diseases of plants sample.Therefore, new quick, simple and direct, sensitive inspection and quarantine technique means is set up extremely urgent.

The diagnosis of the viroses of plant and Pathogen identification, be understanding virus disease, prevent and treat the basis of virus disease, also be an important content of plant virus means of taxonomic research, the substance that plant virus detects comprises several aspect such as biological characteristics, morphological feature, physicochemical property, genome structure, antigenic characteristic of virus, relates to the multiple means such as biological characteristis, determination of serology, physiological and biochemical test, electron microscopy, Protocols in Molecular Biology.At present, the Main Means of the detection technique of China's entry and exit plant quarantine employing mainly: the on-the-spot quarantine in port, Growing season Isolation Quarantine, plant indicator inspection, serologic test, molecular biology inspection.The subject matter that these means exist is: 1: sense cycle is long, does not meet the spirit of " speed passenger flow speed " that State Council proposes; 2: easily occur false positive reaction; 3: detection method very complicated, be unfavorable for grass-roots unit's practical application; 4: main detection reagent or rule of origin are in offshore company, and not only purchase price is high, and the time of ordering is long, and these all can not adapt to the needs of China's rapid economic development.Therefore, new inspection and quarantine technique means is fast and effectively set up extremely urgent.

Current is that Enzyme-linked Immunosorbent Assay (ELISA) detects and inverse transcription polymerase chain reaction (RT-PCR) detects to the major technique detection method of virus.ELISA can virus in qualitative, quantitative assay sample, is one of best means realizing current identifying virus.But, serological reaction must possess efficient antiserum(antisera), low to some viral levels and containing interference measurement material the sample of Serologic detection then can not be made and measuring accurately in addition, the false-positive result of easy appearance, and there is the problem of length detection time, the requirement of current port rapid detection can not be met; RT-PCR technology is one of most widely used molecular biology new technology, and utilize the detection of RT-PCR technology to virus to compare ELISA detection and have highly sensitive feature, detectable minimum viral level reaches fg level.Many new technology are constantly derived at present on RT-PCR technology basis, such as real-time fluorescence RT-PCR technology, biochip technology etc., its range of application is constantly expanded, specificity and susceptibility also improve constantly, but it is loaded down with trivial details and easily degrade that these Protocols in Molecular Biologies exist RNA extracting, PCR reaction easily by the interference of polyphenol, polysaccharide material in host, limits quick, easy, the efficient detection of viral diseases of plants sample.

Nanometer biotechnology uses the theory and means of nanotechnology, on the basis of traditional biological and Modern Molecular Biotechnology, carries out biological study.Nanometer magnetic bead has significant application value in the compartment analysis of biological sample: (1) efficiently concentrating: nanometer magnetic bead specific surface area is large, and significantly increasing reaction interface can efficiently concentrating biological sample; (2) manipulative capability: nanometer magnetic bead has superparamagnetic e ffect, externally-applied magnetic field can move it fast and be separated; (3) scalar nature: biomolecule labeling method is simple, reliable; (4) human-body safety: to human non-toxic, can biological metabolism, biodegradable, good biocompatibility.In a word, magnetic bio is separated the superparamagnetism of both magnetic carrier and the biologic specificity feature of affinity ligand, integrate the highly selective double dominant of the fast and convenient of magnetic resolution and Human serum protein, in the separation and purification of nucleic acid, the enrichment of RNA, DNA molecular, the purifying of albumen, there is application the aspects such as the separation of virus, cell.

About nanometer magnetic bead (magnetic nano particles, MNP) synthesis, the research of Superparamagnetic Iron Oxide ultrafine particle is started abroad at the end of the eighties, bioseparation material based on nanometer magnetic bead has marketable product abroad, nearest nanometer magnetic bead detects at animal virus and is applied, nanometer magnetic bead target acquisition virus from liquid phase of antibody labeling, can detect animal virus in high sensitivity.

And ring mediation nucleic acid isothermal amplification and Loop-mediated isothermalamplification(LAMP) technology is the Viral diagnosis novel method just grown up recent years, a kind of new technological approaches is provided for rapid gene detects, there is the feasibility of successful Application, detect at food safety detection, epidemic virus, clinical diagnosis, and the application in the field such as aquaculture and the existing bibliographical information of research.It does not need expensive test set, and only need an incubator, and its detection sensitivity is more taller than regular-PCR detection method, and is no more than one hour detection time, result is directly estimated, and is particularly suitable for port one line and detects.

The reaction of ring matchmaker isothermal nucleic acid amplification utilizes 4 species-specific primers to rely on a kind of high reactivity strand displacement archaeal dna polymerase to make strand displacement DNA synthesize in ceaselessly oneself's circulation, LAMP amplification point two stages.

1st stage was that initial structure is formed: any one primer to the complementary portions of double-stranded DNA carry out base pairing extend time, another chain will dissociate, and becomes strand.First the F2 sequence of upstream internal primers F IP is combined with template F2c, extends forward and start strand displacement synthesis under the effect of strand displacement type archaeal dna polymerase.External primers F3 is combined with template F3c and extends, and displaces the complementary strand that complete FIP connects.The Fl of F1c on FIP therewith on strand is complementary structure.Oneself's base pairing forms ring texture.With this chain for template.Downstream primer BIP and B3 successively starts the synthesis being similar to FIP and F3, forms the strand of dumbbell structure.Rapidly with the Fl section of 3' end for starting point.With from as template, carry out DNA synthesis and extend to form stem ring texture.

2nd stage was reaction amplification cycles: with stem ring texture for template, FIP is combined with stem Huan F2c district.Begin chain displacement synthesis, the single-chain nucleic acid dissociateed also can form ring texture.Rapidly with the B1 section of 3 ' end for starting point, with from as template.Carry out DNA synthesis to extend and strand displacement. form the DNA of 2 new stem ring texturees different in size, the B2 on BIP primer is hybrid with it.Start new round amplification.And product D NA length doubles.In reaction system, add 2 Loop primer LF and LB, they are also combined with stem ring texture respectively and start strand displacement and synthesize, and go round and begin again.The final product of amplification is the mixture with different number loop-stem structure, different lengths DNA.And product D NA is the alternately inverted repeats of amplified target sequence.

Ring matchmaker isothermal nucleic acid amplification reacts, and is new ideas in biology field, and it under isothermal (60 DEG C ~ 65 DEG C) condition, can will only have the target nucleic acid amplification of several copy to 10 in 30min ~ 60min ⁹level.The one large characteristic of the method to pass through byproduct of reaction---whether the generation of white magnesium pyrophosphate precipitation judges the existence of target gene.Amplification procedure due to ring matchmaker isothermal nucleic acid amplification relies on and identifies target sequence six isolated areas, so have the features such as high-level efficiency, high specific, hypersensitivity for the qualification of microorganism.In addition, the amplification process of ring matchmaker isothermal nucleic acid amplification is carried out under isothermal conditions, ortho-water bath or have the equipment of stable thermal source just can meet to react requirement, testing cost is reduced greatly, PCR then needs expensive PCR instrument to carry out the nucleic acid amplification of certain temperature procedure, and thus, ring matchmaker isothermal nucleic acid amplification is lower to plant and instrument requirement than PCR, testing cost is also lower, have more generalization, be expected as conventional sense instrument.

Summary of the invention

The object of the invention is to overcome weak point of the prior art, there is provided a kind of primer for detecting plumpox virus and comprise test kit and the detection method of this primer, plumpox virus is quick, easy, efficient, accuracy is high, high specificity, susceptibility are high to utilize this primer and detection method to detect.

In order to achieve the above object, the present invention adopts following scheme:

For detecting a primer for plumpox virus, it is characterized in that:

Forward outer primer F3:5'-GATGATGGATGGGGAAACA-3';

Reverse outer primer B3:5'-AGGCTGTAGTCTGTCAGG-3';

Forward inner primer FIP and reverse inner primer BIP;

Wherein said forward inner primer FIP comprises F1c and F2 sequence:

5'-GCCATAATTTGTCTAAAAGTGGGTT-CAAGTGGAGTATCCAATAAAGC-3'；

Wherein F1c:5'-GCCATAATTTGTCTAAAAGTGGGTT-3';

F2：5'-CAAGTGGAGTATCCAATAAAGC-3'；

Described reverse inner primer BIP comprises containing B1c and B2 sequence:

5'-ACATTTCAGTAACGTGGCTGAA-GCTGAATCCCATACCTTGG-3'；

Wherein B1c:5'-ACATTTCAGTAACGTGGCTGAA-3';

B2:5'-GCTGAATCCCATACCTTGG-3'。

Detect a test kit for plumpox virus, it is characterized in that this test kit includes:

Described primer comprises:

Forward outer primer F3:5'-GATGATGGATGGGGAAACA-3';

Reverse outer primer B3:5'-AGGCTGTAGTCTGTCAGG-3';

Forward inner primer FIP and reverse inner primer BIP;

Wherein said forward inner primer FIP comprises F1c and F2 sequence:

5'-GCCATAATTTGTCTAAAAGTGGGTT-CAAGTGGAGTATCCAATAAAGC-3'；

Wherein F1c:5'-GCCATAATTTGTCTAAAAGTGGGTT-3';

F2：5'-CAAGTGGAGTATCCAATAAAGC-3'；

Described reverse inner primer BIP comprises containing B1c and B2 sequence:

5'-ACATTTCAGTAACGTGGCTGAA-GCTGAATCCCATACCTTGG-3'；

Wherein B1c:5'-ACATTTCAGTAACGTGGCTGAA-3';

B2:5'-GCTGAATCCCATACCTTGG-3'。

A kind of test kit detecting plumpox virus as above, is characterized in that described reaction solution damping fluid 2X is composed of the following components:

A kind of test kit detecting plumpox virus as above, is characterized in that described enzyme solution is Bst archaeal dna polymerase and AMV reversed transcriptive enzyme mixed solution.

A kind of test kit detecting plumpox virus as above, is characterized in that described fluorescence dye contains fluorexon.

A detection method for plumpox virus, is characterized in that comprising the following steps:

A, employing nanometer magnetic bead extract the RNA of testing sample;

B, on ice, extract reaction solution damping fluid in proportion, primer, fluorescence dye, enzyme solution, positive control, deionized water put into sterile tube and prepare premixed liquid;

C, add the RNA of appropriate testing sample in step B sterile tube;

D, the test tube in step C is placed in thermostatted, at 63 DEG C, keeps constant temperature 1 hour, at 80 DEG C, then keep constant temperature to make enzyme-deactivating with termination reaction in 5 minutes;

E, use UV irradiation equipment are upwards irradiated bottom test tube, wear ultraviolet protection glasses to carry out sight from test tube side and look into, send green glow if the same with positive control, be then judged to be the positive, do not send fluorescence if the same with negative control, and be judged to be feminine gender;

Wherein said primer comprises:

Forward outer primer F3:5'-GATGATGGATGGGGAAACA-3';

Reverse outer primer B3:5'-AGGCTGTAGTCTGTCAGG-3';

Forward inner primer FIP and reverse inner primer BIP;

Wherein said forward inner primer FIP comprises F1c and F2 sequence:

5'-GCCATAATTTGTCTAAAAGTGGGTT-CAAGTGGAGTATCCAATAAAGC-3'；

Wherein F1c:5'-GCCATAATTTGTCTAAAAGTGGGTT-3';

F2：5'-CAAGTGGAGTATCCAATAAAGC-3'；

Described reverse inner primer BIP comprises containing B1c and B2 sequence:

5'-ACATTTCAGTAACGTGGCTGAA-GCTGAATCCCATACCTTGG-3'；

Wherein B1c:5'-ACATTTCAGTAACGTGGCTGAA-3';

B2:5'-GCTGAATCCCATACCTTGG-3'。

The detection method of a kind of plumpox virus as above, is characterized in that adopting nanometer magnetic bead to extract the concrete steps of testing sample RNA in steps A:

A, sample preparation: the band poison tissue getting 0.1g adds 1mL extraction buffer and grinds;

B, cleaning nanometer magnetic bead: take out immune nanometer magnetic bead in PCR pipe, use ddH ₂o cleans nanometer magnetic bead 3 times repeatedly, under the effect of magnet, suck ddH ₂o;

C, combination: the sample adding 100 μ L, in PCR pipe, mixes sample and nanometer magnetic bead by liquid-transfering gun compressing, absorption at room temperature;

D, cleaning: under the effect of magnet, remove supernatant, nanometer magnetic bead cleans 3 times;

E, cracking: add 50 μ L ddH ₂o in PCR pipe, with liquid-transfering gun compressing mixing nanometer magnetic bead after, 95 DEG C, 10min;

F samples: use magnet adsorption nanometer magnetic bead, and after solution clarification in PCR pipe, supernatant is testing sample RNA.

The detection method of a kind of plumpox virus as above, is characterized in that described immune nanometer magnetic bead is made by following technique:

1. the preparation of nanometer magnetic bead

Get 5.2g FeCl respectively ₃6H ₂o, 2.7g FeSO ₄7H ₂the concentrated hydrochloric acid of O, 0.85mL12.1moL/L is dissolved in 200mL water, ultrasonic deoxidation, after by the instillation of above solution 250mL, 0.75moL/L NaOH solution; Stir at 80 DEG C, after reaction terminates, utilize magnetic separator gained precipitation to be separated from reaction medium, then use washed with de-ionized water 3 times, washes of absolute alcohol 2 times, and be made into the Fe of 5g/mL ₃o ₄magnetic nano-particle ethanol solution; Get 25mL Fe ₃o ₄magnetic nano-particle ethanol solution, then be diluted to 150mL with dehydrated alcohol, sonic oscillation 30min; Drip 0.4mL APTES, stirred at ambient temperature 7h; React complete, Fe APTES modified with magnetic separator ₃o ₄magnetic nano-particle is separated from reaction medium, and cleans 3 times with ethanol solution, is finally made into the amination Fe of 10.5mg/mL ₃o ₄nanometer magnetic bead ethanol solution;

2. the preparation of immunomagnetic beads

Get 0.5mL amination Fe ₃o ₄nanometer magnetic bead ethanol solution, clean 3 times with 1mL PBS, supernatant liquor abandoned by magnetic separator; Add 5% (V/V) glutaraldehyde solution 2mL, shaken at room temperature is cross-linked 2h, Magneto separate, abandons supernatant liquor; Wash 3 times with PBS and after abandoning supernatant liquor, adopt 0.5mL antiviral antibody bag by amination Fe respectively ₃o ₄nanoparticle, the fixing 2h of vibration under room temperature; Magneto separate, washs 3 times respectively with PBS and ultrapure water, then uses PBS constant volume, and 4 DEG C of Refrigerator stores are for subsequent use, obtain immunomagnetic beads.

The detection method of a kind of plumpox virus as above, is characterized in that described ultraviolet shines

The wavelength of injection device is 240-260nm, 350-370nm.

The detection method of a kind of plumpox virus as above, is characterized in that described reaction solution damping fluid 2X is composed of the following components:

Described enzyme solution is Bst archaeal dna polymerase and AMV reversed transcriptive enzyme mixed solution; Described fluorescence dye contains fluorexon.

Some materials used in the present invention:

1.1 strain

Gladiolus mosaic virus Cymbidium mosaic virus (CymMV) and plumpox virus Odontoglossum ringspot virus (ORSV) standard strain are purchased from AGDIA company of the U.S..

1.2 main agents and consumptive material

(1) PCR reaction reagent: 10 × PCR buffer, dNTPs, MgCl2, Taq archaeal dna polymerase, precious biotechnology (Dalian) company limited;

(2) DNA Marker DL1000,6 × loading buffer, precious biotechnology (Dalian) company limited;

(3) LAMP reaction reagent;

(4) RNA amplification reaction kit Loopamp RNA Amplification kit, fluorescence visual detection test kit, fluorescenceDetection Reagent, reaction tube is all purchased in Beijing Lanpu Biological Technology Co., Ltd.,

(5) Bst DNA polymerase large fragment, New England Biolabs company;

(6) trimethyl-glycine (Betaine), the good biological company limited of Guangzhou prestige;

DNTPs, MgCl2, precious biotechnology (Dalian) company limited;

(7) 50 × TAE damping fluids: 242g Tris and 37.2g Na2EDTA2H2O is dissolved in 800mL deionized water, abundant stirring and dissolving, then adds 57.lmL acetic acid, stir, finally with deionized water, solution is settled to 1L, dilute 50 times during use;

(8) agarose, prompt doubly this biological company limited in Guangzhou;

(9) 70% ethanol;

(10) LAMP primer (forward outer primer F3, forward inner primer FIP, oppositely inner primer BIP, oppositely outer primer B3) and PCR primer (upstream primer, downstream primer), precious biotechnology (Dalian) company limited synthesizes;

(11) DNA extraction kit (centrifugal column type), TIANGEN Biotech (Beijing) Co., Ltd..

(12) antiviral antibody One step RT-PCR test kit PrimeScriptTM One-StepVer.2.0 is purchased from precious biotechnology (Dalian) company limited;

(13) nanometer magnetic bead (magnetic nano particles, MNP) test kit is purchased from Beijing Jenner Science and Technology Ltd.;

1.3 key instrument equipment

(1)-80 DEG C of deep freezer;

(2) the freezing desk centrifuge of Labofuge400R high speed;

(3) regular-PCR instrument and AB7500 quantitative real time PCR Instrument;

(4) French UVP gel imaging system;

(5) DYY-7 type electrophoresis apparatus, Beijing 61 bio tech ltd;

(6) micropipet, 1000 μ L, 200 μ L, 100 μ L, 10 μ L, 2.5 μ L;

(7) microwave oven;

(8) analytical balance, plum Teller-Tuo benefit Instrument Ltd.;

(9) autoclave sterilization pot;

(10) ultrapure water system, Milli-Q Academic, Millipore company;

(11) Shimadzu, Japan UV-120-02 type visible-ultraviolet spectrophotometer;

(12) Bechtop;

(13) eddy mixer;

(14) enzyme connection detector.

(15) the real-time turbidimeter of LAMP, LA-320C, Japanese Rong Yan biotech firm;

(16) BIOTEK enzyme joins the equipment such as detector.

Embodiment

Below in conjunction with embodiment, the present invention is described further:

Embodiment 1

Test kit of the present invention:

1. product specification: 45 times

2. feature:

Ring mediated isothermal amplification method (loop-mediated isothermal amplification, LAMP) be a kind of novel gene amplification method, only increase at a constant temperature with a kind of enzyme, owing to using 4 primers that can identify target DNA 6 distinguished sequences, so specificity is high, and amplification efficiency is very high, can increase in a large number in the short period of time, reaction result can estimate judgement.

This test kit adopts ring mediated isothermal amplification method, with Yeast Nucleic Acid (RNA) for template directly carries out the simple and easy test kit of reverse transcription loop-mediated isothermal amplification, this test kit uses the enzyme solution of mixing reversed transcriptive enzyme and archaeal dna polymerase, only the reagent in testing sample and this test kit need to be mixed as requested under being placed on certain temperature (63 DEG C) reaction regular hour can (1 hour), can see and look into reaction result by naked eyes.

3. test kit component content:

1) .LAMP reaction solution damping fluid (0.6mL)

2). primer (250 μ L)

3). fluorescence dye (50 μ L)

4). enzyme solution (50 μ L)

5). positive control (50 μ L)

6). deionized water (1000 μ L)

4. working method:

1). the preparation of reagent:

A. all ingredients preserved at getting-20 DEG C at room temperature thaws, and is placed in immediately and preserves on ice after thawing;

B. the preparation (carrying out on ice) of premixed liquid: get the reacting weight needed for detection, is divided in the sterile tube of the premixed solution preparation prepared in addition respectively according to following table ratio (reaction):

Wherein reaction buffer composition (2X):

Enzyme solution:

Bst archaeal dna polymerase and AMV reversed transcriptive enzyme mixed solution.

Fluorescence dye:

Containing fluorexon, initially be in quenching of fluorescence state with being combined with mn ion, along with the carrying out of LAMP reaction, due to byproduct of reaction pyrophosphate ion capture the mn ion that is combined with fluorexon and make fluorexon recover free out thus send fluorescence, and fluorexon is once the magnesium ion in reaction solution is combined, fluorescence also can be made to be enhanced.

Primer:

PPV universal primer sequence (5 '-3 ')

C. flick after packing and hit test tube and make it mix, the brief centrifugation several seconds makes solution concentrate on bottom test tube;

2). application of sample: the RNA5 μ L adding the detected sample prepared in point test tube installed respectively, total amount is made to reach 25 μ L, negative control reaction uses deionized water 5 μ L as sample, positive control reaction uses the positive control with this PPV viral nucleic acid, then cover test tube cap and touch mixing, brief centrifugation makes reaction soln concentrate on bottom test tube;

3). amplification: test tube is placed in thermostatted, keeps constant temperature 1 hour at 63 DEG C, then keep constant temperature to make enzyme-deactivating with termination reaction in 5 minutes at 80 DEG C;

4). detect: use UV irradiation equipment (wavelength 240-260nm, 350-370nm) upwards to irradiate bottom test tube, wear ultraviolet protection glasses and carry out sight from test tube side and look into.Send green glow if the same with positive control, be then judged to be the positive, do not send fluorescence if the same with negative control, and be judged to be feminine gender.

Embodiment 2

The present invention detects the detection method of plumpox virus, comprises the following steps:

A, employing nanometer magnetic bead extract the RNA of testing sample;

Immune nanometer magnetic bead is made by following technique:

1. the preparation of nanometer magnetic bead

Get 5.2g FeCl respectively ₃6H ₂o, 2.7g FeSO ₄7H ₂the concentrated hydrochloric acid of O, 0.85mL12.1moL/L is dissolved in 200mL water, ultrasonic deoxidation, after by the instillation of above solution 250mL, 0.75moL/L NaOH solution; Stir at 80 DEG C, after reaction terminates, utilize magnetic separator gained precipitation to be separated from reaction medium, then use washed with de-ionized water 3 times, washes of absolute alcohol 2 times, and be made into the Fe of 5g/mL ₃o ₄magnetic nano-particle ethanol solution; Get 25mL Fe ₃o ₄magnetic nano-particle ethanol solution, then be diluted to 150mL with dehydrated alcohol, sonic oscillation 30min; Drip 0.4mL APTES, stirred at ambient temperature 7h; React complete, Fe APTES modified with magnetic separator ₃o ₄magnetic nano-particle is separated from reaction medium, and cleans 3 times with ethanol solution, is finally made into the amination Fe of 10.5mg/mL ₃o ₄nanometer magnetic bead ethanol solution;

2. the preparation of immunomagnetic beads

Get 0.5mL amination Fe ₃o ₄nanometer magnetic bead ethanol solution, clean 3 times with 1mL PBS, supernatant liquor abandoned by magnetic separator; Add 5% (V/V) glutaraldehyde solution 2mL, shaken at room temperature is cross-linked 2h, Magneto separate, abandons supernatant liquor; Wash 3 times with PBS and after abandoning supernatant liquor, adopt 0.5mL antiviral antibody bag by amination Fe respectively ₃o ₄nanoparticle, the fixing 2h of vibration under room temperature; Magneto separate, washs 3 times respectively with PBS and ultrapure water, then uses PBS constant volume, and 4 DEG C of Refrigerator stores are for subsequent use, obtain immunomagnetic beads.

Concrete extracting method:

A, sample preparation: the band poison tissue getting 0.1g adds 1mL extraction buffer and grinds;

B, cleaning nanometer magnetic bead: take out immune nanometer magnetic bead in PCR pipe, use ddH ₂o cleans nanometer magnetic bead 3 times repeatedly, under the effect of magnet, suck ddH ₂o;

C, combination: the sample adding 100 μ L, in PCR pipe, mixes sample and nanometer magnetic bead by liquid-transfering gun compressing, absorption at room temperature;

D, cleaning: under the effect of magnet, remove supernatant, nanometer magnetic bead cleans 3 times;

E, cracking: add 50 μ L ddH ₂o in PCR pipe, with liquid-transfering gun compressing mixing nanometer magnetic bead after, 95 DEG C, 10min;

F samples: use magnet adsorption nanometer magnetic bead, and after solution clarification in PCR pipe, supernatant is testing sample RNA.

B, on ice, extract reaction solution damping fluid in following ratio, primer, fluorescence dye, enzyme solution, positive control, deionized water put into sterile tube and prepare premixed liquid; Wherein premixed liquid primary first-order equation amount adds up to 20 μ L, comprising 12.5 μ L reaction buffers, and 1 μ L enzyme solution, 4 μ L primers, 1 μ L fluorescence dye, 1.5 μ L deionized waters.Wherein include 0.4 μ L forward outer primer F3 in 4 μ L primers, the reverse inner primer BIP of the reverse outer primer B3 of 0.4 μ L, 1.6 μ L forward inner primer FIP and μ L;

Wherein forward outer primer F3:5'-GATGATGGATGGGGAAACA-3';

Reverse outer primer B3:5'-AGGCTGTAGTCTGTCAGG-3';

Forward inner primer FIP and reverse inner primer BIP;

Wherein said forward inner primer FIP comprises F1c and F2 sequence:

5'-GCCATAATTTGTCTAAAAGTGGGTT-CAAGTGGAGTATCCAATAAAGC-3'；

Wherein F1c:5'-GCCATAATTTGTCTAAAAGTGGGTT-3';

F2：5'-CAAGTGGAGTATCCAATAAAGC-3'；

Described reverse inner primer BIP comprises containing B1c and B2 sequence:

5'-ACATTTCAGTAACGTGGCTGAA-GCTGAATCCCATACCTTGG-3'；

Wherein B1c:5'-ACATTTCAGTAACGTGGCTGAA-3';

B2:5'-GCTGAATCCCATACCTTGG-3'。

C, add the RNA of appropriate testing sample in step B sterile tube; Concrete steps: the RNA5 μ L adding the detected sample prepared in point test tube installed respectively, total amount is made to reach 25 μ L, negative control reaction uses deionized water 5 μ L as sample, positive control reaction uses the positive control with this CymMV viral nucleic acid, then cover test tube cap and touch mixing, brief centrifugation makes reaction soln concentrate on bottom test tube;

D, the test tube in step C is placed in thermostatted, at 63 DEG C, keeps constant temperature 1 hour, at 80 DEG C, then keep constant temperature to make enzyme-deactivating with termination reaction in 5 minutes;

E, use UV irradiation equipment are upwards irradiated bottom test tube, wear ultraviolet protection glasses to carry out sight from test tube side and look into, send green glow if the same with positive control, be then judged to be the positive, do not send fluorescence if the same with negative control, and be judged to be feminine gender; The wavelength of described UV irradiation equipment is 240-260nm, 350-370nm.

Claims (1)

1. a detection method for plumpox virus, is characterized in that comprising the following steps:

A, employing nanometer magnetic bead extract the RNA of testing sample;

Immune nanometer magnetic bead is made by following technique:

1. the preparation of nanometer magnetic bead

Get 5.2g FeCl respectively ₃6H ₂o, 2.7g FeSO ₄7H ₂the concentrated hydrochloric acid of O, 0.85mL 12.1moL/L is dissolved in 200mL water, ultrasonic deoxidation, after by the instillation of above solution 250mL, 0.75moL/L NaOH solution; Stir at 80 DEG C, after reaction terminates, utilize magnetic separator gained precipitation to be separated from reaction medium, then use washed with de-ionized water 3 times, washes of absolute alcohol 2 times, and be made into the Fe of 5g/mL ₃o ₄magnetic nano-particle ethanol solution; Get 25mLFe ₃o ₄magnetic nano-particle ethanol solution, then be diluted to 150mL with dehydrated alcohol, sonic oscillation 30min; Drip 0.4mL APTES, stirred at ambient temperature 7h; React complete, Fe APTES modified with magnetic separator ₃o ₄magnetic nano-particle is separated from reaction medium, and cleans 3 times with ethanol solution, is finally made into the amination Fe of 10.5mg/mL ₃o ₄nanometer magnetic bead ethanol solution;

2. the preparation of immunomagnetic beads

Get 0.5mL amination Fe ₃o ₄nanometer magnetic bead ethanol solution, clean 3 times with 1mL PBS, supernatant liquor abandoned by magnetic separator; Add 5% (V/V) glutaraldehyde solution 2mL, shaken at room temperature is cross-linked 2h, Magneto separate, abandons supernatant liquor; Wash 3 times with PBS and after abandoning supernatant liquor, adopt 0.5mL antiviral antibody bag by amination Fe respectively ₃o ₄nanoparticle, the fixing 2h of vibration under room temperature; Magneto separate, washs 3 times respectively with PBS and ultrapure water, then uses PBS constant volume, and 4 DEG C of Refrigerator stores are for subsequent use, obtain immunomagnetic beads;

Concrete extracting method:

A, sample preparation: the band poison tissue getting 0.1g adds 1mL extraction buffer and grinds;

B, cleaning nanometer magnetic bead: take out immune nanometer magnetic bead in PCR pipe, use ddH ₂o cleans nanometer magnetic bead 3 times repeatedly, under the effect of magnet, suck ddH ₂o;

C, combination: the sample adding 100 μ L, in PCR pipe, mixes sample and nanometer magnetic bead by liquid-transfering gun compressing, absorption at room temperature;

D, cleaning: under the effect of magnet, remove supernatant, nanometer magnetic bead cleans 3 times;

E, cracking: add 50 μ L ddH ₂o in PCR pipe, with liquid-transfering gun compressing mixing nanometer magnetic bead after, 95 DEG C, 10min;

F samples: use magnet adsorption nanometer magnetic bead, and after solution clarification in PCR pipe, supernatant is testing sample RNA;

B, on ice, extract reaction solution damping fluid in following ratio, primer, fluorescence dye, enzyme solution, positive control, deionized water put into sterile tube and prepare premixed liquid; Wherein premixed liquid primary first-order equation amount adds up to 20 μ L, comprising 12.5 μ L reaction buffers, and 1 μ L enzyme solution, 4 μ L primers, 1 μ L fluorescence dye, 1.5 μ L deionized waters; Wherein include forward outer primer F3 in primer, reverse outer primer B3, forward inner primer FIP and reverse inner primer BIP;

Wherein forward outer primer F3 sequence is: 5'-GATGATGGATGGGGAAACA-3';

Reverse outer primer B3 sequence is: 5'-AGGCTGTAGTCTGTCAGG-3';

Wherein said forward inner primer FIP comprises F1c and F2, and forward inner primer FIP sequence is: 5'-GCCATAATTTGTCTAAAAGTGGGTT-CAAGTGGAGTATCCAATAAAGC-3';

Wherein F1c sequence is: 5'-GCCATAATTTGTCTAAAAGTGGGTT-3';

F2 sequence is: 5'-CAAGTGGAGTATCCAATAAAGC-3';

Described reverse inner primer BIP comprises containing B1c and B2, and reverse inner primer BIP sequence is:

5'-ACATTTCAGTAACGTGGCTGAA-GCTGAATCCCATACCTTGG-3'；

Wherein B1c sequence is: 5'-ACATTTCAGTAACGTGGCTGAA-3';

B2 sequence is: 5'-GCTGAATCCCATACCTTGG-3';

C, add the RNA of appropriate testing sample in step B sterile tube; Concrete steps: the RNA 5 μ L adding the detected sample prepared in point test tube installed respectively, total amount is made to reach 25 μ L, negative control reaction uses deionized water 5 μ L as sample, positive control reaction uses the positive control with this plumpox virus nucleic acid, then cover test tube cap and touch mixing, brief centrifugation makes reaction soln concentrate on bottom test tube;

D, the test tube in step C is placed in thermostatted, at 63 DEG C, keeps constant temperature 1 hour, at 80 DEG C, then keep constant temperature to make enzyme-deactivating with termination reaction in 5 minutes;

E, use UV irradiation equipment are upwards irradiated bottom test tube, wear ultraviolet protection glasses to carry out sight from test tube side and look into, send green glow if the same with positive control, be then judged to be the positive, do not send fluorescence if the same with negative control, and be judged to be feminine gender; The wavelength of described UV irradiation equipment is 240-260nm, 350-370nm.