CN104450973A - NASBA (nucleic acid sequence based amplification) primer, kit and detection method for detecting hop stunt viroids - Google Patents
NASBA (nucleic acid sequence based amplification) primer, kit and detection method for detecting hop stunt viroids Download PDFInfo
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Abstract
The invention discloses an NASBA (nucleic acid sequence based amplification) primer, a kit and a detection method for detecting hop stunt viroids. The sequences of the upstream primer and the downstream primer of the NASBA primer are SEQ ID NO: 1-2 respectively, and the kit comprises NASBA reaction liquid A and NASBA reaction liquid B. The detection method includes the steps: 1) extracting RNA (ribonucleic acid) of samples; 2) performing NASBA for the hop stunt viroids; 3) detecting electrophoreses. The NASBA primer is applicable to rapidly detecting and confirming the hop stunt viroids, can be widely applied to disease surveillance and control in agricultural production and environments and monitoring and detecting of the viroids in import and export trade and is simple and convenient to operate, the number of the needed samples is small, and the quality requirement for template RNA is low.
Description
technical field:
The present invention relates to a kind of NASBA amplimer for detecting hop stunt viroid, test kit and detection method, belonging to the detection technique field of pathogenic.
background technology:
Viroid is the hitherto known minimum virulence factor causing Plant diseases, is circular RNA molecule, and its full-length gene group is made up of 246 ~ 399 Nucleotide, and many cash crop can be caused to produce serious plant disease.Be divided into 2 groups according to the homology of its sequence and structure, duplication characteristic, be respectively potato spindle tuber viroid group (PSTVd) and avsunviroid viroid group (ASBVd).Hop stunt viroid (Hop stunt vroid) belongs to potato spindle tuber viroid section hop stunt viroid and belongs to, usually containing about 307 Nucleotide.1970, the people such as the Yamamoto of Japan found hop stunt viroid first on the hops downgraded.1985, the Sano of Japan found HSVd first on grape, through the research of 15 years, thinks that the primary source of infection of HSVd is grape, has adapted to new host during evolution gradually.The HSVd host reported both at home and abroad at present comprises draft and the xylophytas such as cucumber, grape, peach, Lee, almond, apricot.China reports HSVd on the plants such as hops, peach, Lee, apricot, grape, almond.Therefore; the Testing and appraisal method general operating specification(GOS) of specification hop stunt viroid; for improve hop stunt viroid quarantine and examination efficiency, prevent the importing into of hop stunt viroid, spread out of; the safety in production of protection China agricultural; promote the smooth outlet of agricultural products in China, tool is of great significance.Studing Plant Viroids infects commonplace due to not dominant, and Symptoms is comparatively large by the impact of envrionment temperature, and the reaction symptom of several differential plant to inhomogeneity virus is similar, therefore is difficult to applying biological method for measuring.Because viroid can not produce any protein, so the Electronic Speculum, the serological method that detect virus can not be used.Therefore, molecular biology method is selected to detect hop stunt viroid.
NASBA(Nuleic and sequence based amplipicain, NASBA) namely " Nucleic acid sequence based amplification " detection technique is a kind of isothermal amplification technology of classics, be applicable to the amplification of singlestranded RNA RNA mono-step and detect, being widely used in the Detection and diagnosis of the mankind and animals and plants cause of disease.NASBA is guided by pair of primers, in the standard reaction system that the various reaction buffers used containing T7 RNA polymerase, RNAseH, ThermoScript II AMV, NTP, dNTP and needs form, realized the isothermal duplication of nucleotide sequence by In-vitro specificity enzymatic reaction homogeneous continuously.
Owing to being rich in a large amount of polysaccharide, aldehydes matter in cell walls, the extraction of plant virus RNA also exists the problems such as poor stability, repeatability is low, efficiency is low, in addition in leaching process RNA itself from signs of degradation, the content of viral RNA is often lower, proposes high requirement to the sensitivity of detection method and stability.Simultaneously because the materials such as polysaccharide are for the restraining effect of Taq polysaccharase, limit the application of RT-PCR technology in the plant virus RNA that the polyphenol contents such as grape, strawberry, Cereals class are higher detects.Because the transcriptive process,reversed of NASBA technology is directly merged in amplified reaction, therefore NASBA has possessed the feature of the amplification being suitable for cause of disease RNA, detection, the analysis of the most applicable various RNA sample, meet the requirement of plant virus quarantine, in view of the seriousness of hop stunt viroid harm, in environmental enhancement monitoring, improve the accuracy of detection efficiency and confirmation, to the propagation effectively controlling hop stunt viroid, in present stage, there is important practical significance.
summary of the invention:
The technical problem to be solved in the present invention is to provide a kind of NASBA amplimer for detecting hop stunt viroid, provides a kind of test kit and detection method of detection simultaneously.Its cardinal principle utilizes pair of primers to guide, in the standard reaction system that the various reaction buffers used containing T7 RNA polymerase, RNAseH, ThermoScript II AMV, NTP, dNTP and needs form, the isothermal duplication NASBA of nucleotide sequence is realized by In-vitro specificity enzymatic reaction homogeneous continuously, through circulation repeatedly, RNA is constantly increased, the hop stunt viroid in sample is detected accurately.
Realization of the present invention, first according to hop stunt viroid whole genome sequence design Auele Specific Primer, extract the Yeast Nucleic Acid (RNA) of testing sample again, then NASBA amplification is carried out with Auele Specific Primer respectively, with agarose gel electrophoresis, amplified production is detected, finally whether judge in sample containing hop stunt viroid according to NASBA amplification.
The technical scheme that the present invention solves the problems of the technologies described above employing is as follows:
The present invention is for detecting the NASBA primer of hop stunt viroid, and its primer sequence is respectively SEQ ID NO:1 ~ 2.
SEQ ID NO:1：NA-P1:5’-aattctaatacgactcactatagggagGAATCCAGCGAGAGGCGTG-3’。
SEQ ID NO:2：NA-P2:5’-aattctaatacgactcactatagggagAGTACCTCCCTGCCTTGTTTT-3’。
Detect the NASBA amplification kit of hop stunt viroid, comprise following component:
(1) preparation of NASBA amplification reaction solution A:
Every 25 μ L comprise 10 × AMV buffer 2.5 μ l, 6.25mmol/L NTPs 3 μ L, 10 mmol/L dNTPs 1.5 μ L, 10 μm of each 0.5 μ L of ol/L primer NA-P1, NA-P2. distilled water 9 μ L.
Wherein damping fluid is for containing 40 mmol/L PH8.5 Tris-HCL, 70 mmol/LKCL, 12 mmol/L MgCL2,5 mmol/L DTT damping fluids.
(2) preparation of NASBA amplification reaction solution B:
0.5 U RNaseH, 32 U T7RNA polysaccharases, 6.4 U AMV ThermoScript II, 2 μ L DMSO, 0.1 μ L1 mol/L dithiothreitol (DTT), 0.25 μ L 10 mg/mL BSA, 20 U RNA enzyme inhibitorss,
(3) NASBA amplification program:
Measuring samples RNA 3 μ L is added 14 μ L reaction solution A, 65 DEG C of temperature bath 5 min on DNA cloning instrument or water-bath, proceed to 41 DEG C of temperature bath 5 min immediately, add rapidly 4 μ L reaction solution B, 41 DEG C of incubation 2 h, 4 DEG C of termination reactions;
(4) NASBA amplified production qualification
Reaction product with 5% agarose gel electrophoresis, observations under ultraviolet lamp also judges.
Invention further provides a kind of method adopting SEQ NO:1, SEQ NQ:2 specificity amplification primer to detect the NASBA of hop stunt viroid, comprise the following steps:
1) extraction of sample RNA
A, get 0.1 g sample, add liquid nitrogen and be ground into powder, abrasive material is moved into rapidly in 1.5 mL centrifuge tubes, add 1 mL Trizol Reagent, put upside down mixing, 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.
B, get supernatant, 15 DEG C ~ 30 DEG C, place 5min; Add 0.2 mL chloroform, use hand concuss, not vortex oscillation, 15s.15 DEG C ~ 30 DEG C, place 2min ~ 3min; 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 15min.
C, carefully absorption are the upper strata aqueous phase of 600 μ L, not disturbance mesophase spherule and lower floor's phase.Add 500 μ L Virahol mixing supernatants, 15 DEG C ~ 30 DEG C, place 10min.2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.
D, removal supernatant liquor, add 1 mL 75% ethanol, washing in precipitation; 2 DEG C ~ 8 DEG C, 7500 g, centrifugal 5min.
E, removal supernatant liquor, after precipitation seasoning, be dissolved in 30 μ L ~ 50 μ L without in the water of RNase, be template ribonucleic acid to be checked.
2) the NASBA amplification of hop stunt viroid is carried out
A, in the reaction tubes that 14 μ L loop-mediated isothermal amplification liquid A are housed, add 3 μ L template ribonucleic acid to be checked,
B, on DNA cloning instrument or water-bath 65 DEG C temperature bath 5 min, proceed to immediately 41 DEG C temperature bath 5 min;
C. in reaction tubes, add rapidly 4 μ L reaction solution B;
D. in 41 DEG C of incubation 2 h;
E. metal bath is transferred to 4 DEG C of stopped reactions, takes out after 3 min;
3) electrophoresis detection
Get 3 g agaroses, heat, fully dissolve in 100 mL electrophoretic buffers, adding ethidium bromide stock solution to final concentration is 0.5 mg/mL, and glue, adds electrophoretic buffer in electrophoresis chamber, makes liquid level just not have gel; 3 mL ~ 6 mL pcr amplification products are mixed with appropriate sample loading buffer respectively, point sample; 9 V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates in the middle part of gel; Electrophoresis result is observed and record under Ultraviolet Detector.If amplified fragments is 262 bp, illustrate that virus to be checked is hop stunt viroid, if there is no appearance 262 bp amplified fragments, then illustrate that virus to be checked is not hop stunt viroid.
The present invention devises two specificity inner primers according to the sequence high conservative region of hop stunt viroid, this conserved genetic sequences is that to have the different strains of hop stunt viroid common, to ensure the reliability of the hop stunt viroid detecting different sources from the level of strain.The present invention is applicable to carry out rapid detection confirmation to hop stunt viroid, can be widely used in the confirmation of this virus in the disease monitoring in production and environment, foreign trade.
Compared with prior art, beneficial effect of the present invention comprises:
The first, convenience.This invention is when carrying out constant-temperature amplification, and do not need expensive nucleic acid amplification reaction device, whole process is carried out at 42 DEG C all the time, and without the need to thermal cycler, only 1 common thermostat water bath just can complete.
The second, tolerance range is high.The cycle number of this invention enzyme circulating reaction is few, does not need high-temperature denatured step, lower relative to RT-PCR mispairing rate, is more suitable for detecting and quantitative special RNA.
3rd, highly sensitive.This invention, compared with round pcr, can just amplify a large amount of goal gene with less circulation, ensure that the hypersensitivity of detection.
4th, shorten the cycle.Because transcriptive process,reversed is directly merged in amplified reaction, PCR approximately needs 20 to take turns circulation could increase 10
6doubly, and NASBA only need circulate and 4 ~ 5 takes turns and can reach 10
6doubly.
5th, reduce the specification of quality to plant RNA template.Owing to being rich in a large amount of polysaccharide, aldehydes matter in cell walls, the extraction of plant virus RNA also exists the problems such as poor stability, repeatability is low, efficiency is low, in addition in leaching process RNA itself from signs of degradation, the content of viral RNA is often lower, proposes high requirement to the sensitivity of detection method and stability.Due to this invention for template be RNA, the product of reaction is also RNA, and the result of reaction is by the impact of DNA in environment.Even if there is external double-stranded DNA to pollute, but because it does not possess T7 promoter sequence, can not be amplified, secondly, this reaction is only carried out under 42 DEG C of constant temperatures, do not need high-temperature denatured step, so NASBA reaction process can not be subject to the pollution of external double-stranded DNA, therefore for template purity and specification of quality lower.
accompanying drawing illustrates:
Fig. 1 be the present invention to NASBA AFLP system in diseased plant sample: wherein M:ssRNA Ladder marker; 1: healthy grape; 2: the susceptible material of hop stunt viroid.
Fig. 2 is specific outcome figure of the present invention: wherein M:ssRNA Ladder marker; 1: hop stunt viroid; 2: apple rust fruit virus; 3: Pear blister canker viroid; 4: peach hides mosaic virus; 5: apple stem pitting virus; 6: grape macula lutea viroid-2.
Fig. 3 is susceptibility results figure of the present invention: wherein M:ssRNA Ladder marker; 1 ~ 7:1 × 10
-1, 1 × 10
-2, 1 × 10
-3, 1 × 10
-4, 1 × 10
-5, 1 × 10
-6, 1 × 10
-7ng/ μ L, the susceptible material total serum IgE of hop stunt viroid.
Fig. 4 is sample detection result figure of the present invention.Wherein M:ssRNA Ladder marker; 1 ~ 24: wherein 1 ~ 6: enter the territory French Grape seedling (S1 ~ S6); 7 ~ 12: Nan Shuili (S7 ~ S12); 13 ~ 18: Italia grape seedling (S13 ~ S18); 19 ~ 24: water-rich areas (S19 ~ S24).
embodiment:
In order to explain implementation method of the present invention more fully, provide the embodiment for detecting hop stunt viroid NASBA test kit.These embodiments are only explain, instead of limit the scope of the invention.Wherein reverse transcription polysaccharase AMV, RNaseH, RNase inhibitor, T7 RNA polysaccharase, dNTP, ssRNA marker, rNTP are all purchased from NEW ENGLAND BIOLAB company; Pcr amplification reagent, DNA marker and etc. be all purchased from Beijing Tian Gen company.
embodiment 1:
1 material
1.1 viral
Hop stunt viroid is the French Grape rootstock seedling isolate that enters the territory, Italy enters the territory grape seedlings isolate, Xinjiang grape seedling and hops isolate, apple rust fruit virus (
apple scar skid viroid, ASSVd), Pear blister canker viroid (
pear Blister Canker Viroid, PBCVd), peach hide embedding line viroid (
peach latent mosaic viroid, PLMVd), apple stem pitting virus (
apple stem pitting virus, ASPV), grape macula lutea viroid-2(
grapevine yellow speckle viroid, GYSVd-2) preserved by the laboratory of applicant.
1.2 reagent
Reverse transcription polysaccharase AMV, RNaseH, RNase inhibitor, T7 RNA polysaccharase, dNTP, ssRNA marker, rNTP are all purchased from NEW ENGLAND BIOLAB company; Pcr amplification reagent, DNA marker and etc. be all purchased from Beijing Tian Gen company;
1.3 primer
According to the full length gene sequence (accession number HM357802.1) of hop stunt viroid in NCBI GenBank, by comparative analysis hop stunt viroid high conservative region under the prerequisite of the degenerate and versatility that ensure amplification, design 5' end band has T7 promoter sequence NASBA to react primer (NA-P1, NA-P2), checking of being compared under the Primer-Blast module of database by primer after having designed.
2 methods
The extraction of 2.1 viral RNAs
Get 0.1 g sample, add liquid nitrogen and be ground into powder, abrasive material is moved into rapidly in 1.5 mL centrifuge tubes, add 1 mL Trizol Reagent, put upside down mixing, 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Get supernatant, 15 DEG C ~ 30 DEG C, place 5min; Add 0.2 mL chloroform, with hand concuss (not vortex oscillation), about 15s.15 DEG C ~ 30 DEG C, place 2min ~ 3min; 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 15min.Careful absorption is about the upper strata aqueous phase of 600 μ L, not disturbance mesophase spherule and lower floor's phase.Add 500 μ L Virahol mixing supernatants, 15 DEG C ~ 30 DEG C, place 10min.2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Remove supernatant liquor, in precipitation, add 1 mL 75% ethanol, washing; 2 DEG C ~ 8 DEG C, 7500 g, centrifugal 5min.Remove supernatant liquor, after precipitation seasoning, be dissolved in 30 μ L ~ 50 μ L without in the water of RNase, be template ribonucleic acid to be checked.
2.2 NASBA amplification systems
A, in the reaction tubes that 14 μ L loop-mediated isothermal amplification liquid A are housed, add 3 μ L template ribonucleic acid to be checked,
B, on DNA cloning instrument or water-bath 65 DEG C temperature bath 5 min, proceed to immediately 41 DEG C temperature bath 5 min;
C. in reaction tubes, add rapidly 4 μ L reaction solution B;
D. in 41 DEG C of incubation 2 h;
E. metal bath is transferred to 4 DEG C of stopped reactions, takes out after 3 min;
2.3 electrophoresis detection
Get 3 g agaroses, heat, fully dissolve in 100 mL electrophoretic buffers, adding ethidium bromide stock solution to final concentration is 0.5 mg/mL, and glue, adds electrophoretic buffer in electrophoresis chamber, makes liquid level just not have gel; 3 mL ~ 6 mL pcr amplification products are mixed with appropriate sample loading buffer respectively, point sample; 9 V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates in the middle part of gel; Electrophoresis result is observed and record under Ultraviolet Detector.Expection product size is 262 bp.See Fig. 1.
embodiment 2: specificity experiments
1, extract hop stunt viroid, RNA that apple rust fruit virus, Pear blister canker viroid, peach hide mosaic virus, apple stem pitting virus, grape macula lutea viroid-2, use NASBA method detects.
2, the extraction of viral RNA
Get 0.1 g sample, add liquid nitrogen and be ground into powder, abrasive material is moved into rapidly in 1.5 mL centrifuge tubes, add 1 mL Trizol Reagent, put upside down mixing, 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Get supernatant, 15 DEG C ~ 30 DEG C, place 5min; Add 0.2 mL chloroform, with hand concuss (not vortex oscillation), about 15s.15 DEG C ~ 30 DEG C, place 2min ~ 3min; 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 15min.Careful absorption is about the upper strata aqueous phase of 600 μ L, not disturbance mesophase spherule and lower floor's phase.Add 500 μ L Virahol mixing supernatants, 15 DEG C ~ 30 DEG C, place 10min.2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Remove supernatant liquor, in precipitation, add 1 mL 75% ethanol, washing; 2 DEG C ~ 8 DEG C, 7500 g, centrifugal 5min.Remove supernatant liquor, after precipitation seasoning, be dissolved in 30 μ L ~ 50 μ L without in the water of RNase, be template ribonucleic acid to be checked.
3, amplified reaction
A, in the reaction tubes that 14 μ L loop-mediated isothermal amplification liquid A are housed, add 3 μ L template ribonucleic acid to be checked,
B, on DNA cloning instrument or water-bath 65 DEG C temperature bath 5 min, proceed to immediately 41 DEG C temperature bath 5 min;
C. in reaction tubes, add rapidly 4 μ L reaction solution B;
D. in 41 DEG C of incubation 2 h;
E. metal bath is transferred to 4 DEG C of stopped reactions, takes out after 3 min;
4, NASBA product electrophoresis detection
Get 3 g agaroses, heat, fully dissolve in 100 mL electrophoretic buffers, adding ethidium bromide stock solution to final concentration is 0.5 mg/mL, and glue, adds electrophoretic buffer in electrophoresis chamber, makes liquid level just not have gel; 3 mL ~ 6 mL pcr amplification products are mixed with appropriate sample loading buffer respectively, point sample; 9 V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates in the middle part of gel; Electrophoresis result is observed and record under Ultraviolet Detector.Electrophoresis result shows, use the RNA that NASBA method carries out detecting hop stunt viroid, apple rust fruit virus, Pear blister canker viroid, peach hide mosaic virus, apple stem pitting virus, grape macula lutea viroid-2, only have hop stunt viroid to obtain the amplified production (see figure 2) of expection 262 bp.
embodiment 3 sensitivity experiments
1, with DEPC water, hop stunt viroid viral RNA template liquid is done 10 times of gradient dilutions downwards, be followed successively by 1 × 10
0, 1 × 10
-1, 1 × 10
-2, 1 × 10
-3, 1 × 10
-4, 1 × 10
-5, 1 × 10
-6, 1 × 10
-7, 1 × 10
-8μ g/ μ L, respectively getting 2 μ L is that template carries out NASBA amplified reaction respectively.
2, the extraction of viral RNA
Get 0.1 g sample, add liquid nitrogen and be ground into powder, abrasive material is moved into rapidly in 1.5 mL centrifuge tubes, add 1 mL Trizol Reagent, put upside down mixing, 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Get supernatant, 15 DEG C ~ 30 DEG C, place 5min; Add 0.2 mL chloroform, with hand concuss (not vortex oscillation), about 15s.15 DEG C ~ 30 DEG C, place 2min ~ 3min; 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 15min.Careful absorption is about the upper strata aqueous phase of 600 μ L, not disturbance mesophase spherule and lower floor's phase.Add 500 μ L Virahol mixing supernatants, 15 DEG C ~ 30 DEG C, place 10min.2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Remove supernatant liquor, in precipitation, add 1 mL 75% ethanol, washing; 2 DEG C ~ 8 DEG C, 7500 g, centrifugal 5min.Remove supernatant liquor, after precipitation seasoning, be dissolved in 30 μ L ~ 50 μ L without in the water of RNase, be template ribonucleic acid to be checked.
3, amplified reaction
A, in the reaction tubes that 14 μ L loop-mediated isothermal amplification liquid A are housed, add 3 μ L template ribonucleic acid to be checked,
B, on DNA cloning instrument or water-bath 65 DEG C temperature bath 5 min, proceed to immediately 41 DEG C temperature bath 5 min;
C. in reaction tubes, add rapidly 4 μ L reaction solution B;
D. in 41 DEG C of incubation 2 h;
E. metal bath is transferred to 4 DEG C of stopped reactions, takes out after 3 min;
4, NASBA product electrophoresis detection
Get 3 g agaroses, heat, fully dissolve in 100 mL electrophoretic buffers, adding ethidium bromide stock solution to final concentration is 0.5 mg/mL, and glue, adds electrophoretic buffer in electrophoresis chamber, makes liquid level just not have gel; 3 mL ~ 6 mL pcr amplification products are mixed with appropriate sample loading buffer respectively, point sample; 9 V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates in the middle part of gel; Electrophoresis result is observed and record under Ultraviolet Detector.NASBA Product Identification: electrophoresis result shows, hop stunt viroid uses NASBA method to obtain 10
-7the template (see figure 3) of extension rate.
embodiment 4 actual sample detects and contrast experiment
Will from Shandong, Deng Di field, Xinjiang gathers the sick sample with typical HSVd class symptom of (2012 to 2014) and laboratory sample retention (enter the territory France (2013,2014), Italia grape seedling (2014) etc.), NASBA, RT-PCR is adopted to detect respectively, the relatively effect of two kinds of methods, to assess the reliability of LAMP method further.
1, actual sample NASBA detects
1) extraction of viral RNA
Get 0.1 g sample, add liquid nitrogen and be ground into powder, abrasive material is moved into rapidly in 1.5 mL centrifuge tubes, add 1 mL Trizol Reagent, put upside down mixing, 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Get supernatant, 15 DEG C ~ 30 DEG C, place 5min; Add 0.2 mL chloroform, with hand concuss (not vortex oscillation), about 15s.15 DEG C ~ 30 DEG C, place 2min ~ 3min; 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 15min.Careful absorption is about the upper strata aqueous phase of 600 μ L, not disturbance mesophase spherule and lower floor's phase.Add 500 μ L Virahol mixing supernatants, 15 DEG C ~ 30 DEG C, place 10min.2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Remove supernatant liquor, in precipitation, add 1 mL 75% ethanol, washing; 2 DEG C ~ 8 DEG C, 7500 g, centrifugal 5min.Remove supernatant liquor, after precipitation seasoning, be dissolved in 30 μ L ~ 50 μ L without in the water of RNase, be template ribonucleic acid to be checked.
2) amplified reaction
A, in the reaction tubes that 14 μ L loop-mediated isothermal amplification liquid A are housed, add 3 μ L template ribonucleic acid to be checked,
B, on DNA cloning instrument or water-bath 65 DEG C temperature bath 5 min, proceed to immediately 41 DEG C temperature bath 5 min;
C. in reaction tubes, add rapidly 4 μ L reaction solution B;
D. in 41 DEG C of incubation 2 h;
E. metal bath is transferred to 4 DEG C of stopped reactions, takes out after 3 min;
3) NASBA Product Identification
Observe electrophoresis result and record under Ultraviolet Detector, electrophoresis result shows, and in 10 increment product, 9 increment product are positive, and other samples are negative (see figure 4).
2, pcr amplification
1) method: conventional RT-PCR reaction conditions is 50 DEG C of reverse transcription 30 min; 94 DEG C of denaturation 2 min; 94 DEG C of 30 s, 53 DEG C of 30 s, 72 DEG C of 30 s, circulating reaction 35 times; 65 DEG C extend 10 min.
2) agarose gel electrophoresis result: in 10 increment product, 9 parts is positive, and all the other are 1 part.
Conclusion: utilize above-mentioned NASBA and PCR method simultaneously to detect actual sample, the coincidence rate 100% of two kinds of methods, but NASBA is lower to equipment requirements, and convenience is higher.
At present, hop stunt viroid is the viroid disease that China is comparatively general, harm is the most serious, and the disease caused often easily is obscured with other virus diseases.Application of the present invention will contribute to the quick discriminating realizing hop stunt viroid in all polymorphic close cause of disease symptoms.The present invention can be applicable in the cultivating process of seedling, by the rapidly and efficiently detection to virus, can guarantee quality and the effect of increasing production of detoxification seedling.The present invention can carry out specificity identification to hop stunt viroid, can be applicable in the processes such as pears, peach, the import and export quarantine of Lee's seedling, the allocation and transportation of domestic interzone and disease survey.
<110> Inspection and Quarantine Technology Center, Shandong Inspection and Quarantine
<120> is for detecting the NASBA amplimer of hop stunt viroid, test kit and detection method
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 48
<212> DNA
<213> hop stunt viroid
<220>
<221> primer_bind
<222> (1)..(48)
<223> is for the upstream primer of the hop stunt viroid that increases
<400> 1
aattctaatacgactcactatagggagAGTACCTCCCTGCCTTGTTTT 48
<210> 2
<211> 46
<212> DNA
<213> hop stunt viroid
<220>
<221> primer_bind
<222> (1)..(46)
<223> is for the downstream primer of the hop stunt viroid that increases
<400> 2
aattctaatacgactcactatagggagGAATCCAGCGAGAGGCGTG 46
Claims (3)
1. detect the NASBA amplimer of hop stunt viroid, the sequence of its upstream primer, downstream primer is respectively SEQ ID NO:1 ~ 2.
2. detect a NASBA amplification kit for hop stunt viroid, comprise following component:
(1) NASBA amplification reaction solution A:
Every 25 μ L comprise 10 × AMV buffer 2.5 μ l, 6.25mmol/L NTPs 3 μ L, 10 mmol/L dNTPs 1.5 μ L, 10 μm of each 0.5 μ L of ol/L primer NA-P1, NA-P2, distilled water 9 Μ l;
Wherein damping fluid is for containing 40 mmol/L PH8.5 Tris-HCL, 70 mmol/LKCL, 12 mmol/L MgCL2,5 mmol/L DTT damping fluids;
(2) NASBA amplification reaction solution B:
0.5 U RNaseH, 32 U T7RNA polysaccharases, 6.4 U AMV ThermoScript II, 2 μ L DMSO, 0.1 μ L1 mol/L dithiothreitol (DTT), 0.25 μ L 10 mg/mL BSA, 20 U RNA enzyme inhibitorss.
3. detect a NASBA method for hop stunt viroid, comprise the following steps:
1) extraction of sample RNA
A, get 0.1 g sample, add liquid nitrogen and be ground into powder, abrasive material is moved into rapidly in 1.5 mL centrifuge tubes, add 1 mL Trizol Reagent, put upside down mixing, 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min;
B, get supernatant, 15 DEG C ~ 30 DEG C, place 5min; Add 0.2 mL chloroform, use hand concuss, not vortex oscillation, 15s; 15 DEG C ~ 30 DEG C, place 2min ~ 3min; 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 15min;
C, draw the upper strata aqueous phase of 600 μ L, not disturbance mesophase spherule and lower floor's phase; Add 500 μ L Virahol mixing supernatants, 15 DEG C ~ 30 DEG C, place 10min; 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min;
D, removal supernatant liquor, add 1 mL 75% ethanol, washing in precipitation; 2 DEG C ~ 8 DEG C, 7500 g, centrifugal 5min;
E, removal supernatant liquor, after precipitation seasoning, be dissolved in 30 μ L ~ 50 μ L without in the water of RNase, be template ribonucleic acid to be checked;
2) the NASBA amplification of HSVd is carried out
A, in the reaction tubes that 14 μ L loop-mediated isothermal amplification liquid A are housed, add 3 μ L template ribonucleic acid to be checked,
B, on DNA cloning instrument or water-bath 65 DEG C temperature bath 5 min, proceed to immediately 41 DEG C temperature bath 5 min;
C. in reaction tubes, add rapidly 4 μ L reaction solution B;
D. in 41 DEG C of incubation 2 h;
E. metal bath is transferred to 4 DEG C of stopped reactions, takes out after 3 min;
3) electrophoresis detection
Get 3 g agaroses, heat, fully dissolve in 100 mL electrophoretic buffers, adding ethidium bromide stock solution to final concentration is 0.5 mg/mL, and glue, adds electrophoretic buffer in electrophoresis chamber, makes liquid level just not have gel; 3 mL ~ 6 mL pcr amplification products are mixed with appropriate sample loading buffer respectively, point sample; 9 V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates in the middle part of gel; Electrophoresis result is observed and record under Ultraviolet Detector.
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Non-Patent Citations (4)
| Title |
|---|
| KIM HR ET AL: "Transmission of apple scar skin viroid by grafting, Using contaminated pruning equipment, and planting infected seeds", 《PLANT PATHIL》, vol. 22, no. 1, 31 December 2006 (2006-12-31) * |
| 张志宏: "利用NASBA技术检测草莓斑驳病毒", 《果树学报》, vol. 24, no. 6, 31 December 2007 (2007-12-31), pages 1 - 5 * |
| 王英超等: "NASBA技术及其在检验检疫中的应用", 《食品安全质量检测学报》, vol. 5, no. 12, 25 December 2014 (2014-12-25) * |
| 赵晓丽等: "啤酒花矮化类病毒实时荧光定量RT_PCR检测方法的建立与应用", 《植物保护学报》, vol. 40, no. 4, 31 August 2013 (2013-08-31) * |
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