CN104450973B - For detecting NASBA amplimer, test kit and the detection method of hop stunt viroid - Google Patents
For detecting NASBA amplimer, test kit and the detection method of hop stunt viroid Download PDFInfo
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Abstract
The invention discloses a kind of NASBA amplimer, test kit and detection method detecting hop stunt viroid, its forward primer, the sequence of downstream primer are respectively SEQ ID NO:1～2, and test kit includes NASBA amplification reaction solution A and NASBA amplification reaction solution B；Detection method includes: the 1) extraction of sample RNA；2) the NASBA amplification and 3 of hop stunt viroid is carried out) electrophoresis detection.The present invention is applicable to hop stunt viroid 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 amplimer for detecting hop stunt viroid, test kit and detection side
Method, belongs to the detection technique field of pathogenic.
Background technology:
Viroid is the hitherto known minimum virulence factor causing plant disease, for circular RNA molecule, its full-length gene
Group is made up of 246～399 nucleotide, and many industrial crops can be caused to produce serious plant disease.Same according to its sequence and structure
Source property, duplication characteristic are divided into 2 groups, respectively potato spindle tuber viroid group (PSTVd) and avsunviroid viroid group
(ASBVd).It is short that hop stunt viroid (Hop stunt vroid) belongs to potato spindle tuber viroid section Flos lupuli (Flos Humuli Lupuli)
Change viroid to belong to, generally containing about 307 nucleotide.1970, Yamamoto of Japan et al. was first at the medicated beer downgraded
Take and be found that hop stunt viroid.1985, the Sano of Japan was found that HSVd, first through 15 years on Fructus Vitis viniferae
Research, it is believed that the primary source of infection of HSVd is Fructus Vitis viniferae, has the most gradually adapted to new host.The most
The HSVd host of report includes draft and the xylophytas such as Fructus Cucumidis sativi, Fructus Vitis viniferae, Fructus Persicae, Lee, almond, Fructus Pruni.China is in Flos lupuli (Flos Humuli Lupuli), Fructus Persicae
, Lee, Fructus Pruni, Fructus Vitis viniferae, report HSVd on the plant such as almond.Therefore, the Testing and appraisal method one of specification hop stunt viroid
As rule of operation, for improve hop stunt viroid quarantine and examination efficiency, prevent hop stunt viroid incoming,
Spreading out of, the safety in production of protection China agricultural, promote the smooth outlet of agricultural products in China, tool is of great significance.Plant
Viroid is infected commonplace due to the most dominant, and Symptoms is affected relatively big by ambient temperature, and several differential plant pair
The reaction symptom of inhomogeneity virus is similar, therefore is difficult to apply the method for bioassay.Owing to viroid can not produce any albumen
Matter, so the Electronic Speculum of detection virus, serological method can not be used.Therefore, select molecular biology method short to Flos lupuli (Flos Humuli Lupuli)
Change viroid to detect.
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 hop stunt viroid harm, strengthen in environment monitoring, improve detection efficiency and the accuracy of confirmation, right
Effectively control the propagation of 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 amplification for detecting hop stunt viroid and draws
Thing, provides test kit and the detection method of a kind of detection simultaneously.Its cardinal principle is to utilize pair of primers to guide, containing T7
RNA polymerase, RNAseH, reverse transcription AMV, NTP, dNTP and the standard needing the various reaction buffers used to be formed are anti-
Answer in system, realized the isothermal duplication NASBA of nucleotide sequence by the most homogeneous In-vitro specificity enzymatic reaction, through following
Ring repeatedly, makes RNA constantly expand, and detects the hop stunt viroid in sample accurately.
The realization of the present invention, is first depending on hop stunt viroid whole genome sequence design specific primer, then carries
Take the ribonucleic acid (RNA) of testing sample, carry out NASBA amplification with specific primer the most respectively, use agarose gel electrophoresis
Amplified production is detected, judges whether sample contains hop stunt viroid finally according to NASBA amplification.
The technical scheme that the present invention solves the employing of above-mentioned technical problem 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’。
The NASBA amplification kit of detection hop stunt viroid, including following component:
(1) preparation of 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) preparation of 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.
Invention further provides one uses SEQ NO:1, SEQ NQ:2 specificity amplification primer detection Flos lupuli (Flos Humuli Lupuli) short
The method changing the NASBA of viroid, 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, carefully absorption are the upper strata aqueous phase of 600 L, not disturbance mesophase and lower floor's phase.Add 500 μ L isopropanol mixing
Supernatant, places 10min by 15 DEG C ~ 30 DEG C.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 hop stunt viroid 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 262 bp, illustrate that virus to be checked is
Hop stunt viroid, without there are 262 bp amplified fragments, then illustrates that virus to be checked is not that Hop stunt class is sick
Poison.
The present invention devises two specificity inner primers according to the sequence high conservative region of hop stunt viroid, should
Conserved genetic sequences is that to have the different strains of hop stunt viroid common, to ensure to detect not from the level of strain
Reliability with the hop stunt viroid in source.The present invention is applicable to be used for quickly detecting hop stunt viroid really
Card, can be widely applied to produce 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.
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 be the present invention to NASBA AFLP system in diseased plant sample: wherein M:ssRNA Ladder marker；1: healthy
Fructus Vitis viniferae；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: Fructus Mali pumilae rust fruit virus；3: Pear blister canker viroid；4: Fructus Persicae is hidden mosaic virus；5: apple stem pitting virus；6: Fructus Vitis viniferae is yellow
Speckle 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, hop stunt viroid susceptible material total serum IgE.
Fig. 4 is the 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).
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 hop stunt viroid NASBA
The embodiment of test kit.These embodiments are only to explain rather than limit the scope of the present invention.Wherein reverse transcription polymerization
Enzyme 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
Hop stunt viroid is the French Grape rootstock seedling separator that enters the territory, Italy enters the territory grape seedlings separator, newly
Boundary grape seedlings and Flos lupuli (Flos Humuli Lupuli) separator, Fructus Mali pumilae rust fruit virus (Apple scar skid viroid, ASSVd), pears blister are burst
Infections viroid (Pear Blister Canker Viroid, PBCVd), Fructus Persicae hide embedding stricture of vagina viroid (Peach latent
Mosaic viroid, PLMVd), apple stem pitting virus (Apple stem pitting virus, ASPV), Fructus Vitis viniferae macula lutea class sick
Poison-2(Grapevine yellow speckle viroid, GYSVd-2) preserved by the laboratory of applicant.
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 hop stunt viroid in NCBI GenBank
HM357802.1), high by comparative analysis hop stunt viroid on the premise of the degenerate ensureing amplification and versatility
Degree conserved region, design 5' end, with T7 promoter sequence NASBA reaction primer (NA-P1, NA-P2), will draw after having designed
Thing is compared checking under the Primer-Blast module of data base.
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 262 bp.See Fig. 1.
Embodiment 2: specificity experiments
1, extract hop stunt viroid, Fructus Mali pumilae rust fruit virus, Pear blister canker viroid, Fructus Persicae hide mosaic disease
Poison, apple stem pitting virus, the RNA of Fructus Vitis viniferae macula lutea viroid-2, 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 detect
Hop stunt viroid, Fructus Mali pumilae rust fruit virus, Pear blister canker viroid, Fructus Persicae hide mosaic virus, apple stem pitting virus,
The RNA of Fructus Vitis viniferae macula lutea viroid-2, only hop stunt viroid obtain expecting the amplified production (see figure 2) of 262 bp.
Embodiment 3 sensitivity experiments
1, with DEPC water, hop stunt viroid viral RNA template liquid is done downwards 10 times of gradient dilutions, it is followed successively by 1 ×
100、 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 takes 2
μ L is that template carries out NASBA amplified reaction respectively.
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, Flos lupuli (Flos Humuli Lupuli) is short
Changing viroid uses NASBA method can obtain 10-7The template (see figure 3) of extension rate.
The detection of embodiment 4 actual sample and contrast experiment
Will from Shandong, Deng Di field, Xinjiang gather the sick sample with typical HSVd class symptom of (2012 to 2014) and experiment
Room keeps sample (enter the territory France (2013,2014), Italia grape Seedling (2014) etc.), is respectively adopted NASBA, RT-PCR and examines
Survey, compare the effect of two kinds of methods, to assess the reliability of LAMP 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 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) 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 10 parts of samples, 9 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 10 parts of samples, 9 parts is positive, and remaining is 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 more universal, harm is the most serious, the disease caused
Often easily obscure with other virosiss.The application of the present invention will assist in and realizes medicated beer in all polymorphic close cause of disease symptoms
The quick discriminating of flower stunt viroid.Present invention can apply in the cultivating process of seedling, by the rapidly and efficiently inspection to virus
Survey, it can be ensured that the quality of detoxification seedling and effect of increasing production.The present invention can carry out specificity identification to hop stunt viroid,
During can be applicable to pears, Fructus Persicae, the import and export quarantine of Lee's seedling, the allocation and transportation of domestic interzone and disease survey etc..
<110>Inspection and Quarantine Technology Center, Shandong Inspection and Quarantine
<120>for detecting NASBA amplimer, test kit and the detection method of hop stunt viroid
<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>for expanding the forward primer of hop stunt viroid
<400> 1
aattctaatacgactcactatagggagAGTACCTCCCTGCCTTGTTTT 48
<210> 2
<211> 46
<212> DNA
<213>hop stunt viroid
<220>
<221> primer_bind
<222> (1)..(46)
<223>for expanding the downstream primer of hop stunt viroid
<400> 2
aattctaatacgactcactatagggagGAATCCAGCGAGAGGCGTG 46
Claims (2)
1. detect a NASBA amplification kit for hop stunt viroid, 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, each 0.5 μ L of 10 μm ol/L forward primer, downstream primer, 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；
Its forward primer, the sequence of downstream primer are respectively SEQ ID NO:1～2.
2. utilize the test kit described in claim 1 to detect a NASBA method for hop stunt viroid, including following step
Rapid:
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, place 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 HSVd is carried out
A, in the reaction tube equipped with the NASBA amplification reaction solution A of 14 μ L, 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 PCR
Amplified production mixes with appropriate sample loading buffer respectively, point sample；9 V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates to coagulate
In the middle part of glue；Electrophoresis result record is observed 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》.2006,第22卷(第1期),摘要、类病毒检测部分. * |
| 张志宏.利用NASBA技术检测草莓斑驳病毒.《果树学报》.2007,第24卷(第6期),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》.2014,第5卷(第12期), * |
| 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》.2013,第40卷(第4期), * |
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