CN108342508A - A kind of passionflower mosaic virus early detection method - Google Patents
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Abstract
The invention discloses a kind of passionflower mosaic virus early detection methods, by designing the specific primer of passionflower mosaic virus coat protein gene segment, using RT round pcr amplifying target genes segments, to realize the accurate detection to virus.The sample requirements of RT round pcrs are few, technical difficulty is small, accuracy is high, is suitable for Mass-analysis of samples.The technology can not only Diagnosis of Suspected passionflower mosaic virus infect the sample of symptom, moreover it is possible to identification virus does not show the sample of manifest symptom in latency stage, can realize the early diagnosis of diseased plant.
Description
Technical field
The present invention relates to biotechnologies, and in particular to a kind of passionflower mosaic virus early detection method.
Background technology
Passionflower plants the accurate poverty alleviation industry as " short, flat, fast ", is promoted in the multiple areas in Guizhou Province, is to promote agriculture
The effective way that the people shake off poverty and set out on the road to prosperity.20th century 80, the nineties, passionflower mosaic virus is to productions such as China Taiwan, Fujian, Guangdong
Area caused larger harm, in August, 2016, Guizhou Province to have also discovered the plant that doubtful mosaic virus infects, and industry development faces
Potential threat.Virus-free seedling-wood breeding and popularization are the bases that guarantee industry continues, develops in a healthy way, and viral diagnosis is nontoxic
Seedling breeding, one of the key technology promoted.
At present it has been reported that passionflower mosaic virus detection method include Biological Detection, Electron microscopy and
Serologic detection.Time-consuming for Biological Detection, and is limited by environmental condition.Electron Microscopy Results are intuitive, accurate, by
In instrument and equipment costliness, film-making and operating technology complexity are not easy to grasp, and require height to operator's technical merit, therefore not
It is used widely.Serological method has the characteristics that quick, intuitive, but the accuracy of its result tends to rely on antiserum
Quality, and unstable virus can not be detected.
Reverse transcriptional PCR (RT-PCR) is a kind of detection plant RNA disease to grow up the 1990s
The method of poison, this method high sensitivity, sampling amount be few, high specificity, is suitable for the batch detection of viral sample.More than being based on
Advantage, RT-PCR are widely used to the detection of various plants virus.Domestic passionflower mosaic virus detection is there is not yet RT-PCR
Method is reported, also has no that related patents disclose.
Invention content
To solve the above problems, the present invention provides a kind of passionflower mosaic virus early detection methods.
To achieve the above object, the technical solution that the present invention takes is：
A kind of passionflower mosaic virus early detection method, includes the following steps：
The extraction for the passionflower total serum IgE that S1, mosaic virus infect
S11, it takes passionflower mosaic virus to infect blade 100mg, is transferred to after liquid nitrogen grinding in 2ml centrifuge tubes, is added
Trizol reagent 1ml, after mixing well, are placed at room temperature for 5min, so that sample is fully cracked by Trizol reagents, obtain lysate；
S12, chlorine is added in the lysate of gained in the ratio for adding 200 μ l chloroforms (chloroform) per 1ml lysates
It is imitative, pipe lid is covered tightly, after acutely rocking 15s up and down, is placed at room temperature for 3min, at 4 DEG C, 12000rpm centrifuges 15min；
After S13, centrifugation, liquid layered in pipe shifts the colourless liquid (about total volume 50% or so) of top layer to one
In the new 1.5ml centrifuge tubes without RNA enzyme, 100% 500 μ l of isopropanol are then added, after being placed at room temperature for 10min, at 4 DEG C
Lower 12000rpm centrifuges 10min；
After the completion of S14, centrifugation, carefully suck supernatant, 75% ethyl alcohol be added, turn upside down 8 times, 7500 rpm, 4 DEG C from
Heart 5min after removing ethyl alcohol, uncaps and is placed at room temperature for 5~10min to get to the RNA of purifying；
The synthesis of the first chain of S2, cDNA
S21,1 μ g of total serum IgE and 100 μM of sequence are added in the PCR pipe of no RNA enzyme as AGATGTGGGAATGCGT's
1 μ l of gene-specific primer are used in combination the processed aqua sterilisas of DEPC to be supplemented to 12 μ l of volume, obtain mixed liquor；
S22, by above-mentioned mixed liquor in 65 DEG C handle 5min after, on ice immediately cool down 1min, then into mixed liquor according to
The secondary dNTP that 4 μ l of 5X reaction buffer, 1 μ l of RiboLock RNase Inhibitor of 20U/ μ l, 10mM is added
The RevertAid M-MuLV Reverse Transcriptase1 μ l of 2 μ l of mix, 200U/ μ l, it is of short duration by its gently mixing
After centrifugation, it is placed in 42 DEG C of incubation 1h in PCR instrument；Then 70 DEG C of incubation 5min, reaction was completed to get the first chains of cDNA；
S3, the RT-PCR detections of passionflower mosaic virus
The design of S31, primer
Special primer design is carried out using 5.0 softwares of Primer Premier：
Sense primer PafMVF：5′-CCGCTCCGCTTCCTCCTC-3′；
Downstream primer PafMVR：5′-TTGAATACACGAGCACGGCG-3′；
S32, PCR reactions are carried out in following PCR reaction systems：PCR-Grade Water15.0μl； 2X Ex taq
Buffer(takara)25.0μl；The 1.0 μ l of dNTP Mix of 10mM；The 1.0 μ l of Ex taq (takara) of 5U/ μ l；CDNA
One chain, 5.0 μ l；10 μM of 1.5 μ l of PafMVF；10 μM of 1.5 μ l of PafMVR；
S4, the PCR product of gained carries out to electrophoretic process on 1.0% Ago-Gel, voltage 120V, electrophoresis 0.5h,
Obtain the gel containing PCR fragment；
S5, the gel containing PCR fragment is cut with blade, is positioned over 1.5ml centrifuge tubes, weighs；It is solidifying that 3 times of volumes are added
The GSB solution of glue weight (mg), 60 DEG C of metal bath 10min；After gel is completely dissolved, the pre- of 1/3 volume GSB solution is added
Cold isopropanol；After solution is cooled to room temperature, is drawn to centrifugal column with liquid-transfering gun, stand 1min, at room temperature, 10000rpm centrifugations
1min；The waste liquid in collecting pipe is outwelled, 650 μ l, 10000rpm centrifugation 1min of WB solution are added, abandon efflux；It centrifuges again,
Thoroughly removal residual solution；Centrifugal column is transferred to 1.5ml centrifuge tubes, uncaps and stands 1min；It is added dropwise among centrifugal column
ddH230 μ l of O stand 1min；10 000rpm centrifuge 1min, elute PCR fragment, obtain the PCR product of purifying；
S6, PCR product after purification are mixed with TA cloning vectors pGM-T and related reagent, include specifically dd H2O 6.0
μl；T4 DNA Ligation 10×Buffer 1.0μl；The 1.0 μ l of T4 DNA Ligase of 3U/ μ l；The pGM-T of 50ng/ μ l
1.0 μ l of carrier；The 1.0 μ l of PCR product of 100ng/ μ l.By above-mentioned mixed solution in PCR instrument, 16 DEG C of incubation 12h must be connected
Product.
S7,5 μ l connection products are taken to import escherichia coli DH5a competent cell, it is solid in the LB of the benzyl mycin of ammonia containing 50mg/l
It is screened on body culture medium, 37 DEG C of culture 12h, picking white single bacterium colony, with M13 primers, PCR verifies its positive, is drawn using general
Object T7 and SP6 carry out sequencing.
Preferably, the PCR response procedures are：
94 DEG C of pre-degeneration 2min, make masterplate cDNA fully be denaturalized.Subsequently into amplification cycles：94 DEG C denaturation 30s, 55 DEG C
Renaturation 30s, 72 DEG C of extension 40s, after 35 recycle, whole extension 5min makes PCR product extend complete, and 4 DEG C preserve.
Preferably, TA clone's coupled reaction systems in the step S5 include：dd H2O 6.0μl； T4 DNA
Ligation 10×Buffer 1.0μl；The T4 DNA Ligase1.0 μ l of 3U/ μ l；The 1.0 μ l of pGM-T carriers of 50ng/ μ l；
The 1.0 μ l of PCR product of 100ng/ μ l.
Preferably, the LB solid culture primary surface coating concentrations in the step S6 are the IPTG10 μ l of 40mg/ml and dense
Degree is the 40 μ l of X-Gal solution of 20mg/ml.
The present invention establishes a set of passionflower mosaic virus molecular detecting method, can be used for the susceptible plant of detoxic seedling and field
The early stage identification of strain, provides technical support for virus-free breeding seedling-wood breeding and popularization, ensures the lasting, strong of passionflower industry
Kang Fazhan；It enriches, improve passionflower mosaic virus detection technique, established to infect viral level and Transport research in plant
Fixed basis；Theoretical foundation is provided for breeding for disease resistance and disease control.
Description of the drawings
Fig. 1 is the result schematic diagram of detected through gel electrophoresis in the embodiment of the present invention.
Specific implementation mode
In order to make objects and advantages of the present invention be more clearly understood, with reference to embodiments to the present invention into advance one
Step is described in detail.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit this
Invention.
An embodiment of the present invention provides a kind of passionflower mosaic virus early detection methods, include the following steps：
The extraction for the passionflower total serum IgE that S1, mosaic virus infect
S11, it takes passionflower mosaic virus to infect blade 100mg, is transferred to after liquid nitrogen grinding in 2ml centrifuge tubes, is added
Trizol reagent 1ml, after mixing well, are placed at room temperature for 5min, so that sample is fully cracked by Trizol reagents, obtain lysate；
S12, chlorine is added in the lysate of gained in the ratio for adding 200 μ l chloroforms (chloroform) per 1ml lysates
It is imitative, pipe lid is covered tightly, after acutely rocking 15s up and down, is placed at room temperature for 3min, 12000rpm centrifuges 15min at 4 DEG C；
After S13, centrifugation, liquid layered in pipe shifts the colourless liquid (about total volume 50% or so) of top layer to one
In the new 1.5ml centrifuge tubes without RNA enzyme, 500 μ l, 100% isopropanols are then added, after being placed at room temperature for 10min, at 4 DEG C
12000rpm centrifuges 10min；
After the completion of S14, centrifugation, carefully suck supernatant, 75% ethyl alcohol be added, turn upside down 8 times, 7500 rpm, 4 DEG C from
Heart 5min after removing ethyl alcohol, uncaps and is placed at room temperature for 5~10min to get to the RNA of purifying；
S2, it is carried out using the RevertAid First Strand cDNA Synthesis Kit of Fermentas companies
The synthesis of the first chains of cDNA；
S21,1 μ g of total serum IgE and 100 μM of sequence are added in the PCR pipe of no RNA enzyme as AGATGTGGGAATGCGT's
Gene-specific primer 1ul is used in combination the processed aqua sterilisas of DEPC to be supplemented to 12 μ l of volume, obtains mixed liquor；
S22, by above-mentioned mixed liquor in 65 DEG C handle 5min after, on ice immediately cool down 1min, then into mixed liquor according to
The secondary dNTP that 4 μ l of 5X reaction buffer, RiboLock RNase Inhibitor1 μ l of 20U/ μ l, 10mM is added
The RevertAid M-MuLV Reverse Transcriptase1 μ l of 2 μ l of mix, 200U/ μ l, it is of short duration by its gently mixing
After centrifugation, it is placed in 42 DEG C of incubation 1h in PCR instrument；Then 70 DEG C of incubation 5min, reaction was completed to get the first chains of cDNA；
S3, the RT-PCR detections of passionflower mosaic virus
The design of S31, primer
According to it has been reported that passionflower mosaic virus coat protein gene sequence, it is soft using Primer Premier 5.0
Part carries out special primer design, is synthesized by Sangon Biotech (Shanghai) Co., Ltd..Primer extension product size
For 506bp, title and sequence are：
Sense primer PafMVF：5′-CCGCTCCGCTTCCTCCTC-3′；
Downstream primer PafMVR：5′-TTGAATACACGAGCACGGCG-3′；
S32, PCR reactions are carried out in following PCR reaction systems：PCR-Grade Water15.0μl； 2X Ex taq
Buffer 25.0μl；The dNTP Mix1.0 μ l of 10mM；The Ex taq1.0 μ l of 5U/ μ l；5.0 μ l of the first chains of cDNA；10 μM
PafMVF 1.5μl；10 μM of PafMVR1.5 μ l；The PCR response procedures are：
94 DEG C of pre-degeneration 2min, make masterplate cDNA fully be denaturalized.Subsequently into amplification cycles：94 DEG C denaturation 30s, 55 DEG C
Renaturation 30s, 72 DEG C of extension 40s, after 35 recycle, whole extension 5min makes PCR products extend complete, and 4 DEG C preserve.
S4, the PCR product of gained carries out to electrophoretic process on 1.0% Ago-Gel, voltage 120V, electrophoresis 0.5h,
Obtain the gel containing PCR fragment；Gel imaging system observation experiment result is simultaneously taken pictures.As shown in Figure 1, positive P, to be measured
1,2 amplified production size about 500bp of sample, consistent with expection, negative control N is not expanded to specific nucleic acid band.
S5, the gel containing PCR fragment is cut with blade, is positioned over 1.5ml centrifuge tubes, weighs；It is solidifying that 3 times of volumes are added
The GSB solution of glue weight (mg), metal bath 10min；After gel is completely dissolved, the precooling that 1/3 volume GSB solution is added is different
Propyl alcohol；It after solution is cooled to room temperature, is drawn to centrifugal column with liquid-transfering gun, stands 1min, 10 000rpm centrifuge 1min；Outwell collection
Waste liquid in pipe is added 650 μ l, 10000rpm centrifugation 1min of WB solution, abandons efflux；It centrifuges again, thoroughly removal residual is molten
Liquid；Centrifugal column is transferred to 1.5ml centrifuge tubes, uncaps and stands 1min；30 μ l deionized waters are added dropwise among centrifugal column, stand
1min；At room temperature, 10 000rpm centrifuge 1min, elute PCR fragment；Recycling segment is connected into TA cloning vectors pGM-T, and 16 DEG C anti-
12h is answered, connection product is obtained；The TA clones coupled reaction system：dd H2O 6.0μl；T4DNA Ligation 10×
Buffer 1.0μl；The 1.0 μ l of T4 DNA Ligase of 3U/ μ l；The pGM-T Vecter1.0 μ l of 50ng/ μ l；100 ng/μl
1.0 μ l of PCR product.
S6,5 μ l connection products are taken to import escherichia coli DH5a competent cell, it is solid in the LB of the benzyl mycin of ammonia containing 50mg/l
It is screened on body culture medium, the IPTG10 μ l and a concentration of 20mg/ml of the LB solid cultures primary surface coating concentration 40mg/ml
40 μ l of X-Gal solution.37 DEG C of culture 12h, picking white single bacterium colony, with M13 primers, PCR verifies its positive, and use is general
Primer T7 and SP6 carry out sequencing, and sequencing sequence is with passionflower mosaic virus CP genetic homologies on ncbi database
97%, it may be determined that the nucleic acid fragment expanded is exactly a part for mosaic virus CP gene orders, illustrates that PCR primer is special
Property is fine.
Application examples
In August, 2016, applicants investigate Guizhou passionflower producing region disease incidence, it is found that doubtful plant virus is infected
Plant.The symptom of passionflower mosaic virus is described according in document report, applicant can only guess that plant may be by west kind
Lotus flower mosaic virus infects.Since marmor upsilon etc. infects passionflower, it can also cause similar flower leaf paresthesia, so can not be true
It is exactly mosaic virus to determine cause of disease.It is exactly mosaic virus less than pathogen is identified once week by the above method.
In April, 2017, applicants analyze plant that passionflower mosaic virus infects in detoxification using the above method
Front and back band poison situation.Testing result shows that the plant before detoxification can expand to virus coat protein gene sequence, and takes off
Plant after poison can not then expand to the gene order.Further to verify the certain detoxification success of the plant after detoxification, applicant
Prolonged observation has been carried out to plant, all not it is observed that symptom after 3 months, and same time artificial infection mosaic virus
Then there is symptom of typically catching an illness in plant.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the principle of the present invention, it can also make several improvements and retouch, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (4)
1. a kind of passionflower mosaic virus early detection method, which is characterized in that include the following steps：
The extraction for the passionflower total serum IgE that S1, mosaic virus infect
S11, it takes passionflower mosaic virus to infect blade 100mg, is transferred to after liquid nitrogen grinding in 2ml centrifuge tubes, Trizol is added
Reagent 1ml after mixing well, is placed at room temperature for 5min, so that sample is fully cracked by Trizol reagents, obtains lysate；
Chloroform is added in the lysate of gained in S12, the ratio that 200 μ l chloroforms are added in every 1ml lysates, covers tightly pipe lid, on
After acutely rocking 15s down, it is placed at room temperature for 3min, 12000rpm centrifuges 15min at 4 DEG C；
After S13, centrifugation, liquid layered in pipe shifts the colourless liquid of top layer to a new 1.5ml centrifugation without RNA enzyme
Then 100% isopropanol, 500 μ l are added in Guan Zhong, after being placed at room temperature for 10min, 12000rpm centrifuges 10min at 4 DEG C；
After the completion of S14, centrifugation, supernatant is carefully sucked, 75% ethyl alcohol 1ml is added, 8 times, 7500rpm, 4 DEG C centrifugations of turning upside down
5min removes ethyl alcohol, is placed at room temperature for 5~10min after uncapping, the sterilizing water dissolution RNA of 20 μ l DEPC processing, spare；
The synthesis of the first chain of S2, cDNA
S21,1 μ g of total serum IgE and a concentration of 100 μM of sequence are added in the PCR pipe of no RNA enzyme as AGATGTGGGAATGCGT's
1 μ l of gene-specific primer are used in combination the processed aqua sterilisas of DEPC to be supplemented to 12 μ l of volume, obtain mixed liquor；
S22, by above-mentioned mixed liquor in 65 DEG C handle 5min after, on ice immediately cool down 1min, then into mixed liquor successively plus
Enter 1 μ l of RiboLock RNase Inhibitor, the 10mM dNTP of 4 μ l of 5X reaction buffer, a concentration of 20U/ μ l
The 1 μ l of RevertAid M-MuLV Reverse Transcriptase of 2 μ l of mix, 200U/ μ l, it is of short duration by its gently mixing
After centrifugation, it is placed in 42 DEG C of incubation 1h in PCR instrument；Then 70 DEG C of incubation 5min, reaction was completed to get the first chains of cDNA；
S3, the RT-PCR detections of passionflower mosaic virus
The design of S31, primer
Special primer design is carried out using 5.0 softwares of Primer Premier：
Sense primer PafMVF：5′-CCGCTCCGCTTCCTCCTC-3′；
Downstream primer PafMVR：5′-TTGAATACACGAGCACGGCG-3′；
S32, PCR reactions are carried out in following PCR reaction systems：PCR-Grade Water 15.0μl；2X Ex taq
Buffer 25.0μl；The 1.0 μ l of dNTP Mix of 10mM；The 1.0 μ l of Ex taq enzymes of 5U/ μ l；5.0 μ l of the first chains of cDNA；10μM
PafMVF1.5 μ l；10 μM of PafMVR1.5 μ l；
S4, the PCR product of gained is carried out to electrophoretic process, voltage 120V on 1.0% Ago-Gel, electrophoresis 0.5h must contain
There is the gel of PCR fragment；
S5, the gel containing PCR fragment is cut with blade, is positioned over 1.5ml centrifuge tubes, weighs；3 times of volume gel weights are added
(mg) GSB solution, 60 DEG C of metal bath 10min；After gel is completely dissolved, the precooling isopropyl of 1/3 volume GSB solution is added
Alcohol；It after solution is cooled to room temperature, is drawn to centrifugal column with liquid-transfering gun, stands 1min, 10000rpm centrifuges 1min；It outwells in collecting pipe
Waste liquid, 650 μ l of WB solution are added, at room temperature, 10000rpm centrifuges 1min, abandons efflux；It centrifuges again, thoroughly removal residual
Solution；Centrifugal column is transferred to 1.5ml centrifuge tubes, uncaps and stands 1min；Sterilizing dd H are added dropwise among centrifugal column230 μ l of O,
Stand 1min；10000rpm centrifuges 1min, elutes PCR fragment；Recycling segment is connected into TA cloning vector pGM-T carriers, and 16 DEG C incubate
12h is educated, connection product is obtained；
S6,5 μ l connection products are taken to import escherichia coli DH5a competent cell, in the LB solid cultures of the benzyl mycin of ammonia containing 50mg/l
It is screened on base, 37 DEG C of culture 12h, picking white single bacterium colony, with M13 primers, PCR verifies its positive；Positive monoclonal in containing
The LB liquid medium of 50mg/l ammonia benzyl mycins, 37 DEG C culture 12h after be used for plasmid extraction, then use universal primer T7 and
SP6 carries out sequencing.
2. a kind of passionflower mosaic virus early detection method as described in claim 1, which is characterized in that the PCR is anti-
The program is answered to be：94 DEG C of pre-degeneration 2min, make masterplate cDNA fully be denaturalized；Subsequently into amplification cycles：94 DEG C denaturation 30s, 55 DEG C
Renaturation 30s, 72 DEG C of extension 40s, after 35 recycle, whole extension 5min makes PCR product extend complete, and 4 DEG C preserve.
3. a kind of passionflower mosaic virus early detection method as described in claim 1, which is characterized in that the step S5
In TA clone coupled reaction system include：dd H2O 6.0μl；T4 DNA Ligation 10X Buffer 1.0μl；3U/μ
The T4 DNA Ligase1.0 μ l of l；The 1.0 μ l of pGM-T carriers of 50ng/ μ l；1.0 μ l of 100ng/ μ l PCR products.
4. a kind of passionflower mosaic virus early detection method as described in claim 1, which is characterized in that in the step S6
LB solid culture primary surface coating concentrations 40mg/ml IPTG10 μ l and a concentration of 20mg/ml 40 μ l of X-Gal solution.
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| CN110358863A (en) * | 2019-06-18 | 2019-10-22 | 贵州省果树科学研究所 | A kind of primer and its application for detecting cucumber mosaic virus passionflower isolate |
| CN110951750A (en) * | 2019-12-25 | 2020-04-03 | 广西壮族自治区农业科学院 | Passion flower internal reference gene PeNADP and screening method and application thereof |
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