CN107918012A - A kind of immunity detection reagent of muskmelon macula lutea virus and its application - Google Patents

A kind of immunity detection reagent of muskmelon macula lutea virus and its application Download PDF

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CN107918012A
CN107918012A CN201610885141.5A CN201610885141A CN107918012A CN 107918012 A CN107918012 A CN 107918012A CN 201610885141 A CN201610885141 A CN 201610885141A CN 107918012 A CN107918012 A CN 107918012A
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muskmelon
macula lutea
virus
mysv
detection reagent
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曾蓉
戴富明
徐丽慧
高士刚
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Shanghai Academy of Agricultural Sciences
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Shanghai Academy of Agricultural Sciences
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses

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  • Food Science & Technology (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Immunity detection reagent and its application the invention discloses a kind of muskmelon macula lutea virus, the kit includes containing the specific polyclonal antibodies of muskmelon macula lutea virus MYSV and ELIAS secondary antibody, has the characteristics that easy to use, easy to operate, rapid sensitive, reliable results, flux are high.Kit of the present invention can detect whether plant leaf blade carries MYSV under laboratory and non-laboratory condition, be adapted to quick detection, the Epidemiology monitor of MYSV of sample, and then take appropriate Prevention method, reduce the loss that disease is brought.

Description

A kind of immunity detection reagent of muskmelon macula lutea virus and its application
Technical field
The invention belongs to technical field of agriculture science, and in particular to a kind of immunity detection reagent of muskmelon macula lutea virus and It is applied.
Background technology
Muskmelon macula lutea virus (Melon Yellow Spot Virus, MYSV) belongs to bunyaviridae, spotted wilt virus Belong to, occur in Japan's report within earliest 1992.The virus mainly infects the important Curcurbitaceae such as watermelon, muskmelon, cucumber, balsam pear and makees Thing.In last decade, there are report in the various regions such as Japan, China, Thailand, Holland, Ecuador, to local ground family crop Production cause serious influence, economic loss is serious.The virus 2009 is on the Guangdong in China, Hainan, Guangxi, Shandong and other places It is found in succession, mainly infects watermelon and muskmelon, incidence is 30%~100%.
2013, on the part cucumber plant of Senior Residents in Fengxian District of Shanghai, find the generation of MYSV viruses, incidence is about 10%.2014, in the Muskmelon Planting area such as Jiading District in Shanghai City, Pudong New District, it has been found that MYSV is in part muskmelon and Hami melon Serious, incidence 50%~80% occurs in kind.2015, on the part cucumber plant in Shanghai, incidence was more than 80%. The viral global spread and outburst, cause great loss to local ground family crop production, become ground family crop One of important threatening factors of production.
After MYSV virus infection plant, plant leaf symptom is very various, is difficult to distinguish with other virus symptoms.Pin at present Mainly there are electron microscopic observation method, RT-PCR, RT-LAMP, Northern blot etc. to the detection method of MYSV, but they are required for Expensive instrument and cumbersome operation, and it is unsuitable for the detection of batch samples.
The content of the invention
Immunity detection reagent and its application it is an object of the invention to provide a kind of muskmelon macula lutea virus, for current Viral diagnosis it is cumbersome, of high cost, with duration the problems such as, there is provided it is a kind of quick, convenient, sensitive, efficient, high specificity The immunity detection reagent of the muskmelon macula lutea virus of non-laboratory condition.
To reach above-mentioned purpose, the technical scheme is that：
A kind of immunity detection reagent of muskmelon macula lutea virus, it includes, macula lutea containing muskmelon virus (MYSV) it is polyclonal Antibody and ELIAS secondary antibody；The polyclonal antibody of the virus of macula lutea containing muskmelon is the muskmelon macula lutea viral capsid using prokaryotic expression Albumen is the antibody that immunogene is prepared.
Further, the preparation method of the polyclonal antibody of the virus of macula lutea containing muskmelon is：By the method for prokaryotic expression, Give expression to the capsid protein (coat protein, CP) of muskmelon macula lutea virus；After SDS-PAGE Purified in electrophoresis, ear vein is immunized Rabbit；Heart extracting blood after 3-5 times immune, collects upper serum, and purifies.
The ELIAS secondary antibody for it is labeled it is any it is common can with the antibody of MYSV antibody bindings, wherein, marker enzyme can , can be with antibody such as goat anti-rabbit igg, horse anti-rabbit IgG, the ox of MYSV antibody bindings for horseradish peroxidase or alkaline phosphatase etc. Anti-rabbit IgG, mouse anti-rabbit IgG etc..The ELIAS secondary antibody is preferably the goat anti-rabbit igg serum of alkali phosphatase enzyme mark.
The immunity detection reagent further includes positive quality control, filter membrane, sample lapping liquid, film washing liquid, confining liquid and colour developing Liquid, the filter membrane are nylon membrane or nitrocellulose filter.
Again, the positive quality control is the prokaryotic expression protein of muskmelon macula lutea virus N-gene (MYSV-CP).
Application of the immunity detection reagent of the present invention in muskmelon macula lutea viral diagnosis.
The application method of the immunity detection reagent of muskmelon macula lutea virus of the present invention, it includes the following steps：
1) sample to be tested is mixed with sample lapping liquid, obtained mixed liquor is dropped on filter membrane, room temperature air-dries, and sets at the same time Put positive and negative control group；
2) dried filter membrane is immersed in confining liquid, when room temperature placement 0.5~2 is small；
3) polyclonal antibody of muskmelon macula lutea virus is included with confining liquid dilution, antibody dilution ratio is：1:2000~1: 20000, volume ratio；The filter membrane that step 2) is obtained is immersed in the polyclonal antibody after dilution, when room temperature placement 1~2 is small, then is used Film washing liquid washes film 5~10 minutes；
4) ELIAS secondary antibody is diluted with confining liquid, ELIAS secondary antibody dilution ratio is 1:4000~1:20000, volume ratio；Will step The rapid filter membrane 3) obtained is immersed in the ELIAS secondary antibody after dilution, when room temperature placement 0.5~1 is small, then washes 5~10 points of film with film washing liquid Clock；
5) nitrite ion is added, room temperature is placed to colour developing under dark condition, observes result：It is shown as purple, sample to be tested Carry MYSV viruses；Do not develop the color or only blade true qualities, sample to be tested do not carry MYSV viruses.
The antibody that the present invention is prepared using the MYSV capsid proteins of prokaryotic expression, the special detection MYSV of energy, with other diseases Poison or healthy plant sample no cross reaction, can be used for the sick detection well.
The specific polyclonal antibodies of MYSV prepared by first Application of the present invention establish a kind of spot immune (Dot Immunobinding Assay, DIBA) detection method, the detection kit of muskmelon macula lutea virus has been built up in this way.The examination Agent box has the characteristics that easy to use, easy to operate, rapid sensitive, reliable results, flux are high, can be applied to laboratory condition or The detection whether plant leaf blade carries MYSV is detected under non-laboratory condition, quick and precisely to judge disease species, so as to formulate For control strategy, reduce the loss that brings of disease, there is very high practical value.
Beneficial effects of the present invention：
(1) specificity is good：The rabbit antibody of CP albumen of the polyclonal antibody of the present invention from MYSV, can specific recognition MYSV.The polyclonal antibody and the confusing CGMMV in field are without immune response, even if the TSWV (tomato spotted wilf virus) belonged to together Without immune response, the sample detection in laboratory or field can be applied to well.
(2) high sensitivity：Kit of the present invention is 1 in ELIAS secondary antibody diluted concentration：25 μ g can be detected when 10000 Contain MYSV in plant sample；It is 1 in ELIAS secondary antibody diluted concentration：It can be detected when 5000 in 3.12 μ g plant samples and contain MYSV.
(3) cost is low：The kit can carry out at ambient temperature, it is not necessary to expensive molecular biology instruments.
(4) kit of the present invention remains to good specific detection in non-laboratory condition and goes out MYSV, easy, accurately may be used Lean on, operated suitable for basic unit plant protection personnel, can find and control in time virus, reduce economic loss.
Brief description of the drawings
Fig. 1 be the embodiment of the present invention 2 in laboratory conditions muskmelon macula lutea virus immunity detection reagent specificity tie Fruit is schemed, wherein ,+it is prokaryotic expression MYSV coat protein antigens, 2-5 arranges respectively MYSV, CGMMV (cucumber green mottle floral leaves Virus), TSWV (tomato spotted wilf virus), Healthy (virus-free infection) sample, experiment is in triplicate.
Fig. 2 is the immunity detection reagent sensitivity knot of the muskmelon macula lutea virus in laboratory conditions of the embodiment of the present invention 3 One of fruit figure, wherein ,+it is prokaryotic expression MYSV coat protein antigens, the 1st row 1-6 is the cucumber leaves for carrying MYSV, and concentration is terraced Spend into 2 times of dilutions, 2.00 × 10-4-6.25×10-6Gram blade；Second behavior negative control CGMMV (cucumber leaves).
Fig. 3 is the immunity detection reagent sensitivity knot of the muskmelon macula lutea virus in laboratory conditions of the embodiment of the present invention 3 The two of fruit figure, wherein ,+it is prokaryotic expression MYSV coat protein antigens, the 1st row 1-6 is the cucumber leaves for carrying MYSV, and concentration is terraced Spend into 2 times of dilutions, 2.00 × 10-4-6.25×10-6Gram blade；Second behavior negative control TSWV (tobacco leaf).
Fig. 4 is the immunity detection reagent sensitivity knot of the muskmelon macula lutea virus in laboratory conditions of the embodiment of the present invention 3 The three of fruit figure, wherein ,+it is prokaryotic expression MYSV coat protein antigens, the 1st row 1-6 is the cucumber leaves for carrying MYSV, and concentration is terraced Spend into 2 times of dilutions, 2.00 × 10-4-6.25×10-6Gram blade；Second behavior negative control cucumber healthy leaves.
Fig. 5 is ELIAS secondary antibody in the embodiment of the present invention 3 in laboratory conditions muskmelon macula lutea virus immunity detection reagent Diluted concentration is 1：Sensitivity results schematic diagram when 5000, wherein ,+it is prokaryotic expression MYSV coat protein antigens；The first row To carry the cucumber leaves of MYSV, concentration gradient dilutes 1-9 lattice into 2 times, and 2.00 × 10-4-7.81×10-7Gram blade, the second row 1-9 lattice Negative compares for negative.
Fig. 6 detects field sample for the embodiment of the present invention 4 in non-experiment condition muskmelon macula lutea virus immunity detection reagent Colour developing result schematic diagram, wherein, A1 is positive quality control, and A2-G6 is field sample, and G7 is healthy cucumber leaves.
Embodiment
With reference to embodiment and attached drawing, the present invention will be further described.It is to be understood that these embodiments are merely to illustrate The present invention rather than limit the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, this area Technical staff can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims Limited range.
In the present embodiment, the immunity detection reagent of muskmelon macula lutea virus includes：
(1) MYSV polyclonal antibodies：The polyclonal antibody of MYSV immunizing rabbits, per 50 secondary response of kit；
(2) ELIAS secondary antibody：The goat anti-rabbit igg of alkaline phosphatase (AP) mark, its work potency are 1:4000-1:20000, Per 50 secondary response of kit, for detecting whether plant tissue carries MYSV.
(3) positive quality control：The prokaryotic expression protein antigen of muskmelon macula lutea virus N-gene (MYSV-CP)；
(4) nylon membrane or nitrocellulose filter of positively charged；
(5) sample lapping liquid：Phosphate buffer (10 × PBS, 0.1M, pH7.5；NaCl 1.37M, KCl 27mM, Na2HPO4-7H2O43mM, KH2PO414mM)；
(6) film washing liquid：Tris-HCl- tweens buffer salt (10 × TBST, 200mM Tri-Cl, pH7.5；NaCl 1.5M； Tween 0.5%)；
(7) confining liquid：5% skimmed milk power (1 × TBST)；
(8) nitrite ion：BCIP/NBT (BCIP (the chloro- 3- indyls-phosphate of the bromo- 4- of 5-) 0.42mg/mL；NBT (tetrazoliums Nitro is blue) 0.21mg/mL, it is dissolved in 0.01M AMP buffer solutions), whether carry MYSV for plant tissue.
The main agents of kit of the present invention provide in the form of working solution or concentrate, which preserves or transport all Should be 4 DEG C or so in low temperature, to ensure the activity of polyclonal antibody and ELIAS secondary antibody in kit.
Pay attention to：Detection all needs to set positive and negative control group every time, if occurring false positive or false negative in detection process, It may be that kit fails, stop being used to detect.
The preparation of embodiment 1MYSV polyclonal antibodies
1) 1mL bacterium after a large amount of induced expressions of the capsid protein of MYSV, will be taken, precipitation abandons supernatant, is suspended with 100 μ L PBST Precipitation, adds 20 μ L 6 × SDS-PAGE Loading buffer, and bacteria suspension is boiled 10min, ice bath 5min by 100 DEG C, 10000r/min centrifuges 5min, and rear supernatant is separated through 12.5% SDS-PAGE, is dyed with 0.25mol/L potassium chloride, is cut mesh Band；
2) gel is pressed 1:1 (W/V) is added after 0.9%NaCl cryogrindings and two rabbit, the 1st note is immunized as antigen Penetrate and add isometric Freund's complete adjuvant, subsequent 5 injections added isometric incomplete Freund's adjuvant, every 7 days immune 1 It is secondary, 3 days heart extracting bloods after the 5th is immunized；
3) 4 DEG C of placement 8h, 14000r/min centrifugation 5min of blood, collect upper serum, purifying.
The specific test of 2 muskmelon macula lutea virus immunity detection reagent of embodiment
1) nylon membrane is drawn to the lattice of 5mm × 5mm with pencil, length and width are respectively 5 × 3 lattice, are cut spare；
2) film washing liquid and sample lapping liquid pure water are diluted 10 times, makes 1 × TBST, 1 × PBS；
3) sample to be tested 0.1g is put into 1.5mL centrifuge tubes, is added the sample lapping liquid 1mL diluted and (is diluted 10 Times), after small grinding rod is smashed to pieces, taking 2 μ L of supernatant fluid, carefully drop is air-dried in nylon membrane lattice center, room temperature；The 1st row of nylon membrane The prokaryotic expression protein of 1st lattice point MYSV-CP, the 1st row 2-5 lattice are respectively MYSV (cucumber leaves), CGMMV (cucumber leafs Piece), TSWV (tobacco leaf), cucumber healthy leaves, sample is in triplicate；
4) dried nylon membrane is dipped in 5mL confining liquids, when 37 DEG C of incubators placements 0.5 are small；
5) 1 μ L MYSV polyclonal antibodies are drawn into fresh 5mL confining liquids, nylon membrane is immersed to clean culture dish, room When temperature placement 1.5 is small；Film 10min is washed with film washing liquid (1 × TBST) shaking table；
6) liquid-transfering gun draws 0.5 μ L ELIAS secondary antibodies into fresh 5mL confining liquids, and nylon membrane is immersed to clean culture dish, When room temperature placement 1 is small；Film 10min is washed with film washing liquid (1 × TBST) shaking table；
7) nitrite ion is added, dark condition room temperature is to developing the color (20-30min), and observed result, concrete outcome is referring to Fig. 1.
In Fig. 1, the 2nd row show purple, and the 3rd, 5 row only have blade true qualities (green), the 4th row not to develop the color, thus Verification：This kit can specifically detect MYSV viruses.
The sensitivity test of 3 muskmelon macula lutea virus immunity detection reagent of embodiment
1) nylon membrane is drawn to the lattice of 5mm × 5mm with pencil, length and width are respectively 5 × 3 lattice, are cut spare；
2) film washing liquid and sample lapping liquid pure water are diluted 10 times, makes 1 × TBST, 1 × PBS；
3) sample to be tested 0.1g is put into 1.5mL centrifuge tubes, adds the sample lapping liquid 1mL diluted, and small grinding rod is smash After broken, taking 2 μ L of supernatant fluid, carefully drop is air-dried in nylon membrane lattice center, room temperature；The protokaryon of the 1st lattice point MYSV-CP of 1st row Expressing protein, the 2-6 lattice respectively cucumber leaves sample of carrying MYSV, 2.00 × 10-4-6.25×10-6Gram blade, concentration Gradient in 2 times dilution after take 2 μ L points in grid center, the 2nd behavior negative control sample, respectively choose CGMMV (cucumber leaves), TSWV (tobacco leaf), three negative controls of cucumber healthy leaves, respectively as in Figure 2-4；
4) dried nylon membrane is dipped in 5mL confining liquids, when 37 DEG C of incubators placements 0.5 are small；
5) 1 μ L MYSV polyclonal antibodies are drawn into fresh 5mL confining liquids, film are immersed to clean culture dish, room temperature is put Put 1.5 it is small when；
6) film 10min is washed with film washing liquid (1 × TBST) shaking table；
7) liquid-transfering gun draws 0.5 μ L ELIAS secondary antibodies into fresh 5mL confining liquids, and film is immersed to clean culture dish, room temperature Place 1 it is small when；
8) film 10min is washed with film washing liquid (1 × TBST) shaking table；
9) nitrite ion is added, for dark condition room temperature to developing the color (20-30min), observed result, display purple is band MYSV diseases Poison, does not develop the color or only blade true qualities are without poison, and observation experiment is as a result, specifically develop the color result as in Figure 2-4.
The 1st row 1-3 lattice show purple in Fig. 2-4, and the 2nd row only has blade true qualities (green), Fig. 3 in Fig. 2, Fig. 4 In the 2nd row do not develop the color.
From Fig. 2-4, the antiserum of MYSV can specifically, sensitively detect the Plant samples containing MYSV, with CGMMV, TSWV, healthy plant and the reaction of grind away buffer solution serum-free, do not show purple, sensitivity, which reaches, can detect 0.25 × 10-4Gram plant Thing blade.
The ELIAS secondary antibody of step 7) is taken into 1 μ L to fresh, in 5mL confining liquids, film is immersed to clean culture dish, room temperature is put Put 1 it is small when, other steps are same as above, colour developing the results are shown in Figure 5.In Fig. 5 ,+it is prokaryotic expression MYSV coat protein antigens；First To carry the cucumber leaves of MYSV, concentration gradient dilutes row 1-9 lattice into 2 times, and 2.00 × 10-4-7.81×10-7Gram blade, second Row 1-9 lattice Negative compares for negative, i.e., response sample is grind away buffer solution.
In Fig. 5, the 1st row 1-7 lattice show purple, and colour developing gradually becomes shallower as；8-9 lattice colour developing unobvious；2nd row is equal Do not develop the color.As shown in Figure 5, the concentration of ELIAS secondary antibody is improved, the detection sensitivity of kit can be greatly improved, can detect 3.12 ×10-6Gram blade contains MYSV, and sensitivity improves 8 times or so, and non-false positive occurs.
The application of the non-test condition of 4 muskmelon macula lutea virus immunity detection reagent of embodiment
1) nylon membrane is drawn to the lattice of 5mm × 5mm with pencil, it is spare according to how much sample is cut；
2) film washing liquid and sample lapping liquid pure water are diluted 10 times, makes 1 × TBST, 1 × PBS；
3) sample to be tested is put into 0.5mL centrifuge tubes, volume is no more than the 1/4 of centrifugation pipe volume, adds what is diluted Sample lapping liquid 1-3 drops, after toothpick is smashed to pieces, dipping liquid above, carefully drop is air-dried in nylon membrane lattice center, room temperature；
4) dried nylon membrane is dipped in 5mL or so confining liquids, when room temperature placement 1 is small；
5) the MYSV polyclonal antibodies that toothpick dips in 1 secondary response immerse film into fresh 5mL confining liquids, and it is small that room temperature places 2 When；
6) film 10min is washed with film washing liquid (1 × TBST)；
7) toothpick dips in 1 secondary response ELIAS secondary antibody into fresh 5mL confining liquids, and film is immersed, when room temperature placement 1 is small；
8) film 10min is washed with film washing liquid (1 × TBST)；
9) nitrite ion is added, dark condition room temperature is to developing the color (20-30min), and observation colour developing is as a result, display purple is band MYSV viruses, do not develop the color or only blade true qualities are without poison.
The results are shown in Figure 6 for specific colour developing.In Fig. 6, A1 is positive quality control, and A2-G6 is field sample, and G7 is healthy cucumber Blade.It will be appreciated from fig. 6 that in 47 detected samples, there are 21 samples to infect MYSV, after tested by RT-PCR, sequencing The reliability of result has been demonstrate,proved, has absolutely proved that the present invention can be widely used in the detection of field sample, reliable results, flux are high.

Claims (5)

  1. A kind of 1. immunity detection reagent of muskmelon macula lutea virus, it is characterised in that including：Macula lutea containing muskmelon virus it is polyclonal Antibody and ELIAS secondary antibody；
    It using the muskmelon macula lutea viral capsid proteins of prokaryotic expression is immune that the polyclonal antibody of the virus of macula lutea containing muskmelon, which is, The antibody that original is prepared.
  2. 2. the immunity detection reagent of muskmelon macula lutea virus according to claim 1, it is characterised in that described yellow containing muskmelon The preparation method of the polyclonal antibody of pinta poison is：The capsid egg of muskmelon macula lutea virus is given expression to by the method for prokaryotic expression In vain；Again after Purified in electrophoresis, ear vein immunizing rabbit；Heart extracting blood after 3-5 times immune, collects upper serum, and purifies to obtain the final product.
  3. 3. the immunity detection reagent of muskmelon macula lutea virus according to claim 1 or 2, it is characterised in that described immune Detection kit further includes positive quality control, filter membrane, sample lapping liquid, film washing liquid, confining liquid and nitrite ion, and the filter membrane is nylon Film or nitrocellulose filter.
  4. 4. application of the immunity detection reagent in muskmelon macula lutea viral diagnosis as described in claim any one of 1-3.
  5. 5. the application method of the immunity detection reagent of muskmelon macula lutea virus as described in claim 1-3, it includes the following steps：
    1) sample to be tested is mixed with sample lapping liquid, obtained mixed liquor is dropped on filter membrane, room temperature air-dries；Meanwhile set Positive and negative control group；
    2) dried filter membrane is immersed in confining liquid, when room temperature placement 0.5~2 is small；
    3) polyclonal antibody of muskmelon macula lutea virus is included with confining liquid dilution, antibody dilution ratio is：1:2000~1: 20000, volume ratio；The filter membrane that step 2) is obtained is immersed in the polyclonal antibody after dilution, when room temperature placement 1~2 is small, then is used Film washing liquid washes film 5~10 minutes；
    4) ELIAS secondary antibody is diluted with confining liquid, ELIAS secondary antibody dilution ratio is 1:4000~1:20000, volume ratio；By step 3) Obtained filter membrane is immersed in the ELIAS secondary antibody after dilution, when room temperature placement 0.5~1 is small, then washes film 5~10 minutes with film washing liquid；
    5) nitrite ion is added, room temperature is placed to colour developing, observation colour developing result under dark condition：It is shown as purple, sample to be tested Carry MYSV viruses；Do not develop the color or only blade true qualities, sample to be tested do not carry MYSV viruses.
CN201610885141.5A 2016-10-11 2016-10-11 A kind of immunity detection reagent of muskmelon macula lutea virus and its application Pending CN107918012A (en)

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CN102495213A (en) * 2011-11-30 2012-06-13 中华人民共和国北京出入境检验检疫局 Kit used for detecting phytopathogenic fungi and detection method thereof
WO2014062775A2 (en) * 2012-10-16 2014-04-24 Monsanto Technology Llc Methods and compositions for controlling plant viral infection
CN104198713A (en) * 2014-09-05 2014-12-10 云南省农业科学院生物技术与种质资源研究所 Method for rapidly detecting tomato spotted wilf virus in thrips

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CHEN,TC等: "Serological relationship between Melon yellow spot virus and Watermelon silver mottle virus and differential detection of the two viruses in cucurbits", 《ARCHIVES OF VIROLOGY》 *
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Application publication date: 20180417