CN102520185A - Serologic detecting kit for potyvirus on sweet potato and detecting method thereof - Google Patents
Serologic detecting kit for potyvirus on sweet potato and detecting method thereof Download PDFInfo
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- CN102520185A CN102520185A CN2011103680094A CN201110368009A CN102520185A CN 102520185 A CN102520185 A CN 102520185A CN 2011103680094 A CN2011103680094 A CN 2011103680094A CN 201110368009 A CN201110368009 A CN 201110368009A CN 102520185 A CN102520185 A CN 102520185A
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Abstract
The invention relates to a serologic detecting kit for potyvirus on a sweet potato and a detecting method thereof. The kit is divided into an upper layer and a lower layer, wherein reagent bottles are arranged on the upper layer; a push-pull drawer is arranged on the lower layer; a nitrocellulose film is placed into the drawer; and a specific antibody for the potyvirus, a mixed antibody for the potyvirus and a goat anti-rabbit IgG, NBT, BCIP and Tris-HCl solution marked with alkaline phospholipase are respectively filled in different reagent bottles. When a sample is detected, the reagent bottle containing the mixed reagent is used for detecting if the sample contains the potyvirus; other specific antibodies are used for detecting when a certain specific virus needs to be confirmed; the detection is convenient; and the cost is saved; and the detecting method is simple, direct, convenient and practical. The serologic detecting kit and the detecting method provided by the invention can be applied to the detection for the detoxified sweet potato stem seedling and all-level potatoes, and the quality and the yield-increasing effect of the detoxified sweet potatoes are ensured by quickly and efficiently detecting the potyviruses.
Description
?
Technical field
The present invention relates to technical field of bioengineering, particularly relate on the sweet potato
HorseBell potato Y virus belongs to the serology detection kit and the detection method thereof of virus.
Background technology
Sweet potato pinniform mottle virus (
Sweet potato feathery mottle virus, SPFMV), the sweet potato cryptovirus (
Sweet potato latent virus, SPLV), sweet potato G virus (
Sweet potato virus G, SPVG) with the sweet potato vein mosaic virus (
Sweet potato vein mosaic virus, SPVMV
)Be the main virus of harm sweet potato, 4 kinds of virus all belong to Potyvirus (
Potyvirus), 4 kinds of virus or with other viruses mixed infection sweet potato usually, cause yield of sweet potato to reduce, article qualitative change bad with plant sexual involution, sweet potato production is caused serious harm.The sweet potato viruses disease is not still had special effective chemical at present prevent and treat method, utilize shoot apical meristem to cultivate, cultivate the detoxification sweet potato and be and prevent and treat the effective method of virosis.Cultivation and virus-free seed potato (seedling) the detoxification sweet potato realize in the industrialization process that the Fast Detection Technique of virus is to guarantee the virus-free seed potato quality and realize the technology that the virus-free seed potato industrialization is very crucial.Have only and utilize virus detection techniques fast and efficiently; Filter out the susceptible seedling of real nontoxic test-tube plantlet and in time superseded different stage; Just can make real virus-free seed potato be used for producing, guarantee the effect of increasing production of virus-free seed potato, and then accelerate the popularization process of virus-free seed potato.Therefore, set up sweet potato viruses efficiently detection technique be the prerequisite and the basis of cultivating the detoxification sweet potato.The common method that is used for the sweet potato viruses detection at present is enzyme linked immunological absorption (ELISA) technology, and nitrocellulose filter enzyme linked immunosorbent assay (ELISA) (NCM-ELISA) technology of setting up on this basis.Owing to infect sweet potato
PotyvirusesViral species is more, when not confirming viral species, only needs to confirm whether contain in the sample
Potyviruses(for example sweet potato detoxification stem sharp is detected) during virus, need detect one by one various viruses when utilizing said method to detect, often waste time and energy, cause waste.
Summary of the invention
The technical matters that the present invention will solve: overcome the problem in the background technology, provide a kind of easy, efficiently and cheaply detect on the sweet potato kits of four kinds of Potyvirus viruses;
The detection method of this kit also is provided in addition.
Technical scheme of the present invention:
Marmor upsilon belongs to the serology detection kit of virus on the sweet potato, and said kit is divided into upper and lower two-layer, upper and lowerly is provided with dividing plate between two-layer; The upper strata also is provided with the reagent bottle jack; Be placed with reagent bottle in the jack, but lower floor is the drawer of push-and-pull, is placed with nitrocellulose filter in the drawer; The specific antibody of Potyvirus virus, the mixed antibody of Potyvirus virus and goat anti-rabbit igg, chlorination nitro blue tetrazolium, 5-bromo-4-chloro-3-indyl-phosphate and the Tris-HCl solution of alkaline phosphatase lipase mark are housed respectively in the different reagent bottles.
Said reagent bottle is nine; Wherein a reagent bottle is equipped with sweet potato pinniform mottle virus SPFMV antibody; No. two reagent bottle is equipped with sweet potato cryptovirus SPLV antibody; No. three reagent bottle is equipped with sweet potato G virus SPVG antibody, and No. four reagent bottle is equipped with sweet potato vein mosaic virus SPVMV antibody, and No. five reagent bottles are equipped with the mixed antibody of above-mentioned four kinds of viruses; No. six reagent bottle is equipped with the goat anti-rabbit igg that working concentration is a 1:1000 alkaline phosphatase lipase mark doubly; No. seven reagent bottle is equipped with the chlorination nitro blue tetrazolium NBT that concentration is 50mg/ml; No. eight reagent bottles are equipped with 5-bromo-4-chloro-3-indyl-phosphate BCIP of concentration 50mg/ml, and it is 7.5 2M Tris-HCl solution that No. nine reagent bottles 9 are equipped with pH.
The specific antibody of said four kinds of Potyviruses virus all is the virus capsid proteins that utilize expression in escherichia coli; Method through purifying protein, immunizing rabbit prepares; The specific antibody and the tiring of mixed antibody of said four kinds of Potyviruses virus are 4096 times, and working concentration is 1:1000 times.
Marmor upsilon belongs to the detection method of the serology detection kit of virus on the sweet potato, may further comprise the steps:
(1) prepare cellulose nitrate NC film: the NC film of the suitable size of clip, with pencil marked on film, so that point sample;
(2) sample preparation: with the quick-frozen in liquid nitrogen of 0.1g sweet potato sample, and grind into powder, add 100 μ l extraction buffers, continue to be ground to thorough cracking and transfer in the centrifuge tube the centrifugal 5min of 5000rpm;
(3) point sample: place TBS to soak 1 min the NC film, inhale the liquid on striping surface with filter paper, get supernatant 5 μ l points on film, room temperature is dried;
(4) sealing: add the 10ml confining liquid in double dish, film is immersed wherein incubated at room 60 min;
(5) wash film: discard confining liquid, wash film once with TBS;
(6) add one anti-: the NC film is placed the antibody that is diluted to working concentration with the antibody damping fluid, under the room temperature with the velocity fluctuation of 50rpm/min, incubated overnight;
(7) wash film: abandon one and resist, wash the NC film 3-4 time with T-TBS solution, 3min/ time;
(8) adding two resists: inhale with filter paper and go excessive solution on the NC film, then the NC film is placed the enzyme labelled antibody that is diluted to working concentration with the antibody damping fluid, with the velocity fluctuation of 50rpm/min, hatch 1 h under the room temperature;
(9) wash film: abandon two and resist, wash the NC film 3-4 time with T-TBS solution, 3min/ time;
(10) colour developing: the NC film is placed the substrate solution of NBT and BCIP, and every 5ml substrate buffer solution adds 33 μ l NBT earlier, adds 16.5 μ l BCIP again, under the room temperature with the speed shaking table vibration of 50 rpm/min, the lucifuge 5-30 min that develops the color;
(11) cessation reaction: the NC film is taken out from substrate solution, wash the NC film 3 times with distillation, 3-4min/ time;
(12) positive judgement: the NC film is dried the back observe color reaction, compare the color reaction of test sample and positive and negative contrast, the sample that purple occurs is positive.
Said extraction buffer is the mixed liquor of TBS and 0.2% sodium sulphite.
The composition of said confining liquid is the potpourri of TBS, 2% skimmed milk power and 2% Triton X-100.
The antibody of said working concentration is SPFMV, SPLV, SPVG, SPVMV antibody or mixed antibody, and the antibody working concentration is 1:1000 doubly; Said enzyme labelled antibody is the goat anti-rabbit igg antibody of alkaline phosphate ester enzyme labeling, and the working concentration of enzyme labelled antibody is 1:1000 times.
Beneficial effect of the present invention:
(1) the invention provides on a kind of detection sweet potato
PotyvirusesEasy, the kit efficiently and cheaply of virus.During test sample, when only needing to confirm whether contain in the sample
PotyvirusesUse the reagent bottle that mixed antibody is housed in the kit to detect during virus; When needs confirm to contain certain specific virus, re-use the specific antibody in other reagent bottles, easy to detect, saved the detection cost.
(2) detection method of the present invention simply, intuitively only need be dried the back with the NC film and observe color reaction, compares the color reaction of test sample and positive and negative contrast, and whether the sample that the purple color reaction occurs is positive, contain thereby judge
PotyvirusesVirus, convenient and practical.
(3) this kit can be applicable to the detection of detoxification sweet potato stem tip seedling and potato seeds at different levels, through right
PotyvirusesThe detection rapidly and efficiently of virus can be guaranteed quality and the effect of increasing production of detoxification sweet potato; Also can carry out specific detection, can be applicable in the processes such as sweet potato potato seed allocation and transportation and disease survey the kind of sweet potato viruses.
(4) this kit is specially adapted to the detection of sweet potato detoxification stem sharp, also can directly be generalized to agrotechnical unit of basic unit and carry out disease generaI investigation and prediction, or be used for the quality monitoring of detoxification sweet potato potato seed, makes real detoxification sweet potato be applied to produce.
Description of drawings
Fig. 1: kit structural representation of the present invention;
Fig. 2: kit of the present invention is to sweet potato sample detection displayed map as a result.
Embodiment
The specific antibody and a kind of mixed antibody that contain four kinds of Potyvirus viruses in the kit; Described kit is divided into upper and lower two-layer; Upper and lowerly be provided with dividing plate between two-layer, the upper strata also is provided with the reagent bottle jack, is placed with reagent bottle in the jack; But lower floor is the drawer of push-and-pull, is placed with nitrocellulose filter in the drawer; The reagent bottle on upper strata is nine; Be respectively reagent bottle No. one to No. nine; The sweet potato pinniform mottle virus SPFMV antibody in reagent bottle wherein, No. two reagent bottles are sweet potato cryptovirus SPLV antibody, No. three reagent bottles are sweet potato G virus SPVG antibody; No. four reagent bottle is a sweet potato vein mosaic virus SPVMV antibody, and No. five reagent bottles are the mixed antibody of above-mentioned four kinds of viruses; No. six reagent bottles are that working concentration is that the goat anti-rabbit igg two of 1:1000 alkaline phosphatase lipase mark doubly is anti-; No. seven reagent bottles are that concentration is the chlorination nitro blue tetrazolium NBT of 50mg/ml; 5-bromo-4-chloro-3-indyl-phosphate BCIP that No. eight reagent bottles are concentration 50mg/ml, No. nine reagent bottles 9 are 7.5 2M Tris-HCl solution for pH.
The antibody of SPFMV, SPLV, SPVG, 4 kinds of viruses of SPVMV all is the virus capsid proteins that are utilized in expression in escherichia coli; Pass through purifying protein; The method of immunizing rabbit prepares, and tiring of the antibody of 4 kinds of viruses and mixed antibody is 4096 times, and working concentration is 1:1000 times.
During with the kit test sample, when only needing to confirm whether to contain Potyvirus virus in the sample, use No. five reagent bottles of mixed antibody to detect; When needs are confirmed to contain certain virus of Potyvirus virus, then use No. one to No. four reagent bottle to detect.
1, the preparation of damping fluid:
（1）TBS：0.02M?Tris+0.50M?NaCl，pH7.5；
(2) extraction buffer: TBS+0.2% sodium sulphite;
(3) confining liquid: TBS+2% skimmed milk power+2% triton X-100;
(4) antibody damping fluid: TBS+2% skimmed milk power;
（5）T-TBS：TBS+0.05%?Tween?20；
(6) substrate buffer solution: 0.1M Tris+ 0.1M NaCl+5mM MgCl
2 . 6H
2O, pH=9.5;
The mol/L of the M unit of being meant wherein.
2, detect step:
(1) prepare nitrocellulose filter (NC film): the NC film of the suitable size of clip, with pencil marked on film, so that point sample.
(2) sample preparation:, add 100 μ l extraction buffers (TBS+ 0.2% sodium sulphite) and continue to be ground to thorough cracking, and transfer in the 1.5ml centrifuge tube the centrifugal 5min of 5000rpm with the quick-frozen and grind and to be powder in liquid nitrogen of 0.1g sweet potato sample.
(3) point sample: the plain film of NC is soaked 1 min in TBS, inhale the striping surface liquid with filter paper, get supernatant 5 μ l points on film, room temperature is dried.
(4) sealing: add 10ml confining liquid (TBS+2% skimmed milk power+2%triton X-100) in double dish, the NC film is immersed wherein incubated at room 60 min.
(5) wash film: discard confining liquid, wash the NC film once with TBS.
(6) add one anti-(SPFMV, SPLV, SPVG, SPVMV antibody or mixed antibody):
The NC film is placed the antibody (SPFMV, SPLV, SPVG, SPVMV antibody or mixed antibody) that is diluted to working concentration with antibody damping fluid (TBS+2% skimmed milk power); Vibration (50rpm/min) incubated overnight under the room temperature, the working concentration of all antibody is 1:1000 doubly.
(7) wash film: abandon one and resist, wash the NC film 3-4 time with T-TBS solution, 3min/ time.
(8) add two anti-(goat anti-rabbit iggs of alkaline phosphate ester enzyme labeling):
Inhale excessive solution on the striping with filter paper, then the NC film is placed the enzyme labelled antibody (goat anti-rabbit igg of alkaline phosphate ester enzyme labeling) that is diluted to working concentration with antibody damping fluid (TBS+2% skimmed milk power), 1 h is hatched in vibration (50rpm/min) under the room temperature; Two anti-working concentrations are 1:1000 times.
(9) wash film: abandon two and resist, wash the NC film 3-4 time with T-TBS solution, 3min/ time.
(10) colour developing: the NC film is placed the NBT/BCIP substrate solution, and every 5ml substrate buffer solution adds 33 μ NBT earlier, adds shaking table vibration (50 rpm/min) lucifuge colour developing 5-30 min (increasing developing time in case of necessity) under the 16.5 μ l BCIP room temperatures again.
(11) cessation reaction: the NC film is taken out from substrate solution, wash the NC film 3 times with distillation, 3-4min/ time.
(12) positive judgement: dry the back and observe color reaction, compare the color reaction of test sample and positive and negative contrast, the sample that the purple color reaction occurs is positive.
Utilize this kit, 24 parts of field sweet potato samples that pick up from the field are detected.Detect the step that step is pressed embodiment 2.The one anti-mixed antibody antiserum (No. five reagent bottles) that adopts.Working concentration is 1:1000 times.The result shows, 10 parts of sweet potato samples (see figure 2) that is positive is arranged in 24 parts of sweet potato samples, and positive rate is 41.7%, and ubiquity on these sweet potato samples is described
PotyvirusesVirus.
Among Fig. 2, on arrange 1,2,3,4,5,6,7,8, and following row's 1,2 positive samples.
Claims (8)
1. marmor upsilon belongs to viral serology detection kit on the sweet potato; It is characterized in that said kit is divided into upper and lower two-layer, upper and lowerly be provided with dividing plate between two-layer; The upper strata also is provided with the reagent bottle jack; Be placed with reagent bottle in the jack, but lower floor is the drawer of push-and-pull, is placed with nitrocellulose filter in the drawer; The specific antibody of Potyvirus virus, the mixed antibody of Potyvirus virus and goat anti-rabbit igg, chlorination nitro blue tetrazolium, 5-bromo-4-chloro-3-indyl-phosphate and the Tris-HCl solution of alkaline phosphatase lipase mark are housed respectively in the different reagent bottles.
2. serology detection kit as claimed in claim 1; It is characterized in that said reagent bottle is nine, wherein a reagent bottle is equipped with sweet potato pinniform mottle virus SPFMV antibody; No. two reagent bottle is equipped with sweet potato cryptovirus SPLV antibody; No. three reagent bottle is equipped with sweet potato G virus SPVG antibody, and No. four reagent bottle is equipped with sweet potato vein mosaic virus SPVMV antibody, and No. five reagent bottles are equipped with the mixed antibody of above-mentioned four kinds of viruses; No. six reagent bottle is equipped with the goat anti-rabbit igg that working concentration is a 1:1000 alkaline phosphatase lipase mark doubly; No. seven reagent bottle is equipped with the chlorination nitro blue tetrazolium NBT that concentration is 50mg/ml; No. eight reagent bottles are equipped with 5-bromo-4-chloro-3-indyl-phosphate BCIP of concentration 50mg/ml, and it is 7.5 2M Tris-HCl solution that No. nine reagent bottles 9 are equipped with pH.
3. like each described serology detection kit of claim 1-3; It is characterized in that; The specific antibody of said four kinds of Potyviruses virus all is the virus capsid proteins that utilize expression in escherichia coli; Method through purifying protein, immunizing rabbit prepares, and the specific antibody and the tiring of mixed antibody of said four kinds of Potyviruses virus are 4096 times, and working concentration is 1:1000 times.
4. marmor upsilon belongs to the detection method of the serology detection kit of virus on the sweet potato, and it is characterized in that: said detection method may further comprise the steps:
(1) prepare cellulose nitrate NC film: the NC film of the suitable size of clip, with pencil marked on film, so that point sample;
(2) sample preparation: with the quick-frozen in liquid nitrogen of 0.1g sweet potato sample, and grind into powder, add 100 μ l extraction buffers, continue to be ground to thorough cracking and transfer in the centrifuge tube the centrifugal 5min of 5000rpm;
(3) point sample: place TBS to soak 1 min the NC film, inhale the liquid on striping surface with filter paper, get supernatant 5 μ l points on film, room temperature is dried;
(4) sealing: add the 10ml confining liquid in double dish, film is immersed wherein incubated at room 60 min;
(5) wash film: discard confining liquid, wash film once with TBS;
(6) add one anti-: the NC film is placed the antibody that is diluted to working concentration with the antibody damping fluid, under the room temperature with the velocity fluctuation of 50rpm/min, incubated overnight;
(7) wash film: abandon one and resist, wash the NC film 3-4 time with T-TBS solution, 3min/ time;
(8) adding two resists: inhale with filter paper and go excessive solution on the NC film, then the NC film is placed the enzyme labelled antibody that is diluted to working concentration with the antibody damping fluid, with the velocity fluctuation of 50rpm/min, hatch 1 h under the room temperature;
(9) wash film: abandon two and resist, wash the NC film 3-4 time with T-TBS solution, 3min/ time;
(10) colour developing: the NC film is placed the substrate solution of NBT and BCIP, and every 5ml substrate buffer solution adds 33 μ l NBT earlier, adds 16.5 μ l BCIP again, under the room temperature with the speed shaking table vibration of 50 rpm/min, the lucifuge 5-30 min that develops the color;
(11) cessation reaction: the NC film is taken out from substrate solution, wash the NC film 3 times with distillation, 3-4min/ time;
(12) positive judgement: the NC film is dried the back observe color reaction, compare the color reaction of test sample and positive and negative contrast, the sample that purple occurs is positive.
5. detection method as claimed in claim 4 is characterized in that: said extraction buffer is the mixed liquor of TBS and 0.2% sodium sulphite.
6. detection method as claimed in claim 4 is characterized in that: the composition of said confining liquid is the potpourri of TBS, 2% skimmed milk power and 2% Triton X-100.
7. like each described detection method of claim 4-6, it is characterized in that: the antibody of said working concentration is SPFMV, SPLV, SPVG, SPVMV antibody or mixed antibody, and the antibody working concentration is 1:1000 doubly.
8. like each described detection method of claim 4-6, it is characterized in that: said enzyme labelled antibody is the goat anti-rabbit igg antibody of alkaline phosphate ester enzyme labeling, and the working concentration of enzyme labelled antibody is 1:1000 times.
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| CN105548544A (en) * | 2016-02-04 | 2016-05-04 | 云南省农业科学院生物技术与种质资源研究所 | Detection kit of hippeastrum chlorotic ringspot viruses (HCRV) as well as detection method and application of detection kit |
| CN105548543A (en) * | 2016-02-04 | 2016-05-04 | 云南省农业科学院生物技术与种质资源研究所 | Kit for detecting yellow spotting virus of peanuts, as well as detection method and application thereof |
| CN109001466A (en) * | 2018-07-19 | 2018-12-14 | 新疆维吾尔自治区人民医院 | It is a kind of for the pulmonary surfactant protein kit of OSA and its application |
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| CN105548544A (en) * | 2016-02-04 | 2016-05-04 | 云南省农业科学院生物技术与种质资源研究所 | Detection kit of hippeastrum chlorotic ringspot viruses (HCRV) as well as detection method and application of detection kit |
| CN105548543A (en) * | 2016-02-04 | 2016-05-04 | 云南省农业科学院生物技术与种质资源研究所 | Kit for detecting yellow spotting virus of peanuts, as well as detection method and application thereof |
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Application publication date: 20120627 |