CN109633153A - A kind of spot immune immunoblot method based on immune colloid gold of detection rice stripe mosaic virus and its application - Google Patents

A kind of spot immune immunoblot method based on immune colloid gold of detection rice stripe mosaic virus and its application Download PDF

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CN109633153A
CN109633153A CN201910081205.XA CN201910081205A CN109633153A CN 109633153 A CN109633153 A CN 109633153A CN 201910081205 A CN201910081205 A CN 201910081205A CN 109633153 A CN109633153 A CN 109633153A
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stripe mosaic
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吴佳瑜
周雪平
洪健
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Zhejiang University ZJU
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Abstract

The invention discloses a kind of spot immune immunoblot method based on immune colloid gold of detection rice stripe mosaic virus (RSMV) and its applications.Using water resistant rice stripe mosaic viral monoclonal antibodies 1D4 be created as rapid sensitive, it is simple and convenient, accurately and efficiently detect rice and pass virus mediator recilia dorsalis body in RSMV the spot immune trace Serologic detection technology based on immune colloid gold, and develop at quick detection kit.Spot immune trace detection technique detection infection RSMV rice disease leaf and the sensitivity for carrying RSMV recilia dorsalis respectively reach 1:40960 (w/v, g/mL) and 1:5120 times of dilution (single head/μ L).This simple and quick, sensitive special, RSMV detection technique of precise and high efficiency and its detection kit provide technology and supporting substances for the quick diagnosis of rice stripe mosaic disease, breeding for disease resistance, prediction and warning and science bridle.

Description

A kind of spot immune print based on immune colloid gold of detection rice stripe mosaic virus Mark method and its application
Technical field
The present invention relates to field of biotechnology more particularly to it is a kind of detection rice stripe mosaic virus based on immune colloid The spot immune immunoblot method of gold and its application.
Background technique
Virus Diseases of Rice is one of important disease of rice plants, causes heavy losses to China's rice yield every year. 2015, a kind of new Rice Virus disease is had found in Luoding City, Guangdong Province field, its symptom is different from having reported various Virus Diseases of Rice is one novel species of Rhabdoviridae cytoplasm Rhabdovirus, is named as rice stripe mosaic virus (Rice stripe mosaic virus,RSMV).As far as is known, which is the cytoplasm of natural infection rice unique so far Rhabdovirus and the first cytoplasm rhabdovirus propagated through recilia dorsalis.RSMV infected plant slightly stunts, and blade is in item Line floral leaf shape, and have the performance distortion of part disease leaf, diseased plant can ear, but incomplete and seed sky of earing is flat.3 leaf phase rice seedlings are through electricity Light leafhopper passes after poison inoculation 10 days, and obvious yellow cord occurs in young leaves, and subsequent blade shows as floral leaf, and blade tip crimps inwardly.
RSMV virion is in the shape of a rod, assembles in mesophyll cell and fibrovascular system cytoplasm and forms a large amount of lattice Shape is almost full with entire cytoplasmic space, and does not observe virion in nucleus.Some virion aggregations are in vesica It is interior.Prediction has 7 ORFs on the complementary strand of RSMV genome, thus it is speculated that the coding albumen of this 7 ORFs and most rhabdoviruses Coding albumen it is similar, ORF1 predictive coding viral N proteins；The P albumen of ORF2 coding virus；It is one that ORF3, which encodes albumen, The memebrane protein of alkalinity, and the albumen may have the function of motor protein；ORF4 speculates coding M albumen；ORF5 predictive coding disease Malicious G albumen；ORF6 speculates one auxilin of coding；The L albumen of ORF7 predictive coding virus.The vector of RSMV is electricity Light leafhopper can propagate RSMV in a manner of high-efficient and lasting.
Currently, judging field rice stripe mosaic virus a situation arises to be still most important side according to Symptom Observation Method, but virus infection rice early period does not have any symptom, and symptom caused by a variety of virosis is very much like, in addition, often ill Malicious Combined Infection situation, therefore lack accuracy and science by the result that Symptom Observation obtains.Electronic Speculum observes virion Expensive instrument is needed, and the size of somewhat different virion and form have similitude, it is caused only to examine as a kind of auxiliary Survey means.The molecular detection technology of based on PCR method is although sensitive, but is unsuitable for the detection of extensive sample, is suitable only for reality Test detection of the room to small sample.And serological method is simple to operation, quick, sensitive, cost is relatively low, and is suitable for field sample Extensive detection, be the detection technique of ideal plant virus at present.For this purpose, the invention patent utilizes the Shen of colloid gold label Patent applied for ask someone application No. is 201810134449.5 monoclonal antibody 1D4, be successfully established detect the virus based on immune glue The spot immune trace detection technique and its detection kit of body gold, so that the detection for China's rice stripe mosaic virus is examined Disconnected, prediction and warning, breeding for disease resistance and science bridle provide substance and technical support.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of detection rice stripe mosaic virus based on exempting from The western dot blot of epidemic disease colloidal gold and its application.
A kind of spot immune immunoblot method based on immune colloid gold detecting rice stripe mosaic virus, specifically: it uses The specific monoclonal antibody 1D4 of colloid gold label water resistant rice stripe mosaic virus is as detection antibody；The water resistant rice stripe mosaic The specific monoclonal antibody 1D4 of virus is secreted by hybridoma cell strain, during which was preserved on December 15th, 2017 State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC No.14898.
Wherein, included the following steps: with colloidal gold labeled monoclonal antibody
1) prepared by colloidal gold solution: 0.01% gold chloride of 100mL being heated to boiling, 1% citric acid three of 1.2mL is added It is mixed after sodium, then gold chloride after boiling 30min is kept to be reduced into colloid gold particle solution；
2) colloidal gold solution described in 50mL is taken, with 0.1M K2CO3Its pH value is adjusted to 8.2；
3) specificity for being added that 2mL IgG concentration is 2mg/mL in the colloidal gold solution of pH value while stirring is mixed up Monoclonal antibody 1D4 stirs 30min；The polyethylene glycol i.e. PEG solution that 1mL 25M molecular weight is 20000 is added dropwise again, is stirred for 15min；
4) then by mixed liquor obtained in 3), 15min is centrifuged in 4 DEG C of 20000rpm, abandon supernatant, 10mL pH is added The 0.01M PBS buffer solution of 7.4 and PEG containing 0.4M 20000 suspends, and is centrifuged 15min in 4 DEG C of 20000rpm, supernatant is gone to obtain Precipitating；
5) precipitating is repeated to be suspended with 0.01M PBS buffer solution, centrifugation；
6) with 10mL pH 7.5 and containing heavy obtained in the dissolution 5) of the PBS buffer solution of 2%BSA and 0.02% sodium azide It forms sediment, after being filtered with 0.22 μm of sterilizing filter, filtrate is colloidal gold labeled monoclonal antibody solution, and 4 DEG C save backup.
Wherein, it is specifically included using the method that above-mentioned colloidal gold labeled monoclonal antibody solution carries out the detection of rice stripe mosaic virus Following steps:
1) nitrocellulose (NC) film prepares: it is crossed on NC film outer layer paper with pencil, NC film is made to stamp grid stria mark, Every lattice specification 0.3 × 0.3cm, it is smooth to be put on clean filter paper；
2) sample preparation:
A) preparation of plant sample: rice or wheat plant tissue weighing are placed in mortar with liquid nitrogen grinding into powder End, in 0.1g plant tissue add 2mL ratio the 0.01M PBS of pH 7.4 is added after continue grinding homogenate 1min in mortar, obtain To homogenate；Or it will add the ratio of 2mL that 0.01M is added in 0.1g plant tissue after rice or wheat plant tissue weighing Grinding is homogenized 2min in mortar after PBS, obtains homogenate；Homogenate is moved in 1.5mL centrifuge tube, 5000rpm centrifugation 3min, or 5min is stood, supernatant is the crude extract of rice or wheat plant tissue；
B) pass the preparation of virus mediator recilia dorsalis sample: a recilia dorsalis adds after being put into the centrifuge tube of a 0.5ml Enter 50-100 μ L 0.01M PBS, mashes leafhopper in homogenate with toothpick butt end or 200 μ L pipette tips；
3) point sample: drawing each 3 μ L of crude extract and recilia dorsalis homogenate of rice or wheat plant tissue with pipettor, Point sample dries 10min in the grid center of NC film at room temperature；
4) it closes: adding the ratio of 100mL PBST buffer to prepare to obtain 5% skimmed milk power using in 5g skimmed milk power Confining liquid, the PBST buffer are the 0.01mol/L PBS containing 0.05%Tween-20；NC film in confining liquid at room temperature Close 0.5-1h；
5) wash: deblocking liquid washes film 2 times with PBST buffer, each 3min；
6) colloidal gold labeled monoclonal antibody is incubated for: it is anti-that NC film is put into the colloid gold label for diluting 2000 times with 0.01mol/L PBS In liquid solution, 1h is incubated at room temperature in slowly shaking on horizontal shaker；
7) it washs: removing colloidal gold labeled monoclonal antibody solution, wash film 4-5 times with PBST buffer, each 3min；
8) it observes judging result: the sample of punctation occurs for the positive, the sample of colourless or green spot is feminine gender.
Above method detection infection RSMV rice disease leaf sensitivity reaches 1:40960 times and dilutes, unit w/v, g/mL, The sensitivity that detection single head carries RSMV recilia dorsalis reaches 1:5120 times and dilutes, and unit is single head/μ L；
The rice and recilia dorsalis tissue of above method detection infection rice stripe mosaic virus are in strong positive reaction, and are examined Survey infection rice stripe virus, rice black-streaked dwarf virus, southern rice black-streaked dwarf virus, rice sawtooth dwarf virus, water Rice tumor dwarf virus, fractilinea oryzae and healthy rice plants tissue and non-to take malicious recilia dorsalis tissue negative.
The present invention also provides a kind of applications of above-mentioned spot immune immunoblot method, it is characterised in that utilizes this method or root According to the kit that this method designs, checkout and diagnosis is carried out to field rice and the malicious situation of band for passing virus mediator recilia dorsalis, is determined The malicious rate of the band of rice and recilia dorsalis, happening and prevelence trend of the prediction and warning rice stripe mosaic disease in field.
The present invention has the advantages that 1) present invention provides a kind of detection rice stripe mosaic disease compared with prior art The spot immune immunoblot method based on immune colloid gold and its detection kit energy high special of poison, it is accurate, delicately detect Rice stripe mosaic virus；2) using a kind of detection rice stripe mosaic virus provided by the present invention based on immune colloid gold Spot immune immunoblot method does not need the equipment such as expensive electron microscope, PCR instrument；3) detection water provided by the invention is utilized The spot immune immunoblot method and its kit based on immune colloid gold of rice stripe mosaic virus, are effectively used for field water Rice and the detection and diagnosis for passing RSMV in virus mediator recilia dorsalis body, it can also be used to the epidemiological survey of the virosis, virus Genome functions analysis, resistance breeding, science bridle etc.；4) provide a kind of detection rice stripe mosaic virus based on exempting from The spot immune immunoblot method of epidemic disease colloidal gold does not need the enzyme mark of the immunological methods such as traditional dot-ELISA and ACP-ELISA Antibody and substrate chromogenic reaction, thus, detecting step is simpler and detection time greatly shortens.
Detailed description of the invention
Fig. 1 is the sensitivity point for detecting the western dot blot based on immune colloid gold of rice stripe mosaic virus Analysis；
Fig. 2 is the specificity point for detecting the western dot blot based on immune colloid gold of rice stripe mosaic virus Analysis: 1,2 be respectively the rice plants of infection RSMV and the recilia dorsalis for infecting RSMV；3-6 is respectively to infect rice stripe disease Poison, rice black-streaked dwarf virus, southern rice black-streaked dwarf virus, rice sawtooth dwarf virus, Rice Gall Dwarf In Guangdong Province, rice are short The rice disease leaf texture for the virus that contracts；9,10 are respectively the rice plants tissue of health and non-take malicious recilia dorsalis tissue.
Fig. 3 is the western dot blot detection field sample based on immune colloid gold for detecting rice stripe mosaic virus The result of RSMV in product.
Specific embodiment
Below by drawings and examples, further the present invention is described in detail.Used kit in the present invention Material, if can use commercial product without specified otherwise.Percentage in the present invention, if referring both to matter without specified otherwise Measure percentage.
A kind of spot immune immunoblot method based on immune colloid gold detecting rice stripe mosaic virus, which utilize The specific monoclonal antibody 1D4 of colloid gold label water resistant rice stripe mosaic virus detects rice stripe floral leaf as detection antibody Virus.Specific monoclonal antibody 1D4 is secreted by secretion water resistant rice stripe mosaic virus monoclonal antibody hybridoma, is recorded in patent application Number for 201810134449.5 Chinese invention patent application in.The secretion water resistant rice stripe mosaic virus monoclonal antibody hybridoma It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: BeiChen West Road, Chaoyang District, BeiJing City 1 Institute 3, postcode: 100101, preservation day is on December 15th, 2017, and deposit number is CGMCC No.14898.
In the spot immune immunoblot method based on immune colloid gold of the detection rice stripe mosaic virus, colloidal gold mark Remember the markers step of antibody:
1) prepared by colloidal gold solution: 0.01% gold chloride of 100mL being heated to boiling, 1% citric acid three of 1.2mL is added It is mixed after sodium, then gold chloride after boiling 30min is kept to be reduced into colloid gold particle solution；
2) 50mL colloidal gold solution is taken, with 0.1M K2CO3Its pH value is adjusted to 8.2；
3) the above-mentioned water resistant rice item for being added that 2mL IgG concentration is 2mg/mL in the colloidal gold solution of pH value while stirring is mixed up The monoclonal antibody 1D4 of line mosaic virus stirs 30min；The polyethylene glycol i.e. PEG that 1mL 25M molecular weight is 20,000 is added dropwise again Solution is stirred for 15min；
4) by previous step mixed liquor in 4 DEG C 20,000rpm is centrifuged 15min, abandons supernatant, and 10mL pH 7.4 is added and contains The 0.01M PBS buffer solution of 0.4M PEG 20,000 suspends, and 4 DEG C of 20,000rpm are centrifuged 15min, removes supernatant；
5) precipitating is suspended with 0.01M PBS buffer solution, is centrifuged, and is repeated 1 times；
6) it is dissolved and is precipitated containing the PBS buffer solution of 2%BSA and 0.02% sodium azide with 10mL pH 7.5, with 0.22 μm After sterilizing filter filtering, filtrate is colloidal gold labeled monoclonal antibody solution, and 4 DEG C save backup；
In the spot immune immunoblot method based on immune colloid gold for detecting rice stripe mosaic virus, specific detection step It is rapid as follows:
1) nitrocellulose (NC) film prepares: it is crossed on NC film outer layer paper with pencil, NC film is made to stamp grid stria mark, Every lattice specification 0.3 × 0.3cm, it is smooth to be put on clean filter paper；
2) sample preparation:
A) preparation of plant sample: old rice, wheat plant tissue weighing are placed in mortar with liquid nitrogen grinding into powder End, in 0.1g plant tissue add 2mL ratio the 0.01M PBS of pH 7.4 is added after continue grinding homogenate 1min in mortar；It is tender Rice, add the ratio of 2mL to be added after 0.01M PBS in 0.1g plant tissue after wheat plant tissue weighing to grind in mortar It is homogenized 2min；Homogenate moves in 1.5mL eppendorf centrifuge tube, 5,000rpm centrifugation 3min, or stands 5min, supernatant The as crude extract of rice or wheat plant tissue；
B) pass the preparation of virus mediator recilia dorsalis sample: a recilia dorsalis be put into the eppendorf of a 0.5ml from 50-100 μ L 0.01M PBS is added in heart Guan Zhonghou, mashes leafhopper in homogenate with toothpick butt end or 200 μ L pipette tips；
3) each 3 μ of crude extract and recilia dorsalis homogenate of rice or wheat plant tissue point sample: is drawn with pipettor L, point sample dry 10min in the grid center of NC film at room temperature；
4) it closes: adding the 100mL PBST buffer (0.01mol/ containing 0.05%Tween-20 using by 5g skimmed milk power L PBS) ratio prepare to obtain 5% skimmed milk power confining liquid, NC film closes 0.5-1h at room temperature in confining liquid；
5) wash: deblocking liquid washes film 2 times with PBST buffer, each 3min；
6) colloidal gold labeled monoclonal antibody is incubated for: NC film is put into the colloid gold label with 1:2000 times of 0.01mol/L PBS dilution In antibody-solutions in slowly shaken on horizontal shaker incubation at room temperature 1h；
7) it washs: removing colloidal gold labeled monoclonal antibody solution, wash film 4-5 times with PBST buffer, each 3min；
8) it visually observes judging result: the sample of punctation occurs for the positive, the sample of colourless or green spot is yin Property.
Detect the western dot blot detection infection RSMV water based on immune colloid gold of rice stripe mosaic virus Rice disease leaf sensitivity reaches 1:40960 times and dilutes, unit w/v, g/mL, and detection single head carries the sensitivity of RSMV recilia dorsalis Reach 1:5120 times to dilute, unit is single head/μ L；
Detect the western dot blot detection infection rice item based on immune colloid gold of rice stripe mosaic virus The rice and recilia dorsalis tissue of line mosaic virus are in strong positive reaction, and it is short to detect infection rice stripe virus, rice secret note Contract virus, southern rice black-streaked dwarf virus, rice sawtooth dwarf virus, Rice Gall Dwarf In Guangdong Province, fractilinea oryzae and be good for Health rice plants tissue and non-to take malicious recilia dorsalis tissue negative.
The western dot blot based on immune colloid gold and its kit for detecting rice stripe mosaic virus are to field Between rice and pass the malicious situation of band of virus mediator recilia dorsalis and carry out checkout and diagnosis, determine the malicious rate of the band of rice and recilia dorsalis, in advance Early warning rice stripe mosaic disease is surveyed in the happening and prevelence trend in field and takes scientific prevention and cure measure in time.
The western dot blot based on immune colloid gold of detection rice stripe mosaic virus provided by the invention and Its detection kit energy high special accurately, delicately detects field rice and passes rice stripe in virus mediator recilia dorsalis body Mosaic virus, it can also be used to the epidemiological survey of the virosis, prediction and warning, viral genome functional analysis, resistance breeding, Science bridle etc., and do not need the equipment such as expensive electron microscope, PCR instrument, do not need traditional dot-ELISA and The enzyme labelled antibody and substrate chromogenic reaction of the immunological methods such as ACP-ELISA, thus detecting step is simpler and detection time It greatly shortens.To provide substance and technical support for the checkout and diagnosis, prediction and warning and science bridle of RS in China MV.
Below with reference to embodiment and attached drawing, the invention will be further described.
One, the preparation of colloidal gold labeled monoclonal antibody
1. the preparation of monoclonal antibody ascites and purifying
(1) 8 week old BALB/c mouses are taken, 0.3mL norphytane, Intraperitoneal injection 6 × 10 after 7-10d is injected intraperitoneally5It is a to divide The hybridoma (the application Chinese patent 201810134449.5 from applicant) of anti-RSMV monoclonal antibody is secreted, is injected The visible mouse web portion of 7-10d obviously expands afterwards, and syringe needle takes ascites, and 3000 rpm are centrifuged 1min, and collecting supernatant is Dan Ke Grand antibody ascites.
(2) saturated ammonium sulfate method purified monoclonal antibody ascites (by taking 1mL as an example)
1) 5 000rpm of 1mL ascites, 4 DEG C of centrifugation 5min are taken, supernatant is taken, 2mL normal saline dilution is added；
2) pH value is added dropwise in the saturated ammonium sulfate solution 3mL of 7.0-7.4, stirring while adding, room temperature after being all added Continue to stir 10min；4 DEG C of static 2-3h；
3) 12 000rpm, 4 DEG C of centrifugation 10min abandon supernatant, precipitating 2mL physiological saline solution；
4) antibody is packed into bag filter, and for 24 hours, during which every 2h changes a PBS solution for 4 DEG C of dialysis in 0.01M PBS；
5) antibody in bag filter, 12 4 DEG C of 000rpm centrifugation 10min are taken out, supernatant is the antibody purified；It takes on 1 μ L IgG content is measured with NanoDrop ultraviolet spectrometer clearly, antibody is saved in -80 DEG C of refrigerators after measurement.
2. prepared by colloidal gold solution
0.01% gold chloride of 100mL is heated to boiling, is mixed after 1% trisodium citrate of 1.2mL is added, then keep boiling Gold chloride is reduced into colloid gold particle solution after rising 30min.
3. the preparation of colloidal gold labeled monoclonal antibody
1) 50mL colloidal gold solution is taken, with 0.1M K2CO3Its pH value is adjusted to 8.2；
2) the above-mentioned water resistant rice item for being added that 2mL IgG concentration is 2mg/mL in the colloidal gold solution of pH value while stirring is mixed up The monoclonal antibody 1D4 of line mosaic virus stirs 30min；The polyethylene glycol i.e. PEG that 1mL 25M molecular weight is 20,000 is added dropwise again Solution is stirred for 15min；
3) by previous step mixed liquor in 4 DEG C 20,000rpm is centrifuged 15min, abandons supernatant, and 10mL pH 7.4 is added and contains The 0.01M PBS buffer solution of 0.4M PEG 20,000 suspends, and 4 DEG C of 20,000rpm are centrifuged 15min, removes supernatant；
4) precipitating is suspended with 0.01M PBS buffer solution, is centrifuged, and is repeated 1 times；
5) it is dissolved and is precipitated containing the PBS buffer solution of 2%BSA and 0.02% sodium azide with 10mL pH 7.5, with 0.22 μm After sterilizing filter filtering, filtrate is colloidal gold labeled monoclonal antibody solution, and 4 DEG C save backup；
Two, the foundation of the western dot blot based on immune colloid gold of rice stripe mosaic virus is detected
1. detecting the operating procedure 1 of the western dot blot based on immune colloid gold of rice stripe mosaic virus) Nitrocellulose (NC) film prepares: being crossed on NC film outer layer paper with pencil, NC film is made to stamp grid stria mark, every lattice specification 0.3 × 0.3cm, it is smooth to be put on clean filter paper；
2) sample preparation:
A) preparation of plant sample: old rice, wheat plant tissue weighing are placed in mortar with liquid nitrogen grinding into powder End, in 0.1g plant tissue add 2mL ratio the 0.01M PBS of pH 7.4 is added after continue grinding homogenate 1min in mortar；It is tender Rice, add the ratio of 2mL to be added after 0.01M PBS in 0.1g plant tissue after wheat plant tissue weighing to grind in mortar It is homogenized 2min；Homogenate moves in 1.5mL eppendorf centrifuge tube, 5,000rpm centrifugation 3min, or stands 5min, supernatant The as crude extract of rice or wheat plant tissue；
B) pass the preparation of virus mediator recilia dorsalis sample: a recilia dorsalis be put into the eppendorf of a 0.5ml from 50-100 μ L 0.01M PBS is added in heart Guan Zhonghou, mashes leafhopper in homogenate with toothpick butt end or 200 μ L pipette tips；
3) each 3 μ of crude extract and recilia dorsalis homogenate of rice or wheat plant tissue point sample: is drawn with pipettor L, point sample dry 10min in the grid center of NC film at room temperature；
4) it closes: adding the 100mL PBST buffer (0.01mol/ containing 0.05%Tween-20 using by 5g skimmed milk power L PBS) ratio prepare to obtain 5% skimmed milk power confining liquid, NC film closes 0.5-1h at room temperature in confining liquid；
5) wash: deblocking liquid washes film 2 times with PBST buffer, each 3min；
6) colloidal gold labeled monoclonal antibody is incubated for: NC film is put into the suitably diluted colloidal gold labeled monoclonal antibody of 0.01mol/L PBS In solution in slowly shaken on horizontal shaker incubation at room temperature 1-3h；
7) it washs: removing colloidal gold labeled monoclonal antibody solution, wash film 4-5 times with PBST buffer, each 3min；
8) it visually observes judging result: the sample of punctation occurs for the positive, the sample of colourless or green spot is yin Property.
2. the determination of colloidal gold antibody working concentration
Using the rice and recilia dorsalis for infecting RSMV as positive control, nontoxic rice and virus-free recilia dorsalis are negative right According to, using 0.01mol/L PBS to colloidal gold antibody carry out from 1:500-1:128000 times of doubling dilution, take each dilution Colloidal gold antibody carries out the detection of spot immune trace, determines the spot based on immune colloid gold of detection rice stripe mosaic virus The best effort concentration of colloidal gold antibody in immunoblot assay, 3 repetitions the experimental results showed that, it is dilute when colloidal gold antibody When degree of releasing is 1:2000 times, the detection effect of the technology is best, therefore, dense for the work of the technology colloidal gold antibody with this concentration Degree.
3. detecting the sensitivity and specificity analysis of RSMV western dot blot
The rice plants tissue crude extract of RSMV is infected with 0.01M PBS from 1:160-81920 times of doubling dilution (w/v, g/ ML), single head recilia dorsalis homogenate takes each dilution crude extract or homogenate from 1:160-20480 times of doubling dilution (head/μ L) The western dot blot detection that liquid is established respectively, testing result discovery, western dot blot detection infection RSMV rice disease leaf sensitivity reaches 1:40960 times and dilutes, unit w/v, g/mL, and detection single head carries RSMV recilia dorsalis Sensitivity reaches 1:5120 times and dilutes, and unit is single head/μ L (Fig. 1).
With infection rice stripe virus (RSV), rice black-streaked dwarf virus (RBSDV), southern rice black-streaked dwarf respectively The disease of viral (SRBSDV), rice sawtooth dwarf virus (RRSV), Rice Gall Dwarf In Guangdong Province (RGDV), fractilinea oryzae (RDV) Leaf crude extract is test sample, is to infect rice plants tissue crude extract and the infection RSMV recilia dorsalis homogenate of RSMV Positive control is coated with respectively using healthy rice plants tissue crude extract and virus-free recilia dorsalis homogenate as negative control Elisa plate carries out spot immune print using the healthy leaves crude extract of rice, the non-homogenate for taking malicious recilia dorsalis as negative control Mark detection, as a result, it has been found that, the rice and recilia dorsalis tissue of the technology detection infection rice stripe mosaic virus of the foundation are in strong Positive reaction, and detect infection rice stripe virus, rice black-streaked dwarf virus, southern rice black-streaked dwarf virus, rice saw Tooth dwarf virus, Rice Gall Dwarf In Guangdong Province, fractilinea oryzae and healthy rice plants tissue and non-take malicious recilia dorsalis tissue Negative (Fig. 2).
4. detecting the Field information of RSMV western dot blot
The water that rice field using the western dot blot of foundation in August, 2018 in Luoding City, Guangdong Province acquires Totally 27 samples are detected for rice and recilia dorsalis, and testing result discovery has 16 sample infection RSMV (Fig. 3).It uses simultaneously It is completely quasi- that the nucleic acid sequencing and sequence alignment of RT-PCR detection and PCR product demonstrate the western dot blot testing result Really.
Two, rice stripe mosaic virus spot immune Blot Detection kits
1. rice stripe mosaic virus spot immune prints detection kit main component:
The above reagent is stored in 4 DEG C
2. detecting the operating procedure of rice sample:
1) nitrocellulose (NC) film prepares: it is crossed on NC film outer layer paper with pencil, NC film is made to stamp grid stria mark, Every lattice specification 0.3 × 0.3cm, it is smooth to be put on clean filter paper；
2) sample preparation:
A) preparation of plant sample: old rice, wheat plant tissue weighing are placed in mortar with liquid nitrogen grinding into powder End, in 0.1g plant tissue add 2mL ratio the 0.01M PBS of pH 7.4 is added after continue grinding homogenate 1min in mortar；It is tender Rice, add the ratio of 2mL to be added after 0.01M PBS in 0.1g plant tissue after wheat plant tissue weighing to grind in mortar It is homogenized 2min；Homogenate moves in 1.5mL eppendorf centrifuge tube, 5,000rpm centrifugation 3min, or stands 5min, supernatant The as crude extract of rice plants tissue；
B) pass the preparation of virus mediator recilia dorsalis sample: a recilia dorsalis be put into the eppendorf of a 0.5ml from 50-100 μ L 0.01M PBS is added in heart Guan Zhonghou, mashes leafhopper in homogenate with toothpick butt end or 200 μ L pipette tips；
3) point sample: the 3 μ L of crude extract or recilia dorsalis homogenate of rice plants tissue is drawn with pipettor, point sample is in NC The grid center of film, dries 10min at room temperature；
4) it closes: adding the 100mL PBST buffer (0.01mol/ containing 0.05%Tween-20 using by 5g skimmed milk power L PBS) ratio prepare to obtain 5% skimmed milk power confining liquid, NC film closes 0.5-1h at room temperature in confining liquid；
5) wash: deblocking liquid washes film 2 times with PBST buffer, each 3min；
6) colloidal gold labeled monoclonal antibody is incubated for: NC film is put into the colloid gold label with 1:2000 times of 0.01mol/L PBS dilution In antibody-solutions in slowly shaken on horizontal shaker incubation at room temperature 1h；
7) it washs: removing colloidal gold labeled monoclonal antibody solution, wash film 4-5 times with PBST buffer, each 3min；
8) it visually observes judging result: the sample of punctation occurs for the positive, the sample of colourless or green spot is yin Property.
3) preservation and validity period:
It is kept in dark place in 2-8 DEG C, validity period 12 months.
4) buffer formulation:
A. phosphate buffer (PBS, 0.01mol/L, pH7.4): NaCl 8g, KCl 0.2g, KH2PO4 0.2g, Na2HPO4·12H2O 3g adjusts pH to 7.4 after adding distilled water 950mL to dissolve, is settled to 1000mL.
B.ELISA cleaning solution (0.01M PBST): add 0.5mL Tween-20 in 1000mL 0.01M PBS.
C.ELISA confining liquid: skimmed milk power is added in 0.01M PBST to final concentration 5% (w/v, g/mL).

Claims (6)

1. a kind of spot immune immunoblot method based on immune colloid gold for detecting rice stripe mosaic virus, it is characterised in that use The specific monoclonal antibody 1D4 of colloid gold label water resistant rice stripe mosaic virus is as detection antibody；The water resistant rice stripe mosaic The specific monoclonal antibody 1D4 of virus is secreted by hybridoma cell strain, during which was preserved on December 15th, 2017 State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC No.14898.
2. the spot immune trace side based on immune colloid gold of detection rice stripe mosaic virus as described in claim 1 Method, it is characterised in that included the following steps: with colloidal gold labeled monoclonal antibody
1) prepared by colloidal gold solution: 0.01% gold chloride of 100mL being heated to boiling, after 1% trisodium citrate of 1.2mL is added It mixes, then gold chloride after boiling 30min is kept to be reduced into colloid gold particle solution；
2) colloidal gold solution described in 50mL is taken, with 0.1M K2CO3Its pH value is adjusted to 8.2；
3) specific monoclonal antibody for being added that 2mL IgG concentration is 2mg/mL in the colloidal gold solution of pH value while stirring is mixed up 1D4 stirs 30min；The polyethylene glycol i.e. PEG solution that 1mL 25M molecular weight is 20000 is added dropwise again, is stirred for 15min；
4) then by mixed liquor obtained in 3), 15min is centrifuged in 4 DEG C of 20000rpm, abandon supernatant, 10mL pH 7.4 is added And the 0.01M PBS buffer solution of PEG containing 0.4M 20000 suspends, and is centrifuged 15min in 4 DEG C of 20000rpm, supernatant is gone to be sunk It forms sediment；
5) precipitating is repeated to be suspended with 0.01M PBS buffer solution, centrifugation；
6) it with 10mL pH 7.5 and containing being precipitated obtained in the dissolution 5) of the PBS buffer solution of 2%BSA and 0.02% sodium azide, uses After 0.22 μm of sterilizing filter filtering, filtrate is colloidal gold labeled monoclonal antibody solution, and 4 DEG C save backup.
3. the spot immune trace side based on immune colloid gold of detection rice stripe mosaic virus as claimed in claim 2 Method, it is characterised in that specifically included using the method that the colloidal gold labeled monoclonal antibody solution carries out the detection of rice stripe mosaic virus Following steps:
1) nitrocellulose (NC) film prepares: being crossed on NC film outer layer paper with pencil, NC film is made to stamp grid stria mark, every lattice Specification 0.3 × 0.3cm, it is smooth to be put on clean filter paper；
2) sample preparation:
A) preparation of plant sample: rice or wheat plant tissue weighing are placed in mortar with liquid nitrogen grinding into powder, pressed 0.1g plant tissue continues grinding homogenate 1min in mortar after adding the ratio of 2mL that the 0.01M PBS of pH 7.4 is added, and is homogenized Liquid；Or add Yu Yan after the ratio addition 0.01M PBS of 2mL after rice or wheat plant tissue are weighed in 0.1g plant tissue Grinding homogenate 2min, obtains homogenate in alms bowl；Homogenate is moved in 1.5mL centrifuge tube, 5000rpm is centrifuged 3min, or stands 5min, supernatant are the crude extract of rice or wheat plant tissue；
B) pass the preparation of virus mediator recilia dorsalis sample: 50- is added after being put into the centrifuge tube of a 0.5ml in a recilia dorsalis 100 μ L 0.01M PBS mash leafhopper in homogenate with toothpick butt end or 200 μ L pipette tips；
3) each 3 μ L of crude extract and recilia dorsalis homogenate of rice or wheat plant tissue, point sample point sample: are drawn with pipettor In the grid center of NC film, 10min is dried at room temperature；
4) it closes: adding the ratio of 100mL PBST buffer to prepare to obtain 5% skimmed milk power closing using in 5g skimmed milk power Liquid, the PBST buffer are the 0.01mol/L PBS containing 0.05%Tween-20；NC film is closed at room temperature in confining liquid 0.5-1h；
5) wash: deblocking liquid washes film 2 times with PBST buffer, each 3min；
6) colloidal gold labeled monoclonal antibody is incubated for: it is molten that NC film is put into the colloidal gold labeled monoclonal antibody for diluting 2000 times with 0.01mol/L PBS In liquid, 1h is incubated at room temperature in slowly shaking on horizontal shaker；
7) it washs: removing colloidal gold labeled monoclonal antibody solution, wash film 4-5 times with PBST buffer, each 3min；
8) it observes judging result: the sample of punctation occurs for the positive, the sample of colourless or green spot is feminine gender.
4. the spot immune trace side based on immune colloid gold of detection rice stripe mosaic virus as described in claim 1 Method, it is characterised in that its detection infection RSMV rice disease leaf sensitivity reaches 1:40960 times and dilutes, unit w/v, g/mL, inspection The sensitivity for surveying single head carrying RSMV recilia dorsalis reaches 1:5120 times and dilutes, and unit is single head/μ L.
5. the spot immune trace side based on immune colloid gold of detection rice stripe mosaic virus as described in claim 1 Method, it is characterised in that its rice for detecting infection rice stripe mosaic virus and recilia dorsalis tissue are examined in strong positive reaction Survey infection rice stripe virus, rice black-streaked dwarf virus, southern rice black-streaked dwarf virus, rice sawtooth dwarf virus, water Rice tumor dwarf virus, fractilinea oryzae and healthy rice plants tissue and non-to take malicious recilia dorsalis tissue negative.
6. a kind of application of spot immune immunoblot method as described in claim 1, it is characterised in that using this method or according to this The kit of method design carries out checkout and diagnosis to field rice and the malicious situation of band for passing virus mediator recilia dorsalis, determines rice With the malicious rate of band of recilia dorsalis, happening and prevelence trend of the prediction and warning rice stripe mosaic disease in field.
CN201910081205.XA 2019-01-28 2019-01-28 A kind of spot immune immunoblot method based on immune colloid gold of detection rice stripe mosaic virus and its application Pending CN109633153A (en)

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