CN1824800A - Process for testing rose mosaic virus - Google Patents
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- CN1824800A CN1824800A CN 200510048772 CN200510048772A CN1824800A CN 1824800 A CN1824800 A CN 1824800A CN 200510048772 CN200510048772 CN 200510048772 CN 200510048772 A CN200510048772 A CN 200510048772A CN 1824800 A CN1824800 A CN 1824800A
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Abstract
This invention provides a method of immune catching reverse transcription PCR detecting plum necrosis ring spot viruses (PNRSV), it belongs to plant protection field. The procedures are that antigen is captured through antibody, RT-PCR and electrophoresis detecting. The PNRSV infected in the plant is amplified and separated and detected through gel electrophoresis. This invention has all the advantages in existing RT-PCR, and it mainly realized unite of serology and molecular biology technique. First, low concentration virus in plant is collected through special absorbing of virus antibody to antigen virus, then they are amplified through PCR, so detecting sensitivity and specificity can be great enhanced, and virus omission factor is reduced.
Description
Technical field
The invention provides a kind of method, platymiscium protection field by immunocapture reverse transcription PCR technology for detection rose mosaic virus.
Background technology
Rose mosaic virus (PNRSV) belongs to the member for the Bromoviridae Ilarvirus, can propagate by modes such as mechanical friction, insect amboceptor, bud grafting and graftings.After plant infects rose mosaic virus, whole strain band poison, harm throughout one's life.The rose mosaic virus host range is wide, can infect 21 sections in the dicotyledons, causes that many plants produce downright bad and the ring spot.Rose mosaic virus causes that yield and quality descends after infecting fruit tree.It is reported, the germination rate that infects the cherry of rose mosaic virus reduces greatly, for another example, the Chinese rose that have the laudatory title of " spending middle queen " always is subjected to liking of the people of various countries, have very high ornamental value and economic worth, but in case after infecting rose mosaic virus, plant is downgraded, enter and bloom hardly behind the generative growth phase or bloom too small and deformity etc., seriously reduced commodity and the sight of plant.Up to the present, still the special effect agent that does not have the treatment virus disease in the world, therefore the plant to the infective virus disease carries out cause of disease detection and monitoring, propagation for identification virus disease and its disease of control has important and practical meanings, is to produce to go up one of important measures of virus disease being carried out the comprehensive regulation.
Usually the method that detects virus has plant indicator method, electron microscopy, enzyme linked immunological adsorption technology and Protocols in Molecular Biology etc.Comparatively speaking, though the plant indicator method is simple, sense cycle is long, the shortlyest also take 10～20 days, and sensitivity is very low, some plant materials contains virus, also be subjected to the influence of aspects such as season, environment and viral character, therefore seldom be used for the detection of virus.The appearance of immuno-electron microscope has promoted the application of electron microscopy in viral context of detection greatly, but electron microscope costs an arm and a leg, and the sample making technology complexity is difficult for grasping, and is not suitable as the usual way that virus detects.The method of the detection virus that is most widely used at present is enzyme-linked immunosorbent assay (ELISA) and Protocols in Molecular Biology.Than plant indicator method and electron microscopy, ELISA method sense cycle is shorter, and is highly sensitive, can detect the disease of nanogram (ng) level level, but for the lower virus of plant in-vivo content, tend to take place the phenomenon of omission.Protocols in Molecular Biology, particularly the RT-PCR technology is not only simple to operate, and specificity, sensitivity can detect the virus that flies gram (fg) level level than ELISA method height.Rose mosaic virus infects xylophyta more, and plant body inner virus content is low, and plant tissue is rich in polysaccharide and phenols, has had a strong impact on sensitivity and the specificity of common RT-PCR, is easy to take place the situation of omission.
Summary of the invention
The present invention has overcome traditional method in the deficiency that detects rose mosaic virus on the plant, and the method for a kind of sensitivity, special detection rose mosaic virus is provided.
Step of the present invention is:
1. design primer:
Design detects the primer of rose mosaic virus:
| The primer title | Primer sequence | The amplified fragments size |
| PNRSV-F PNRSV-R | 5-GTGAAGACCACTTGGACCG-3 5-GGTCCTCATCGACCAGCA -3 | 483bp |
2. immunocapture:
1) get the susceptible tissue of 100mg, after adding the 1mL grinding buffer solution and fully grinding lapping liquid is changed in the 1.5mL sterilization centrifuge tube, the centrifugal 4min of 4000rpm, supernatant liquor are the thick juice of diseased tissues;
2) add 150 μ L in the 0.5mL PCR pipe and be cushioned the rose mosaic virus antiserum(antisera) of 200 times of liquid dilutions, hatch 3h or 4 ℃ for 37 ℃ and spend the night with bag;
3) abandon coating buffer in the PCR pipe,, add the thick juice of 150 μ L, hatch 3h or 4 ℃ under 37 ℃ and spend the night with PBST washing 3 times;
4) manage ddH 3 times with PBST washing PCR
2O washing 1 time, low-speed centrifugal 15sec abandons residual liquid;
3.RT-PCR：
1) in the PCR pipe that the bag quilt is crossed, adds downstream primer PNRSV-R 1 μ L, 9.5 μ L ddH
2O, behind the mixing that slightly vibrates in 70 ℃ of incubation 10min, rapid ice bath 5min afterwards;
2) reverse transcription: in above-mentioned PCR pipe, add reagent by following system, the mixing that fully vibrates, room temperature is placed 10min, and 42 ℃ of insulation 1hr obtain cDNA;
| Reagent | Volume |
| AMV Buffer dNTPs(10mmol/L) AMV(5u/μL) RNase Inhibitor(40u/μL) | 4μL 1.5μL 1μL 0.5μL |
3) PCR: get a new PCR pipe, the according to the form below system adds each reagent:
| Reagent | Volume |
| ddH 2O 10×PCR Buffer MgCl 2(25mmol/L) dNTPs (10mmol/L) PNRSV-F (20 μ mol/L) PNRSV-R (20 μ mol/L) cDNA Taq enzyme (5u/ μ L) | 33.5μL 5μL 3μL 1μL 1μL 1μL 5μL 0.5μL |
| Cumulative volume | 50μL |
The PCR program is set is: 94 ℃ of 4min, 94 ℃ of 1min, 50 ℃～56 ℃ 1min, 72 ℃ of 2min, 30～38 circulations of increasing, 4 ℃ of preservations behind 72 ℃ of extension 10min;
4. electrophoresis detection: the sepharose of preparation 0.8%～2%, in 0.5 * tbe buffer pendular ring border, 150V voltage stabilizing electrophoresis 90min takes out gel afterwards and is immersed in the 10min that dyes in the 0.5 μ g/mL ethidium bromide, observes under ultraviolet lamp then and the record result;
5. interpretation of result: according to amplified band on the running gel have or not and size judges in the plant detected whether infect rose mosaic virus, if promptly have the nucleic acid fragment of 483bp in the gel then contain rose mosaic virus in the interpret sample.
Wherein, described PCR circulation can be 30～38 circulations, also can be 31～34 circulations; Described PCR annealing temperature can be 50 ℃～56 ℃, also can be 51 ℃～54 ℃; Described agarose concentration can be 0.8%～2%, also can be 0.9%～1.2%.
The invention has the beneficial effects as follows immunology detection technology and molecular Biological Detection technology are organically combined, can be applicable to the detection of disease plant lower concentration virus.Save the loaded down with trivial details step of extracting total RNA among the common RT-PCR on the one hand, reduced the detection cost; Because antibody combines with antigenic specificity, improved the specificity that detects on the other hand.Simultaneously can directly from viral juice, catch virus particle, play the effect that concentrates with purified virus, utilize round pcr to amplify the sensitivity that detects again, can effectively prevent the situation of viral omission.In addition, utilize immunocapture PCR can also avoid that polysaccharide and phenols reduce the omission odds to the interference of RT-PCR in the dicotyledons.
Description of drawings
Fig. 1 is the electrophoresis detection figure that detects the rose mosaic virus on the xylophyta Chinese rose.
Embodiment
Embodiment one
With the known Chinese rose blade that has infected rose mosaic virus is material, with present method and common RT-PCR method rose mosaic virus is detected respectively, with the negative contrast of healthy Chinese rose blade, and positive control is set.
1. design primer:
Design detects the primer of rose mosaic virus:
| The primer title | Primer sequence | The amplified fragments size |
| PNRSV-F PNRSV-R | 5-GTGAAGACCACTTGGACCG-3 5-GGTCCTCATCGACCAGCA -3 | 483bp |
2. immunocapture:
1) get the susceptible tissue of 100mg, after adding the 1mL grinding buffer solution and fully grinding lapping liquid is changed in the 1.5mL sterilization centrifuge tube, the centrifugal 4min of 4000rpm, supernatant liquor are the thick juice of diseased tissues;
2) add 150 μ L in the 0.5mL PCR pipe and be cushioned the rose mosaic virus antiserum(antisera) of 200 times of liquid dilutions, hatch 3h or 4 ℃ for 37 ℃ and spend the night with bag;
3) abandon coating buffer in the PCR pipe,, add the thick juice of 150 μ L, hatch 3h or 4 ℃ under 37 ℃ and spend the night with PBST washing 3 times;
4) manage ddH 3 times with PBST washing PCR
2O washing 1 time, low-speed centrifugal 15sec abandons residual liquid;
3.RT-PCR：
1) in the PCR pipe that the bag quilt is crossed, adds downstream primer PNRSV-R 1 μ L, 9.5 μ L ddH
2O in 70 ℃ of incubation 10min, puts ice bath 5min rapidly behind the mixing that slightly vibrates afterwards;
2) reverse transcription: in above-mentioned PCR pipe, add reagent by following system, the mixing that fully vibrates, room temperature is placed 10min, and 42 ℃ of insulation 1hr obtain cDNA;
| Reagent | Volume |
| AMV Buffer dNTPs(10mmol/L) AMV(5u/μL) RNase Inhibitor(40u/μL) | 4μL 1.5μL 1μL 0.5μL |
3) PCR: get a new PCR pipe, the according to the form below system adds each reagent:
| Reagent | Volume |
| ddH 2O 10×PCR Buffer MgCl 2(25mmol/L) dNTPs (10mmol/L) PNRSV-F (20 μ mol/L) PNRSV-R (20 μ mol/L) cDNA Taq enzyme (5u/ μ L) | 33.5μL 5μL 3μL 1μL 1μL 1μL 5μL 0.5μL |
| Cumulative volume | 50μL |
The PCR program is set is: 94 ℃ of 4min, 94 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 2min, 30 circulations of increasing, 4 ℃ of preservations behind 72 ℃ of extension 10min;
4. electrophoresis detection: the sepharose of preparation 0.8%, in 0.5 * tbe buffer pendular ring border, 150V voltage stabilizing electrophoresis 90min takes out gel afterwards and is immersed in the 10min that dyes in the 0.5 μ g/mL ethidium bromide, observes under ultraviolet lamp then and the record result;
5. interpretation of result: detect the downright bad Orbivirus of Lee with present method, have the nucleic acid band of 483bp on gel, band is clear special, and is consistent with the stripe size of positive control, illustrates that present method can be used for the detection of rose mosaic virus; Do not detect any nucleic acid band in the negative control, illustrate not contain rose mosaic virus in the negative control.Method with common RT-PCR detects the nucleic acid band of finding can detect sometimes 483bp, but band brightness is faint, then can not detect the nucleic acid band sometimes, and result's stability and repeatability are relatively poor.
Embodiment two
With the known Chinese rose blade that has infected rose mosaic virus is material, with present method and common RT-PCR method rose mosaic virus is detected respectively, with the negative contrast of healthy Chinese rose blade, and positive control is set.
1. design primer:
Design detects the primer of rose mosaic virus:
| The primer title | Primer sequence | The amplified fragments size |
| PNRSV-F PNRSV-R | 5-GTGAAGACCACTTGGACCG-3 5-GGTCCTCATCGACCAGCA -3 | 483bp |
2. immunocapture:
1) get the susceptible tissue of 100mg, after adding the 1mL grinding buffer solution and fully grinding lapping liquid is changed in the 1.5mL sterilization centrifuge tube, the centrifugal 4min of 4000rpm, supernatant liquor are the thick juice of diseased tissues;
2) add 150 μ L in the 0.5mL PCR pipe and be cushioned the rose mosaic virus antiserum(antisera) of 200 times of liquid dilutions, hatch 3h or 4 ℃ for 37 ℃ and spend the night with bag;
3) abandon coating buffer in the PCR pipe,, add the thick juice of 150 μ L, hatch 3h or 4 ℃ under 37 ℃ and spend the night with PBST washing 3 times;
4) manage ddH 3 times with PBST washing PCR
2O washing 1 time, low-speed centrifugal 15sec abandons residual liquid;
3.RT-PCR：
1) in the PCR pipe that the bag quilt is crossed, adds downstream primer PNRSV-R 1 μ L, 9.5 μ L ddH
2O, behind the mixing that slightly vibrates in 70 ℃ of incubation 10min, rapid ice bath 5min afterwards;
2) reverse transcription: in above-mentioned PCR pipe, add reagent by following system, the mixing that fully vibrates, room temperature is placed 10min, and 42 ℃ of insulation 1hr obtain cDNA;
| Reagent | Volume |
| AMV Buffer dNTPs(10mmol/L) AMV(5u/μL) RNase Inhibitor(40u/μL) | 4μL 1.5μL 1μL 0.5μL |
3) PCR: get a new PCR pipe, the according to the form below system adds each reagent:
| Reagent | Volume |
| ddH 2O 10×PCR Buffer MgCl 2(25mmol/L) dNTPs (10mmol/L) PNRSV-F (20 μ mol/L) PNRSV-R (20 μ mol/L) cDNA Taq enzyme (5u/ μ L) | 33.5μL 5μL 3μL 1μL 1μL 1μL 5μL 0.5μL |
| Cumulative volume | 50μL |
The PCR program is set is: 94 ℃ of 4min, 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 2min, 38 circulations of increasing, 4 ℃ of preservations behind 72 ℃ of extension 10min;
4. electrophoresis detection: the sepharose of preparation 2%, in 0.5 * tbe buffer pendular ring border, 150V voltage stabilizing electrophoresis 90min takes out gel afterwards and is immersed in the 10min that dyes in the 0.5 μ g/mL ethidium bromide, observes under ultraviolet lamp then and the record result;
5. interpretation of result: detect the downright bad Orbivirus of Lee with present method, have the nucleic acid band of 483bp on gel, band is clear special, and is consistent with the stripe size of positive control, illustrates that present method can be used for the detection of PNRSV; Do not detect any nucleic acid band in the negative control, illustrate not contain PNRSV in the negative control.Method with common RT-PCR detects the nucleic acid band of finding can detect sometimes 483bp, but band brightness is faint, then can not detect the nucleic acid band sometimes, and result's stability and repeatability are relatively poor.
Embodiment three
With the known Chinese rose blade that has infected the downright bad Orbivirus of Lee is material, with present method and common RT-PCR method rose mosaic virus is detected respectively, with the negative contrast of healthy Chinese rose blade, and positive control is set.
1. design primer:
Design detects the primer of rose mosaic virus:
| The primer title | Primer sequence | The amplified fragments size |
| PNRSV-F PNRSV-R | 5-GTGAAGACCACTTGGACCG-3 5-GGTCCTCATCGACCAGCA -3 | 483bp |
2. immunocapture:
1) get the susceptible tissue of 100mg, after adding the 1mL grinding buffer solution and fully grinding lapping liquid is changed in the 1.5mL sterilization centrifuge tube, the centrifugal 4min of 4000rpm, supernatant liquor are the thick juice of diseased tissues;
2) add 150 μ L in the 0.5mL PCR pipe and be cushioned the rose mosaic virus antiserum(antisera) of 200 times of liquid dilutions, hatch 3h or 4 ℃ for 37 ℃ and spend the night with bag;
3) abandon coating buffer in the PCR pipe,, add the thick juice of 150 μ L, hatch 3h or 4 ℃ under 37 ℃ and spend the night with PBST washing 3 times;
4) manage ddH 3 times with PBST washing PCR
2O washing 1 time, low-speed centrifugal 15sec abandons residual liquid;
3.RT-PCR：
1) in the PCR pipe that the bag quilt is crossed, adds downstream primer PNRSV-R 1 μ L, 9.5 μ L ddH
2O, behind the mixing that slightly vibrates in 70 ℃ of incubation 10min, rapid ice bath 5min afterwards;
2) reverse transcription: in above-mentioned PCR pipe, add reagent by following system, the mixing that fully vibrates, room temperature is placed 10min, and 42 ℃ of insulation 1hr obtain cDNA;
| Reagent | Volume |
| AMV Buffer dNTPs(10mmol/L) AMV(5u/μL) RNase Inhibitor(40u/μL) | 4μL 1.5μL 1μL 0.5μL |
3) PCR: get a new PCR pipe, the according to the form below system adds each reagent:
| Reagent | Volume |
| ddH 2O 10×PCR Buffer MgCl 2(25mmol/L) dNTPs (10mmol/L) PNRSV-F (20 μ mol/L) PNRSV-R (20 μ mol/L) cDNA Taq enzyme (5u/ μ L) | 33.5μL 5μL 3μL 1μL 1μL 1μL 5μL 0.5μL |
| Cumulative volume | 50μL |
The PCR program is set is: 94 ℃ of 4min, 94 ℃ of 1min, 53 ℃ of 1min, 72 ℃ of 2min, 32 circulations of increasing, 4 ℃ of preservations behind 72 ℃ of extension 10min;
4. electrophoresis detection: the sepharose of preparation 1%, in 0.5 * tbe buffer pendular ring border, 150V voltage stabilizing electrophoresis 90min takes out gel afterwards and is immersed in the 10min that dyes in the 0.5 μ g/mL ethidium bromide, observes under ultraviolet lamp then and the record result;
5. interpretation of result: detect the downright bad Orbivirus of Lee with present method, have the nucleic acid band of 483bp on gel, band is clear special, and is consistent with the stripe size of positive control, illustrates that present method can be used for the detection of PNRSV; Do not detect any nucleic acid band in the negative control, illustrate not contain PNRSV in the negative control.Method with common RT-PCR detects the nucleic acid band of finding can detect sometimes 483bp, but band brightness is faint, then can not detect the nucleic acid band sometimes, and result's stability and repeatability are relatively poor.
Utilize this method under the differential responses condition to plant in rose mosaic virus detect.Find out from experimental result, by present method all can be from the plant that contains rose mosaic virus the special nucleic acid band that amplifies 483bp, and negative control does not have non-specific result and takes place.And compare common RT-PCR, it is highly sensitive that present method detects, and the situation of omission seldom appears in stability and good reproducibility as a result.Therefore the detection of this method rose mosaic virus that can be applied to taking place on the plant.
Claims (4)
1, a kind of method that detects rose mosaic virus, its step is as follows:
1) design primer:
Design detects the primer of rose mosaic virus:
The primer title Primer sequence The amplified fragments size
PNRSV-F PNRSV-R 5-GTGAAGACCACTTGGACCG-3 5-GGTCCTCATCGACCAGCA-3 483bp
2) immunocapture:
(a) get the susceptible tissue of 100mg, after adding the 1mL grinding buffer solution and fully grinding lapping liquid is changed in the 1.5mL centrifuge tube, the centrifugal 4min of 4000rpm, supernatant liquor are the thick juice of diseased tissues;
(b) add 150 μ L in the 0.5mL PCR pipe and be cushioned the rose mosaic virus antiserum(antisera) of 200 times of liquid dilutions, hatch 3h or 4 ℃ for 37 ℃ and spend the night with bag;
(c) discard coating buffer,, add the thick juice of 150 μ L, hatch 3h or 4 ℃ under 37 ℃ and spend the night with PBST washing 3 times;
(d) manage ddH 3 times with PBST washing PCR
2O washing 1 time, low-speed centrifugal 15sec abandons residual liquid; 3) RT-PCR:
(a) in the PCR pipe that the bag quilt is crossed, add downstream primer PNRSV-R1 μ L, 9.5 μ L ddH
2O, behind the mixing that slightly vibrates in 70 ℃ of incubation 10min, rapid ice bath 5min afterwards;
(b) reverse transcription: in above-mentioned PCR pipe, add reagent by following system, the mixing that fully vibrates, room temperature is placed 10min, and 42 ℃ of insulation 1hr obtain cDNA;
Reagent Volume
AMV Buffer dNTPs(10mmol/L) AMV(5u/μL) RNase Inhibitor(40u/μL) 4μL 1.5μL 1μL 0.5μL
(c) PCR: get a new PCR pipe, the according to the form below system adds each reagent:
Reagent Volume
ddH
2O 10×PCR Buffer MgCl
2(25mmol/L) dNTPs, (10mmol/L) PNRSV-F, (20 μ mol/L) PNRSV-R, (20 μ mol/L) cDNA Taq enzyme, (5u/ μ L)
33.5μL 5μL 3μL 1μL 1μL 1μL 5μL 0.5μL
Cumulative volume 50μL
The PCR program is set is: 94 ℃ of 4min, 94 ℃ of 1min, 50 ℃～56 ℃ 1min, 72 ℃ of 2min, 30～38 circulations of increasing, 4 ℃ of preservations behind 72 ℃ of extension 10min;
4) electrophoresis detection: the sepharose of preparation 0.8%～2%, in 0.5 * tbe buffer pendular ring border, 150V voltage stabilizing electrophoresis 90min takes out gel afterwards and is immersed in the 10min that dyes in the 0.5 μ g/mL ethidium bromide, observes under ultraviolet lamp then and the record result;
5) interpretation of result: according to amplified band on the running gel have or not and size judges in the plant detected whether infect rose mosaic virus, if promptly have the nucleic acid fragment of 483bp in the gel then contain rose mosaic virus in the interpret sample.
2, the method for detection rose mosaic virus according to claim 1 is characterized in that annealing temperature is 51 ℃～54 ℃.
3, the method for detection rose mosaic virus according to claim 1,31～34 circulations is characterized in that increasing.
4, the method for detection rose mosaic virus according to claim 1 is characterized in that the concentration of the sepharose prepared is 0.9%～1.2%.
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| CN 200510048772 CN1824800A (en) | 2005-12-27 | 2005-12-27 | Process for testing rose mosaic virus |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925589A (en) * | 2012-11-13 | 2013-02-13 | 新疆农业大学 | Establishment of reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection method for Prunus necrotic ringspot virus (PNRSV) |
| CN105527424A (en) * | 2015-12-19 | 2016-04-27 | 中国科学院寒区旱区环境与工程研究所 | Lily prunus necrotic ringspot virus semi-quantitative detection colloidal gold card and preparation method thereof |
| CN109423527A (en) * | 2017-08-23 | 2019-03-05 | 河南省农业科学院植物保护研究所 | Using the method for reverse transcription loop-mediated isothermal amplification technique detection Prunus necrotic ring spot virus (PNRSV) |
-
2005
- 2005-12-27 CN CN 200510048772 patent/CN1824800A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925589A (en) * | 2012-11-13 | 2013-02-13 | 新疆农业大学 | Establishment of reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection method for Prunus necrotic ringspot virus (PNRSV) |
| CN105527424A (en) * | 2015-12-19 | 2016-04-27 | 中国科学院寒区旱区环境与工程研究所 | Lily prunus necrotic ringspot virus semi-quantitative detection colloidal gold card and preparation method thereof |
| CN109423527A (en) * | 2017-08-23 | 2019-03-05 | 河南省农业科学院植物保护研究所 | Using the method for reverse transcription loop-mediated isothermal amplification technique detection Prunus necrotic ring spot virus (PNRSV) |
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