CN104450972B - NASBA primer, test kit and the method for detection Fructus Mali pumilae rust fruit virus - Google Patents
NASBA primer, test kit and the method for detection Fructus Mali pumilae rust fruit virus Download PDFInfo
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Abstract
The invention discloses a kind of NASBA primer, test kit and method detecting Fructus Mali pumilae rust fruit virus, forward primer, the sequence of downstream primer are respectively SEQ ID NO:1～2；NASBA amplification kit, including following component: (1) NASBA amplification reaction solution A；(2) NASBA amplification reaction solution B；Detection method is as follows: the 1) extraction of sample RNA；2) the NASBA amplification of ASSVd is carried out；3) electrophoresis detection.The present invention is applicable to Fructus Mali pumilae rust fruit virus is used for quickly detecting confirmation, can be widely applied to monitoring and the detection of this viroid in the epidemic situation monitoring in agricultural production and environment and foreign trade, operation is extremely easy, and little and for template ribonucleic acid the prescription of required sample size is relatively low.
Description
Technical field:
The present invention relates to a kind of NASBA primer, test kit and method for detecting Fructus Mali pumilae rust fruit virus, belong to plant
The detection technique field of cause of disease.
Background technology:
Fructus Mali pumilae rust fruit virus (Apple scar skid viroid, ASSVd) is attributed to apscaviroid
(Apscaviroid), plastochondria is made up of 330 nucleotide.The host that infects of Fructus Mali pumilae rust fruit virus is Fructus Mali pumilae and pears.Susceptible
Fructus Mali pumilae shows 5 kinds of dominant diseases because of the change of kind and environmental condition: become rusty fruit type, Hua Lianxing, rust fruit-paint face's type, ring speckle type
With green point-type, susceptible pears great majority show as recessiveness, and minority is malformed fruit.Owing to viroid is answered in host plant cell core
System and accumulation, all can detect that in the leaf of susceptible host, stem, skin, stock, and the epidermis of fruit, sarcocarp and seed class is sick
Poison.Therefore, infect the fruit tree lifelong band poison of viroid, and the approach infected by grafting and pruning tool makes viroid disease climing
Prolong.The apple tree of many countries and pear tree all find the harm of ASSVd, China northeast, northwest, North China, and Jiangsu, peace
Also there is generation on the ground such as emblem, Shanxi and Shandong.Detection technique in terms of the current constant-temperature amplification not yet having Fructus Mali pumilae rust fruit virus
Invention report.Therefore, the simple and practical Fructus Mali pumilae rust fruit novel Testing and appraisal method of virus is set up, for improving Fructus Mali pumilae rust fruit
Virus quarantine and examination efficiency, prevent Fructus Mali pumilae rust fruit virus incoming, spread out of, protection China agricultural safety in production, promote
The smooth outlet of agricultural products in China, tool is of great significance.Studing Plant Viroids due to the most dominant infect commonplace, symptom
Show and affected relatively greatly by ambient temperature, and several differential plant is similar to the reaction symptom of inhomogeneity virus, therefore be difficult to answer
By the method for bioassay.Owing to viroid can not produce any protein, so the Electronic Speculum of detection virus, blood can not be used
Learn clearly method.Therefore, select molecular biology method that Fructus Mali pumilae rust fruit virus is detected.
NASBA(Nuleic and sequence based amplipicain, NASBA) i.e. " Nucleic acid sequence expansion
Increasing " detection technique is the isothermal amplification technology of a kind of classics, it is adaptable to the amplification of singlestranded RNA RNA mono-step and detection, the most extensively
General it is applied to the mankind and the detection of animals and plants cause of disease and diagnosis.NASBA is guided by pair of primers, is being polymerized containing T7 RNA
The standard reaction system that the various reaction buffers that enzyme, RNAseH, reverse transcription AMV, NTP, dNTP and needs use are formed
In, the isothermal duplication of nucleotide sequence is realized by the most homogeneous In-vitro specificity enzymatic reaction.
Due in cell wall rich in substantial amounts of polysaccharide, aldehydes matter, the extraction of plant virus RNA also exist poor stability,
Problem that repeatability is low, efficiency is low etc., in addition during extracting RNA itself from signs of degradation, the content of viral RNA is the most relatively
Low, sensitivity and stability to detection method propose high requirement.Simultaneously because the materials such as polysaccharide are for Taq polymerase
Inhibitory action, limits RT-PCR technology in the plant virus RNA detection that the polyphenol contents such as Fructus Vitis viniferae, Fructus Fragariae Ananssae, Cereals class are higher
Application.Owing to the transcriptive process,reversed of NASBA technology is directly merged in amplified reaction, therefore NASBA has possessed and has been suitable for
The amplification of cause of disease RNA, the feature of detection, be best suitable for the analysis of various RNA sample, meets the requirement of plant virus quarantine, in view of
The seriousness of Fructus Mali pumilae rust fruit virus harm, strengthen in environment monitoring, improve detection efficiency, for effectively controlling Fructus Mali pumilae rust
The propagation of fruit virus has important practical significance in present stage.
Summary of the invention:
The technical problem to be solved in the present invention is to provide a kind of NASBA primer for detecting Fructus Mali pumilae rust fruit virus, examination
Agent box and method.Its cardinal principle is to utilize pair of primers to guide, containing T7 RNA polymerase, RNAseH, reverse transcription
In the standard reaction system that the various reaction buffers that AMV, NTP, dNTP and needs use are formed, by the most homogeneous body
Outer specificity enzymatic reaction realizes the isothermal duplication NASBA of nucleotide sequence.Through circulation repeatedly, RNA is made constantly to expand, to sample
Fructus Mali pumilae rust fruit virus in product detects accurately.
The realization of the present invention, is first depending on Fructus Mali pumilae rust fruit virus whole genome sequence design specific primer；Extract again
The ribonucleic acid (RNA) of testing sample, carries out NASBA amplification with specific primer the most respectively；With agarose gel electrophoresis pair
Amplified production detects, and judges whether contain Fructus Mali pumilae rust fruit virus in sample finally according to NASBA amplification.
The present invention is for detecting the NASBA primer of Fructus Mali pumilae rust fruit virus, and its primer sequence is respectively SEQ ID NO:1
～2.
SEQ ID NO:1:
NA-P1:5’-aattctaatacgactcactatagggag TGGGTTCGCCTACAAGAACG-3’
SEQ ID NO:2:
NA-P2:5’-aattctaatacgactcactatagggag ATTTACCCTGGGAACCCACC-3’
The NASBA amplification kit of the detection Fructus Mali pumilae rust fruit virus that the present invention provides, including following component:
(1) NASBA amplification reaction solution A:
Every 25 μ L include 10 × AMV buffer 2.5 μ l, 6.25mmol/L NTPs 3 μ L, 10 mmol/L
The each 0.5 μ L of dNTPs 1.5 μ L, 10 μm ol/L primer NA-P1, NA-P2. distilled water 9 μ L.
Wherein buffer is containing 40 mmol/L PH8.5 Tris-HCL, 70 mmol/LKCL, 12 mmol/L
MgCL2,5 mmol/L DTT buffer.
(2) NASBA amplification reaction solution B:
0.5 U RNaseH, 32 U T7RNA polymerases, 6.4 U AMV reverse transcription, 2 μ L DMSO, 0.1 μ L1
Mol/L dithiothreitol, DTT, 0.25 μ L 10 mg/mL BSA, 20 U RNA enzyme inhibitors.
(3) NASBA amplification program:
Measuring samples RNA 3 μ L is added 14 μ L reactant liquor A, 65 DEG C of temperature baths 5 on DNA cloning instrument or water-bath
Min, proceeds to 41 DEG C of temperature bath 5 min immediately, is rapidly added 4 μ L reactant liquor B, 41 DEG C of incubation 2 h, and 4 DEG C terminate reaction；
(4) NASBA amplified production is identified
Product with 5% agarose gel electrophoresis, observed result judging under uviol lamp.
A kind of method that present invention also offers NASBA detecting Fructus Mali pumilae rust fruit virus, is carried out by the steps:
1) extraction of sample RNA
A, take 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, reverse mixing, 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.
B, take supernatant, 15 DEG C ~ 30 DEG C, place 5min；Add 0.2 mL chloroform, acutely shake (not vortex oscillation) with hands,
About 15s.15 DEG C ~ 30 DEG C, place 2min ~ 3min；2 DEG C ~ 8 DEG C, 12000 g, centrifugal 15min.
C, the careful upper strata aqueous phase drawing about 600 L, not disturbance mesophase and lower floor's phase.Add 500 μ L isopropanols to mix
Close clear liquid, 15 DEG C ~ 30 DEG C, places 10min.2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.
D, removal supernatant, add 1 mL 75% ethanol, washing in precipitation；2 DEG C ~ 8 DEG C, 7500 g, centrifugal 5min.
E, remove supernatant, after precipitation natural drying, be dissolved in 30 μ L ~ 50 μ L without in the water of RNase, be and treat
Inspection template ribonucleic acid.
2) the NASBA amplification of Fructus Mali pumilae rust fruit virus is carried out
A, in the reaction tube equipped with 14 μ L loop-mediated isothermal amplification liquid A, add 3 μ L template ribonucleic acids 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 tube, it is rapidly added 4 μ L reactant liquor 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
Take 3 g agaroses, heat in 100 mL electrophoretic buffers, fully dissolve, add ethidium bromide stock solution to end
Concentration is 0.5 mg/mL, glue, adds electrophoretic buffer, make liquid level just not have gel in electrophoresis tank；By 3 mL～6 mL
Pcr amplification product mixes 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 record is observed under Ultraviolet Detector.If amplified fragments is 157 bp, illustrate that virus to be checked is
Fructus Mali pumilae rust fruit virus, without there are 157 bp amplified fragments, then illustrates that virus to be checked is not Fructus Mali pumilae rust fruit virus.
The present invention according to Fructus Mali pumilae rust fruit virus sequence high conservative region devise two specificity inner primers, this guarantor
Keeping gene order is that the different strains having Fructus Mali pumilae rust fruit viral are common, to ensure that detection difference is come from the level of strain
The reliability of the Fructus Mali pumilae rust fruit virus in source.The present invention is applicable to Fructus Mali pumilae rust fruit virus is used for quickly detecting confirmation, can be wide
General being applied to produces and the confirmation of this virus in the disease monitoring in environment, foreign trade.
Compared with prior art, the beneficial effect comprise that
First, convenience.This invention is when carrying out constant-temperature amplification, it is not necessary to expensive nucleic acid amplification reaction device, whole
Process is carried out at 42 DEG C all the time, it is not necessary to thermal cycler, and only 1 common thermostat water bath just can complete.
Second, degree of accuracy is high.The period of this invention enzyme circular response is few, is not required to high-temperature denatured step, relative to RT-
PCR mismatch rate is relatively low, is more suitable for detection and quantitative special RNA.
3rd, highly sensitive.This invention, compared with round pcr, can just amplify substantial amounts of genes of interest with less circulation,
Ensure that the hypersensitivity of detection, be 10 times of round pcr sensitivity.
4th, shorten the cycle.Owing to transcriptive process,reversed is directly merged in amplified reaction, PCR takes around 20 repeating queries
Ring could expand 106Times, and NASBA only need to circulate 4 ~ 5 and takes turns and i.e. can reach 106Times.
5th, reduce the prescription to plant RNA template.Due in cell wall rich in substantial amounts of polysaccharide, phenolic material
Matter, the extraction of plant virus RNA also exists the problems such as poor stability, repeatability is low, efficiency is low, in addition RNA during extracting
Itself from signs of degradation, the content of viral RNA is the most relatively low, and the sensitivity of detection method and stability propose higher wanting
Ask.Due to this invention for template be RNA, the product of reaction is also RNA, and the result of reaction is not affected by DNA in environment.
Even if there is external double-stranded DNA to pollute, but do not possess T7 promoter sequence due to it, it is impossible to be amplified, secondly, this reaction
Only carry out under 42 DEG C of constant temperatures, be not required to high-temperature denatured step, so NASBA reaction process will not be by external double-stranded DNA
Pollution, therefore relatively low for template purity and prescription.
Accompanying drawing illustrates:
Fig. 1 is that the present invention is to NASBA AFLP system in diseased plant sample.Wherein M:ssRNA Ladder marker；1: healthy
Fructus Vitis viniferae；2: blank；The 3 Fructus Mali pumilae rust fruit susceptible materials of virus.
Fig. 2 is specific outcome figure of the present invention.Wherein M:ssRNA Ladder marker；1: Fructus Mali pumilae rust fruit virus；2:
Hop stunt viroid；3: Pear blister canker viroid；4: Fructus Persicae is hidden mosaic virus；5: apple stem pitting virus.
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、1×10-6Ng/ μ L, the Fructus Mali pumilae rust fruit susceptible material total serum IgE of virus.
Fig. 4 is the actual sample detection figure of the embodiment of the present invention 4.Wherein M:ssRNA Ladder marker；1 ~ 20: its
In 1,4,6,8,9: water-rich areas sample S1, S4, S6, S8, S9；2,3,5,7: Kuerle delicious pear S2, S3, S5, S7；11,12,13:
South water pears S11, S12, S13；10,14,16,19: Chinese pear S10, S14, S16, S19；15,17,18,20: golden pear S15,
S17、S18、S20。
Detailed description of the invention
In order to explain the implementation of the present invention more fully, it is provided that be used for detecting Fructus Mali pumilae rust fruit virus N ASBA examination
The embodiment of agent box.These embodiments are only to explain rather than limit the scope of the present invention.Wherein reverse transcription polymerase
AMV, RNaseH, RNase inhibitor, T7 RNA polymerase, dNTP, ssRNA marker, rNTP are all purchased from NEW
ENGLAND BIOLAB company;PCR amplifing reagent, DNA marker and etc. be all purchased from Beijing Tian Gen company;.
Embodiment 1
1 material
1.1 it is viral
Fructus Mali pumilae rust fruit virus provides for Jian Ke institute of China, hop stunt viroid (Hop stunt vroid,
HSVd), Pear blister canker viroid (Pear Blister Canker Viroid, PBCVd), Peach latent mosaic viroid
(Peach latent mosaic viroid, PLMVd), apple stem pitting virus (Apple stem pitting virus,
ASPV) preserved by this laboratory.
1.2 reagent
Reverse transcription polymerase AMV, RNaseH, RNase inhibitor, T7 RNA polymerase, dNTP, ssRNA
Marker, rNTP are all purchased from NEW ENGLAND BIOLAB company;PCR amplifing reagent, DNA marker and etc. be all purchased from north
Jing Tiangen company;
1.3 primer
According to the full length gene sequence (accession number of Fructus Mali pumilae rust fruit virus in NCBI GenBank
EU825613.1), by comparative analysis Fructus Mali pumilae rust fruit virus height on the premise of the degenerate ensureing amplification and versatility
Conserved region, design 5' end is with T7 promoter sequence NASBA reaction primer (NA-P1, NA-P2), by primer after having designed
Compare under the Primer-Blast module of data base checking.
2 methods
The extraction of 2.1 viral RNAs
Take 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, reverse mixing, 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Take supernatant, 15 DEG C ~ 30 DEG C, place
5min；Add 0.2 mL chloroform, acutely shake (not vortex oscillation), about 15s with hands.15 DEG C ~ 30 DEG C, place 2min ~ 3min；2
DEG C ~ 8 DEG C, 12000 g, centrifugal 15min.The careful upper strata aqueous phase drawing about 600 L, not disturbance mesophase and lower floor's phase.Add
500 μ L isopropanol mixing supernatants, place 10min by 15 DEG C ~ 30 DEG C.2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Remove supernatant
Liquid, adds 1 mL 75% ethanol in precipitation, washing；2 DEG C ~ 8 DEG C, 7500 g, centrifugal 5min.Removing supernatant, precipitation nature is done
After dry, it 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 tube equipped with 14 μ L loop-mediated isothermal amplification liquid A, add 3 μ L template ribonucleic acids 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 tube, it is rapidly added 4 μ L reactant liquor 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
Take 3 g agaroses, heat in 100 mL electrophoretic buffers, fully dissolve, add ethidium bromide stock solution to end
Concentration is 0.5 mg/mL, glue, adds electrophoretic buffer, make liquid level just not have gel in electrophoresis tank；By 3 mL～6 mL
Pcr amplification product mixes 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 record is observed under Ultraviolet Detector.Expection product size is 157 bp.See Fig. 1.
Embodiment 2 specificity experiments
1, extract Fructus Mali pumilae rust fruit virus, hop stunt viroid, Pear blister canker viroid, Fructus Persicae hide mosaic disease
Poison, the RNA of apple stem pitting virus, use NASBA method to detect.
2, the extraction of viral RNA
Take 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, reverse mixing, 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Take supernatant, 15 DEG C ~ 30 DEG C, place
5min；Add 0.2 mL chloroform, acutely shake (not vortex oscillation), about 15s with hands.15 DEG C ~ 30 DEG C, place 2min ~ 3min；2
DEG C ~ 8 DEG C, 12000 g, centrifugal 15min.The careful upper strata aqueous phase drawing about 600 L, not disturbance mesophase and lower floor's phase.Add
500 μ L isopropanol mixing supernatants, place 10min by 15 DEG C ~ 30 DEG C.2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Remove supernatant
Liquid, adds 1 mL 75% ethanol in precipitation, washing；2 DEG C ~ 8 DEG C, 7500 g, centrifugal 5min.Removing supernatant, precipitation nature is done
After dry, it 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 tube equipped with 14 μ L loop-mediated isothermal amplification liquid A, add 3 μ L template ribonucleic acids 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 tube, it is rapidly added 4 μ L reactant liquor 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
Take 3 g agaroses, heat in 100 mL electrophoretic buffers, fully dissolve, add ethidium bromide stock solution to end
Concentration is 0.5 mg/mL, glue, adds electrophoretic buffer, make liquid level just not have gel in electrophoresis tank；By 3 mL～6 mL
Pcr amplification product mixes 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 record is observed under Ultraviolet Detector.Electrophoresis result shows, uses NASBA method to carry out simultaneously
Detection Fructus Mali pumilae rust fruit virus, hop stunt viroid, Pear blister canker viroid, Fructus Persicae are hidden mosaic virus, stem of Fructus Mali pumilae pox
The RNA of virus, only Fructus Mali pumilae rust fruit virus obtain expecting the amplified production (see figure 2) of 157 bp.
Embodiment 3 sensitivity experiments
1, do downwards 10 times of gradient dilutions with the DEPC water fruit viral RNA template liquid that become rusty by Fructus Mali pumilae, be followed successively by 1 × 10-1、1
×10-2、1×10-3、1×10-4、1×10-5、1×10-6Ng/ μ L, respectively taking 2 μ L is that template carries out NASBA amplification instead respectively
Should..
2, the extraction of viral RNA
Take 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, reverse mixing, 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Take supernatant, 15 DEG C ~ 30 DEG C, place
5min；Add 0.2 mL chloroform, acutely shake (not vortex oscillation), about 15s with hands.15 DEG C ~ 30 DEG C, place 2min ~ 3min；2
DEG C ~ 8 DEG C, 12000 g, centrifugal 15min.The careful upper strata aqueous phase drawing about 600 L, not disturbance mesophase and lower floor's phase.Add
500 μ L isopropanol mixing supernatants, place 10min by 15 DEG C ~ 30 DEG C.2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min.Remove supernatant
Liquid, adds 1 mL 75% ethanol in precipitation, washing；2 DEG C ~ 8 DEG C, 7500 g, centrifugal 5min.Removing supernatant, precipitation nature is done
After dry, it 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 tube equipped with 14 μ L loop-mediated isothermal amplification liquid A, add 3 μ L template ribonucleic acids 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 tube, it is rapidly added 4 μ L reactant liquor 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
Take 3 g agaroses, heat in 100 mL electrophoretic buffers, fully dissolve, add ethidium bromide stock solution to end
Concentration is 0.5 mg/mL, glue, adds electrophoretic buffer, make liquid level just not have gel in electrophoresis tank；By 3 mL～6 mL
Pcr amplification product mixes 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 record is observed under Ultraviolet Detector.NASBA product is identified: electrophoresis result shows, Fructus Mali pumilae rust fruit
Viroid uses NASBA method can obtain 10-4The template (see figure 3) of extension rate.
The detection of embodiment 4 actual sample and contrast experiment
By the sick sample with typical apple rust fruit virus symptoms gathered from Xinjiang and laboratory sample retention (outlet Taiwan pears
Scion), water-rich areas (S1, S4, S6, S8, S9), Kuerle delicious pear (S2, S3, S5, S7) Nan Shuili (S11, S12, S13), snowflake
Pears (S10, S14, S16, S19), golden pear (S15, S17, S18, S20) are respectively adopted NASBA, RT-PCR and detect, and compare
The effect of two kinds of methods, to assess the reliability of NASBA method further.
1, actual sample NASBA detection
1) extraction of viral RNA
Take 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, reverse mixing, 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10 min.Take supernatant, 15 DEG C ~ 30 DEG C, place 5
min；Add 0.2 mL chloroform, acutely shake (not vortex oscillation), about 15s with hands.15 DEG C ~ 30 DEG C, place 2 min ~ 3 min；2
DEG C ~ 8 DEG C, 12000 g, centrifugal 15 min.The careful upper strata aqueous phase drawing about 600 L, not disturbance mesophase and lower floor's phase.
Add 500 μ L isopropanol mixing supernatants, 15 DEG C ~ 30 DEG C, place 10 min.2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10 min.Remove
Supernatant, adds 1 mL 75% ethanol in precipitation, washing；2 DEG C ~ 8 DEG C, 7500 g, centrifugal 5 min.Removing supernatant, precipitation is certainly
So dried, it is dissolved in 30 μ L ~ 50 μ L without in the water of RNase, is template ribonucleic acid to be checked.
2) amplified reaction
A, in the reaction tube equipped with 14 μ L loop-mediated isothermal amplification liquid A, add 3 μ L template ribonucleic acids 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 tube, it is rapidly added 4 μ L reactant liquor 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 is identified
Observing electrophoresis result record under Ultraviolet Detector, electrophoresis result shows, in 20 parts of samples, 8 parts of samples are sun
Property, other samples are negative (see figure 4).
2, PCR amplification
1) method: conventional RT-PCR reaction condition is 50 DEG C of reverse transcription 30 min；94 DEG C of denaturation 2 min; 94 ℃
30 s, 53 DEG C of 30 s, 72 DEG C of 30 s, circular response 35 times；65 DEG C extend 10 min.
2) agarose gel electrophoresis result: in 20 parts of samples, 8 parts is positive, and remaining 12 parts of sample is negative.
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, Fructus Mali pumilae rust fruit virus is the pears viroid disease that China is more universal, harm is the most serious, the disease caused
Often easily obscure with virosis.The application of the present invention will assist in and realizes Fructus Mali pumilae rust fruit in all polymorphic close cause of disease symptoms
The quick discriminating of viroid.Present invention can apply in the cultivating process of Fructus Mali pumilae, pears seedling, by viroid rapidly and efficiently
Detection, it can be ensured that the quality of detoxification pears and effect of increasing production.The present invention can carry out specificity identification to Fructus Mali pumilae rust fruit virus, can
It is applied to Fructus Mali pumilae, the import and export quarantine of pears seedling, the allocation and transportation of domestic interzone and disease survey.
Nucleotides sequence list
<110>Inspection and Quarantine Technology Center, Shandong Inspection and Quarantine
<120>NASBA primer, test kit and the method for detection Fructus Mali pumilae rust fruit virus
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 47
<212> DNA
<213>Fructus Mali pumilae rust fruit virus
<220>
<221> primer_bind
<222> (1)..(47)
<223>for expanding the forward primer of Fructus Mali pumilae rust fruit virus
<400> 1
aattctaatacgactcactatagggagTGGGTTCGCCTACAAGAACG 47
<210> 2
<211> 47
<212> DNA
<213>Fructus Mali pumilae rust fruit virus
<220>
<221> primer_bind
<222> (1)..(47)
<223>for expanding the downstream primer of Fructus Mali pumilae rust fruit virus
<400> 2
aattctaatacgactcactatagggagATTTACCCTGGGAACCCACC 47
Claims (2)
1. the NASBA amplification kit of detection Fructus Mali pumilae rust fruit virus, including following component:
(1) NASBA amplification reaction solution A:
Every 25 μ L include 10 × AMV buffer 2.5 μ L, 6.25mmol/L NTPs 3 μ L, 10 mmol/L dNTPs 1.5 μ
L, 10 μm ol/L forward primer NA-P1, downstream primer NA-P2 each 0.5 μ L, distilled water 9 μ L；
Wherein buffer is containing 40 mmol/L pH 8.5 Tris-HCl, 70 mmol/L KCl, 12 mmol/L MgCl2, 5
Mmol/L DTT buffer；
(2) NASBA amplification reaction solution B:
0.5 U RNaseH, 32 U T7RNA polymerases, 6.4 U AMV reverse transcription, 2 μ L DMSO, 0.1 μ L 1 mol/L
Dithiothreitol, DTT, 0.25 μ L 10 mg/mL BSA, 20 U RNA enzyme inhibitors；
The sequence of primer NA-P1, NA-P2 is respectively SEQ ID NO:1～2.
2. the NASBA method of detection Fructus Mali pumilae rust fruit virus, comprises the following steps:
1) extraction of sample RNA
A, take 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, reverse mixing, 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min；
B, take supernatant, 15 DEG C ~ 30 DEG C, place 5min；Add 0.2 mL chloroform, acutely shake with hands, 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 L, not disturbance mesophase and lower floor's phase, add 500 μ L isopropanol mixing supernatants, 15 DEG C
~ 30 DEG C, placement 10min, 2 DEG C ~ 8 DEG C, 12000 g, centrifugal 10min；
D, removal supernatant, add 1 mL 75% ethanol, washing in precipitation；2 DEG C ~ 8 DEG C, 7500 g, centrifugal 5min；
E, remove supernatant, after precipitation natural drying, be dissolved in 30 μ L ~ 50 μ L without in the water of RNase, be mould to be checked
Plate RNA；
2) the NASBA amplification of ASSVd is carried out
A, in the reaction tube equipped with 14 μ L NASBA amplification reaction solution A, add 3 μ L template ribonucleic acids 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 tube, it is rapidly added 4 μ L reactant liquor 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
Take 3 g agaroses, heat in 100 mL electrophoretic buffers, fully dissolve, add ethidium bromide stock solution to final concentration
It is 0.5 mg/mL, glue, electrophoresis tank adds electrophoretic buffer, makes liquid level just not have gel；By 3 mL～6 mL
NASBA amplified production mixes with appropriate sample loading buffer respectively, point sample；9 V/cm constant voltage electrophoresis, until bromophenol blue indicator moves
Move in the middle part of gel；Electrophoresis result record is observed under Ultraviolet Detector.
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