CN109061161B - For detecting the detection kit and its application of capsicum arteries and veins flavivirus - Google Patents
For detecting the detection kit and its application of capsicum arteries and veins flavivirus Download PDFInfo
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Abstract
The invention discloses a kind of for detecting the detection kit and its application of capsicum arteries and veins flavivirus, wherein including PeVYV P4 protein polyclone antibody for detecting the detection kit of capsicum arteries and veins flavivirus.Using indirect elisa method, PeVYV P4 protein polyclone antibody is subjected to the capsicum arteries and veins flavivirus in sample and is detected, has many advantages, such as high specificity, high sensitivity.Application of the above-mentioned detection kit in detection capsicum arteries and veins flavivirus is also disclosed, quick, high-throughput detection may be implemented, instrument is simple, easy to operate.
Description
Technical field
The present invention relates to nucleic acid detection technique fields more particularly to a kind of for detecting the detection reagent of capsicum arteries and veins flavivirus
Box and its application.
Background technique
Capsicum arteries and veins flavivirus (Peppervein yellows virus,It PeVYV) is yellow sign Viraceae
(Luteoviridae) Polerovirus (Polerovirus) newcomer can be infected a variety of heavy by aphis propagation
The solanaceous crops wanted.Infecting capsicum causes the yellow of capsicum vein, leaf roll bent, influences capsicum normal growth.Therefore, the virus is established
Fast Detection Technique system provide technological means for the generation, distribution and harm for monitoring the virus, be the Scientific evaluation disease
Poison provides scientific basis to the harm of agricultural production.
The detection method of the PeVYV reported at present only has RT-PCR and RT-LAMP technology, these methods require have compared with
The operator of good technical background and instrument such as high-speed refrigerated centrifuge, PCR instrument, transmissometer and the gel imaging system of valuableness
System etc., while detection process needs complicated sample handling processes, such as total serum IgE extracting, reverse transcription；And the amplification more than 2 h
Process and cumbersome electrophoresis ultraviolet observe process.Lack a kind of side that quick, high-throughput detection capsicum arteries and veins flavivirus may be implemented
Method.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art, provide a kind of quickly detection capsicum arteries and veins jaundice
Quick, high-throughput detection may be implemented in the serological method of poison, this method；Serologic detection is being carried out using preparation
PeVYV P4 protein polyclone antibody carries out the capsicum arteries and veins flavivirus in detection sample using indirect elisa method.
It is a kind of for detecting the detection kit of capsicum arteries and veins flavivirus, including PeVYV P4 protein polyclone antibody.
Above-mentioned detection kit, it is preferred that further include confining liquid, horseradish peroxidase-labeled goat antirabbit secondary antibody,
Developing solution and colour developing terminate liquid.
Above-mentioned detection kit, it is preferred that the confining liquid is 10 × PBST buffer containing 1% BSA；
And/or the developing solution is TMB tetramethyl biphenyl diamines；
And/or the sulfuric acid solution that the colour developing terminate liquid is 2 mol/L.
Above-mentioned detection kit, it is preferred that the PeVYV P4 protein polyclone antibody is prepared into using following methods
It arrives:
S1, using PeVYV P4 gene as template design primer, to infection PeVYV capsicum cDNA carry out PCR amplification obtain
Amplified production；
S2, pDEST17-P4 will be obtained in amplified production recombination to pDEST17 carrier；
S3, it the pDEST17-P4 is converted to competent cell obtains converted product；
S4, it induces converted product progress arabinose to obtain induced product；
S5, the induced product is cracked, purifies to obtain PeVYV P4 albumen；
S6, PeVYV P4 albumen progress animal immune is obtained into PeVYV P4 protein polyclone antibody.
Above-mentioned detection kit, it is preferred that the primer includes upstream primer and downstream primer composition, and the upstream is drawn
The amino acid sequence of object is as shown in SEQ ID NO.1, and the amino acid sequence of shown downstream primer is as shown in SEQ ID NO.2.
As a total technical concept, the present invention also provides a kind of above-mentioned detection kits in detection capsicum capsicum arteries and veins
Application in flavivirus.
Above-mentioned application, it is preferred that the application method the following steps are included:
(1) it is coated with: the supernatant of plant sample being added in ELISA Plate and is coated with；
(2) it closes: by coated ELISA Plate confining liquid, 37 DEG C of 1 h of incubation；
(3) add primary antibody: primary antibody dilution being added in the ELISA Plate closed, in 37 DEG C of 1 h of incubation, the primary antibody dilution
Liquid is the solution that primary antibody dilutes 5000 times；
(4) add secondary antibody: secondary antibody diluent, 37 DEG C of 1 h of incubation is added in every hole, and the secondary antibody diluent is secondary antibody dilution 5000
Solution again；
(5) add developing solution: every hole is added TMB tetramethyl biphenyl diamines Chromogenic Substrate Solution and develops the color；
(6) terminate reaction: colour developing terminate liquid is added in every hole, and microplate reader measures 450 nm light absorption values；450 nm light absorption values
>=2 .5 times be that positive i.e. sample has infected capsicum arteries and veins flavivirus, sample 450 nm light absorption value < 2 .5 times be feminine gender.
Above-mentioned application, it is preferred that the primary antibody dilution is that PeVYV P4 polyclonal antibody is pressed with antibody diluent
The volume ratio of PeVYV P4 Duo clone's Kang Ti ︰ antibody diluent is the mixed liquor that 1 ︰ 5000 is mixed, the antibody diluent
For 10 × PBST buffer containing 1% BSA.
Above-mentioned application, it is preferred that the secondary antibody diluent is horseradish peroxidase-labeled goat antirabbit secondary antibody and resists
For body dilution with the solution after the dilution of 1 ︰, 5000 volume ratio, the antibody diluent is 10 × PBST buffer containing 1% BSA.
Compared with the prior art, the advantages of the present invention are as follows:
(1) the present invention provides a kind of for detecting the detection kit of capsicum arteries and veins flavivirus, and Huang sign Viraceae includes three
A category: Huang sign Tobamovirus (Luteovirus), Polerovirus (Plerovirus) and pea enation mosaic virus category
(Enamovirus), whereinLuteovirusWithPlerovirusEncoding viral P4 albumen, andEnamovirusVirus does not encode
P4 albumen.LuteovirusWithPlerovirusEncoding viral P4 albumen exists obvious in sequence homology and molecular size range
Difference, therefore, the P4 albumen encoded by molecular biology method great expression PeVYV, and in this, as antigen, prepare more grams
Grand antibody carries out the capsicum arteries and veins flavivirus in detection sample using indirect elisa method, establishes the Serologic detection of detection PeVYV
Method has many advantages, such as easy to operate, quick, high specificity, high sensitivity, can be widely used in the detection of plant virus
With disease screening.
(2) detection kit that the present invention provides a kind of for detecting capsicum arteries and veins flavivirus is just by survey capsicum arteries and veins jaundice
Application in poison, compared with the molecular biology methods such as RT-PCR and RT-LAMP, big, the quick, high specificity with detection flux
The advantages of.Meanwhile instrument is simple, easy to operate, the infection rate that can be used for solanaceous crops field sample P eVYV is advised greatly
Mould investigation, early warning.
Detailed description of the invention
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, the technical scheme in the embodiment of the invention is clearly and completely described.
The protein induced expression SDS-PAGE figure of Fig. 1 PeVYV P4.In figure, M is albumen Marker, and swimming lane 1 is PeVYV P4
Recombinant protein；Swimming lane 2 is BSA(0.5 mg/mL).
Fig. 2 PeVYV P4 polyclonal antibody sensitivity technique result.
Specific embodiment
Below in conjunction with Figure of description and specific preferred embodiment, the invention will be further described, but not therefore and
It limits the scope of the invention.
Embodiment
Material employed in following embodiment and instrument are commercially available.
Embodiment 1:
A kind of detection kit for being used to detect capsicum arteries and veins flavivirus of the invention, including PeVYVP4 protein polyclone are anti-
Body, horseradish peroxidase (Horseradish Peroxidase, HRP) label goat-anti rabbit secondary antibody, TMB(3,3 ', 5,5
'-tetramethyl benzidine), the sulfuric acid solution of the PBST buffer containing 1% BSA and 2 M.
Wherein PeVYVP4 protein polyclone antibody is prepared using following methods:
(1) preparation of PeVYV P4 proteantigen
1.1 specific primer designs and synthesis
PeVYV P4 gene is capsicum arteries and veins flavivirus (the Pepper vein yellows to report on GenBank
Virus, PeVYV) the P4 gene completed in (accession number: KP326573) genome is template, it is set online using Primer 5.0
Primer is counted, primer is made of upstream primer and downstream primer, and the base sequence of upstream primer is as shown in SEQ ID NO.1, downstream
The base sequence of the base sequence of primer is as shown in SEQ ID NO.2.Primer sequence is as follows:
F:GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGAAATGGTGGATCACGTAAC (SEQ ID
NO.1)；
R:GGGGACCACTTTGTACAAGAAAGCTGGGTCCCCCGTTAATCTGCGAAGC(SEQ ID NO.2).
1.2 Prokaryotic expression vector construction
1.2.1 PCR amplification is carried out using the capsicum cDNA of above-mentioned primer pair infection PeVYV, obtains complete P4 gene
Amplified production.
Specific amplification system are as follows: 10 × PCR buffer:5 μ L；DNTP(10 mM): 1 μ L；Upstream primer/downstream
Each 1 μ L of primer (10 μM)；ddH2O 12 μL；Total volume is 20 μ L.
Amplification program is 94 DEG C of initial denaturation 5 min, 94 DEG C of denaturation 45 s, 58 DEG C of renaturation 30 s, 72 DEG C of extension 60 s, 30
A circulation, 10 min of last 72 DEG C of extensions.
1.2.2 amplified production is detected through 1.0 % agarose gel electrophoresis, being shown in 500 bp has single band, and pre-
Phase sequence size is consistent.
1.2.3 the building of recombinant plasmid uses Gateway technology, carries out referring to specification: P4 gene PCR product is led to
BP reaction recombination is crossed to entry vector pDONR221, the recombination of P4 gene PCR product is then reacted to by LR and is carried to pDEST17
Body obtains recombinant expression carrier pDEST17-P4.
The expression and analysis of 1.3 recombinant proteins
1.3.1 in 42 DEG C of 50 S of heat shock, make recombinant vector convert toE. coliCompetent cell Rosetta is converted
Product (method for transformation reference [beauty] J. Pehanorm Brooker etc., Molecular Cloning:A Laboratory guide (third edition), Science Press,
2002).
1.3.2, obtained converted product is coated on to the LB plate of the ampicillin containing 100 μ g/L, 37 DEG C overnight
Culture.
1.3.3 the positive single colonie on picking plate is verified using bacterium colony PCR.
1.3.4 the engineering bacteria Rosetta-pDEST17-P4 containing pDEST17-P4 recon is containing 100 μ g/mL ammonia
37 DEG C of overnight incubations obtains culture solution in the LB culture medium of parasiticin.
1.3.5 above-mentioned culture solution is inoculated in the LB culture medium of 20 mL with 1: 100 volume ratio and is cultivated to OD600It is 0.6
~0.7(incubation time is generally 2~4 h), and inducer arabinose is added to final concentration of 0.5 mM, continues to train in 37 DEG C
It supports, and is to take bacterium solution to detect in 4~6 h in incubation time.1 mL bacterium solution is taken, thalline were collected by centrifugation.1 mL bacterium is taken before induction
Liquid is as control.
1.3.6 the 400 μ L lysate (groups of lysate are added in the thallus of thallus and control group by induction respectively
It is divided into: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazol (pH8.0), 10 μ g/ml lysozymes), boil 10
Then min is centrifuged 5 min with 12000 rpm, collects supernatant respectively.
1.3.7 by supernatant with 15 %(v/v) PAGE gel 5 h of electrophoresis at 100 V, the deposition condition of 25 mA,
Then with Coomassie brilliant blue dye liquor (ingredient of Coomassie brilliant blue dye liquor includes: 0.1 %(w/v) Coomassie brilliant blue, 45 %(v/v)
Methanol, 10 %(v/v) glacial acetic acid, surplus ddH2O 15 min) are dyed, finally (ingredient of destainer includes: 40 with destainer
V/v% methanol, 10 v/v% glacial acetic acid, 50 v/v% sterile waters) it decolourizes to observe electrophoresis result after 1 h.
Electrophoresis result is referring to Fig. 1.Known in the electrophoresis result of Fig. 1: having soluble protein expression at about 25 kDa, this is big
It is small almost the same plus the amino acid molecular size of label H is gene fusion proteins with P4 protein amino acid sequence, it is believed that this egg
White is amalgamation and expression P4 albumen.
1.4 recombinant protein purifications and concentration mensuration
His and P4 fusion protein is purified from total protein using the Ni-NTA agarose kit of GE company；The kit
His can be specifically bound, specific binding His feature purified fusion albumen is utilized.The P4 protein of expression contains His
Label, convenient for purifying, the induction expression protein purifying of soluble form.
Purifying protein 15%(v/v) PAGE gel electrophoresis detection, the expression egg of visible single band at 25 kDa
White (Fig. 1), it was demonstrated that this albumen is the PeVYV P4 of the is containing label H, as PeVYV P4 proteantigen, and concentration is 200 μ g/ml.
(2) prepared by PeVYVP4 protein polyclone antibody
It takes purified protein 3-4mg to be packaged with dry ice, it is immune strong to mail Hangzhou Huaan Bio-Tech. Co., Ltd. to
The large ear rabbit (two) of health finally obtains polyclonal antibody and antiserum, and the concentration of resulting PeVYV P4 polyclonal antibody is
500 μg/ml。
Embodiment 2:
A kind of application of the detection kit of embodiment 1 in detection capsicum arteries and veins flavivirus, is detected using ID-ELISA method
The sensitivity of PeVYV P4 polyclonal antibody.Specific experimentation is as follows:
(1) it is coated with: being coated with PeVYV antigen with 10 × PBST buffer, be 200 μ g/ by concentration in 1 step 1.4 of embodiment
Ml PeVYV P4 proteantigen, carries out gradient dilution with antibody diluent, be successively diluted to 200 times, 1000 times, 5000 times,
10000 times, 20000 times, 60000 times, 240000 times of each PeVYV antigen gradient dilution liquid, in each hole of ELISA Plate successively
The aforementioned each PeVYV antigen gradient dilution liquid of 100 μ l, 4 DEG C of overnight or 37 DEG C of 2 h of incubation are added.With 10 × PBST containing 1%BSA
Buffer (7 .4 of pH) is used as negative control.
The BSA is bovine serum albumin(BSA) (Albumin from bovine serum, BSA).
(2) close: after coated ELISA Plate washs 3 times with cleaning solution, 100 μ l confining liquids, 37 DEG C of temperature are added in every hole
It educates
Then 1h is washed 3 times with cleaning solution.10 × PBST buffer (7 .4 of pH) that the cleaning solution is；The closing
Liquid be containing 1% BSA PBST buffer.
(3) add primary antibody: the ELISA Plate after step (2) closing is by each of the former PeVYV antigen added with different extension rates
200 μ l primary antibody dilutions are added in hole, then 37 DEG C of incubation 1h are washed 3 times with cleaning solution.Cleaning solution is 10 × PBST buffering
Liquid；Primary antibody dilution is that the concentration for obtaining embodiment 1 is the PeVYV P4 polyclonal antibody of 500 μ g/ml, uses antibody diluent
Be diluted to 2000 times PeVYV P4 polyclonal antibody (i.e. concentration be 0.25 μ g/ml PeVYV P4 polyclonal antibody), antibody
Dilution is 10 × PBST buffer (7 .4 of pH) containing 1% BSA.
(4) add secondary antibody: secondary antibody diluent 200 μ l, 37 DEG C of incubation 1h, with 10 × PBST buffer (pH 7.4) is added in every hole
Washing 4 times.Secondary antibody diluent is that horseradish peroxidase (Horseradish Peroxidase, HRP) is marked goat-anti rabbit two
Anti- to be diluted to 5000 times with antibody diluent, the antibody diluent is the PBST buffer containing 1% BSA.
(5) develop the color: TMB(3,3 ', 5,5 '-tetramethyl benzidines be added) 100 hole μ l/ of substrate solution, it shows at room temperature
5 min of color.
(6) it terminates: 50 hole μ l/ of terminate liquid, microplate reader OD450nm light absorption value, with the OD 450nm light absorption value in each hole is added
Fig. 2 is as a result seen using the dilution of PeVYV antigen gradient dilution liquid in the present embodiment step (2) as abscissa for ordinate.It takes
Negative control OD 450nm light absorption value 2 .5 times be critical value .5 times of light absorption value >=2 reacting hole OD 450nm be it is positive, instead
Answer .5 times of hole OD 450nm light absorption value < 2 be it is negative, colour developing terminate liquid be 2 M with the concentration that sterile water is prepared sulfuric acid it is molten
Liquid.
Fig. 2 shows (concentration is 0.25 μ g/ml), the maximum of antigen when PeVYV P4 polyclonal antibody dilutes 2000 times
Extension rate is 240000 times, i.e., the concentration of antigen is 0.83 ng/ml, and PeVYV polyclonal antibody remains to the knowledge of specificity at this time
Other PeVYV antigen shows the high sensitivity of its PeVYV P4 polyclonal antibody.
Embodiment 3:PeVYV P4 protein polyclone antibody specific detection
This experiment detects the specificity of PeVYV P4 polyclonal antibody using ID-ELISA method.Test following 3 kinds of viruses:
PeVYV, capsicum arteries and veins turn round viral (Tobacco vein distorting virus, TVDV) and soybean chlorisis virus (Beet
Chlorosis virus, BCV).PeVYV, TVDV and BCV positive sample are to infect the capsicum sample (leaf of these three viruses respectively
Piece is stored in -80oC).The experimentation difference from Example 2 of ID-ELISA method is: PeVYV, TVDV and BCV positive sample
Product are plant leaf, add liquid nitrogen grinding, double gauze filtering.Subsequent step is same as Example 2.
Test result shows PeVYV P4 antibody, only infects PeVYV plant leaf with identification, shows that its PeVYV P4 is more
The high specificity of clonal antibody.
Embodiment 4: field capsicum sample detection
According to the method for embodiment 2,30 parts of Pepper Leaves samples that 2012-2017 is acquired in provinces such as Hunan, Shandong
It is detected.
Wherein 18 parts of Hunan collecting sample, positive rate 44.4%, 12 parts of Shandong collecting sample, positive rate 25.0%.
Above embodiments show: the present invention can be used for quickly detecting capsicum arteries and veins Huang, detect specific good, high sensitivity,
Detection large batch of to sample (general 96 orifice plates, once can at least make several hundred a samples) may be implemented simultaneously, shorten inspection
Time and testing cost (detection time is about 6-8h, and if cost, a sample only needs about 1-2 first) is surveyed, is advantageously implemented
Quick detection to capsicum arteries and veins flavivirus.
The above described is only a preferred embodiment of the present invention, being not intended to limit the present invention in any form.Though
So the present invention is disclosed as above with preferred embodiment, and however, it is not intended to limit the invention.It is any to be familiar with those skilled in the art
Member, in the case where not departing from Spirit Essence of the invention and technical solution, all using in the methods and techniques of the disclosure above
Appearance makes many possible changes and modifications or equivalent example modified to equivalent change to technical solution of the present invention.Therefore,
Anything that does not depart from the technical scheme of the invention are made to the above embodiment any simple according to the technical essence of the invention
Modification, equivalent replacement, equivalence changes and modification, all of which are still within the scope of protection of the technical scheme of the invention.
<110>Huang Heqing
<120>for detecting the detection kit and its application of capsicum arteries and veins flavivirus
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 54
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ggggacaagt ttgtacaaaa aagcaggctt catggaaatg gtggatcacg taac 54
<210> 2
<211> 49
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggggaccact ttgtacaaga aagctgggtc ccccgttaat ctgcgaagc 49
Claims (9)
1. a kind of for detecting the detection kit of capsicum arteries and veins flavivirus, which is characterized in that including PeVYV P4 protein polyclone
Antibody.
2. detection kit according to claim 1, which is characterized in that further include confining liquid, horseradish peroxidase mark
Remember goat antirabbit secondary antibody, developing solution and colour developing terminate liquid.
3. detection kit according to claim 2, which is characterized in that the confining liquid is 10 × PBST containing 1% BSA
Buffer；
And/or the developing solution is TMB tetramethyl biphenyl diamines；
And/or the sulfuric acid solution that the colour developing terminate liquid is 2 mol/L.
4. detection kit according to any one of claim 1 to 3, which is characterized in that the PeVYV P4 albumen is more
Clonal antibody is prepared using following methods:
S1, using PeVYV P4 gene as template design primer, to infection PeVYV capsicum cDNA carry out PCR amplification expanded
Product；
S2, pDEST17-P4 will be obtained in amplified production recombination to pDEST17 carrier；
S3, it the pDEST17-P4 is converted to competent cell obtains converted product；
S4, it induces converted product progress arabinose to obtain induced product；
S5, the induced product is cracked, purifies to obtain PeVYV P4 albumen；
S6, PeVYV P4 albumen progress animal immune is obtained into PeVYV P4 protein polyclone antibody.
5. detection kit according to claim 4, which is characterized in that the primer is by upstream primer and downstream primer group
At the amino acid sequence of the upstream primer is as shown in SEQ ID NO.1, the amino acid sequence of shown downstream primer such as SEQ ID
Shown in NO.2.
6. a kind of application of the detection kit described in any one of claims 1 to 5 in detection capsicum arteries and veins flavivirus.
7. application according to claim 6, which is characterized in that it is described application the following steps are included:
(1) it is coated with: the supernatant of plant sample being added in ELISA Plate and is coated with；
(2) it closes: by coated ELISA Plate confining liquid, 37 DEG C of 1 h of incubation；
(3) add primary antibody: primary antibody dilution being added in the ELISA Plate closed, in 37 DEG C of 1 h of incubation, the primary antibody dilution is
Primary antibody dilutes 5000 times of solution；
(4) add secondary antibody: secondary antibody diluent, 37 DEG C of 1 h of incubation is added in every hole, and the secondary antibody diluent is that secondary antibody dilutes 5000 times
Solution；
(5) add developing solution: every hole is added TMB tetramethyl biphenyl diamines Chromogenic Substrate Solution and develops the color；
(6) terminate reaction: colour developing terminate liquid is added in every hole, and microplate reader measures 450 nm light absorption values；450 light absorption value >=2 nm
.5 again for it is positive be that sample has infected capsicum arteries and veins flavivirus, sample 450 nm light absorption value < 2 .5 times be negative.
8. application according to claim 7, which is characterized in that the primary antibody dilution be PeVYV P4 polyclonal antibody with
Antibody diluent is by the mixed liquor that the volume ratio of PeVYV P4 Duo clone's Kang Ti ︰ antibody diluent is that 1 ︰ 5000 is mixed, institute
Stating antibody diluent is 10 × PBST buffer containing 1% BSA.
9. application according to claim 7, which is characterized in that the secondary antibody diluent is horseradish peroxidase-labeled mountain
With the solution after the dilution of 1 ︰, 5000 volume ratio, the antibody diluent is 10 containing 1% BSA for goat-anti rabbit secondary antibody and antibody diluent
× PBST buffer.
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