CN104059999B - A kind of napiform root Rhizoma Iridis Tectori Viral diagnosis primer and method - Google Patents
A kind of napiform root Rhizoma Iridis Tectori Viral diagnosis primer and method Download PDFInfo
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Abstract
The present invention, according to the light mosaic virus of Rhizoma Iridis Tectori, narcissus cryptovirus and the nucleotide sequence of three kinds of viral coat protein genes of bean virus 2, separately designs and has synthesized three to corresponding specific primer；And also disclose and utilize this three pairs of specific primers method that mosaic virus light to the Rhizoma Iridis Tectori in napiform root Iris Leaves, narcissus cryptovirus and bean virus 2 detect simultaneously.The present invention is established and a kind of just can be detected 3 kinds of napiform root Rhizoma Iridis Tectori virus light mosaic viruss of i.e. Rhizoma Iridis Tectori, narcissus cryptovirus and the detection method of bean virus 2 by primary first-order equation simultaneously, have that detection efficiency is high, the feature of required low cost, even and if the rna content of 3 kinds of napiform root Rhizoma Iridis Tectori viruses is the lowest in sample, the present invention also can detect, thus the present invention has the highest detection sensitivity.
Description
[technical field]
The present invention relates to a kind of plant virus detection method of field of biology, be specifically related to a kind of napiform root kite
Tail Viral diagnosis primer and method.
[background technology]
The light mosaic virus of Rhizoma Iridis Tectori (Iris mild mosaic virus, IMMV), narcissus cryptovirus (Narcissus
Latent virus, NLV) and bean virus 2 (Bean yellow mosaic virus, BYMV) be
Three kinds of main viruses of harm napiform root Rhizoma Iridis Tectori.
At field these three virus usually Combined Infection, cause napiform root flag flower mottle to refute, blade produce flower
Leaf moves back quality and quantity green, cut-flower and is decreased obviously, thus has a strong impact on the quality of napiform root Rhizoma Iridis Tectori.Work as kind
After ball plants several years continuously, virus runs up to a certain degree, can cause the degeneration of kind of ball, lose recycling
Value, the Combined Infection of these several viruses have become as the major limitation of napiform root Rhizoma Iridis Tectori large-scale production because of
Element.At present, napiform root Rhizoma Iridis Tectori virus there is no particularly effective chemical prevention and control method, therefore, to napiform root Rhizoma Iridis Tectori
Carry out viruses indentification, make kind of a ball rejuvenation in conjunction with Shoot-tip Culture detoxification, it appears particularly important, but multiple at kind of ball
During Zhuan, it is often necessary to it is carried out Viral diagnosis.
At present, plant virus detection uses mostly based on sero-fast Enzyme-linked Immunosorbent Assay technology (i.e. ELISA
Method), but antiserum not only price antiserum higher, a kind of can only detect, and one is viral, detection program is numerous
Trivial, but also there is the problems such as a certain degree of false positive reaction, detection sensitivity be relatively low.In recent years
Coming, in order to improve Viral diagnosis efficiency, the virus detection techniques of PCR-based technology develops rapidly,
But substance PCR detection technique, a reaction can only detect a kind of virus, if being applied to the multiple virus in field
The detection of the napiform root Rhizoma Iridis Tectori virus of Combined Infection, then also exist time-consuming, effort, problem costly.
[summary of the invention]
The technical problem to be solved is to provide a kind of napiform root Rhizoma Iridis Tectori Viral diagnosis primer and side
Method.
The present invention is to solve above-mentioned technical problem by the following technical programs: a kind of napiform root Rhizoma Iridis Tectori virus inspection
Surveying primer, it specifically includes following three primer pair:
Rhizoma Iridis Tectori light mosaic virus primer pair: forward primer 5'-GAAGGTAAAGCACCATACAT-3',
Reverse primer 5'-CGTGCTAGTGACATATCGGTAA-3'；
Narcissus cryptovirus primer pair: forward primer 5'-AATTGGTGCATTTGGTGC-3', reversely
Primer 5'-TCAAAAGCGTAGGGGATCAT-3'；
Bean virus 2 primer pair: forward primer 5'-AGACACACAGCAGGAGATGT-3',
Reverse primer 5'-GCTTACCCTGTCAGAGTAGA-3'.
Further, mosaic virus PCR light to Rhizoma Iridis Tectori is amplified by described Rhizoma Iridis Tectori light mosaic virus primer
The product of 770bp；Described narcissus cryptovirus primer amplifies 266bp to narcissus cryptovirus PCR
Product；Described bean virus 2 primer amplifies 186bp's to bean virus 2 PCR
Product.
Further, above three primer is utilized to detect to napiform root Rhizoma Iridis Tectori virus, its detection method
As follows: according to the light mosaic virus of Rhizoma Iridis Tectori, narcissus cryptovirus and bean virus 2 three kinds viral outside
The nucleotide sequence of coat protein gene separately designs and synthesizes the light mosaic disease of Rhizoma Iridis Tectori as described in the appended claim 1
Poison primer to, narcissus cryptovirus primer to and bean virus 2 primer pair；Then kite it is utilized respectively
Mosaic virus light to the Rhizoma Iridis Tectori in napiform root Iris Leaves, narcissus cryptovirus are drawn by tail light mosaic virus primer
Thing to the narcissus cryptovirus in napiform root Iris Leaves, bean virus 2 primer to napiform root Rhizoma Iridis Tectori
Bean virus 2 in blade carries out mRT-PCR reaction, finally product is carried out 1% agar
Sugar gel electrophoresis analysis and cloning and sequencing comparison.
Further, reaction system and the response procedures of described mRT-PCR reaction are as follows:
Reaction system: the cumulative volume of reaction system is 50 μ L, the Taq enzyme containing 0.3 μ L5U/ μ L, 5 μ L
10 × Taq enzyme buffer, the dNTPs of 4 μ L2.5mmol/L, 1 μ L template, 0.12-0.32 μm ol/L
Rhizoma Iridis Tectori light mosaic virus forward primer, the Rhizoma Iridis Tectori light mosaic virus reverse primer of 0.12-0.32 μm ol/L,
The narcissus cryptovirus forward primer of 0.08-0.24 μm ol/L, the narcissus cryptovirus of 0.08-0.24 μm ol/L
Reverse primer, the bean virus 2 forward primer of 0.04-0.24 μm ol/L, 0.04-0.24 μm ol/L
Bean virus 2 reverse primer, remaining composition is sterilizing distilled water；Wherein, every described primer centering
Forward primer and the concentration of reverse primer the most equal, described Rhizoma Iridis Tectori light mosaic virus forward primer, Rhizoma Iridis Tectori
Light mosaic virus reverse primer, narcissus cryptovirus forward primer, narcissus cryptovirus reverse primer, dish
Glycine max (L.) Merr. mosaic virus forward primer, bean virus 2 reverse primer concentration all with reaction system
On the basis of cumulative volume 50 μ L；
Response procedures: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 48-58 DEG C of annealing 30s, 72 DEG C
Extend 1min, circulate 35 times；72 DEG C extend 10min；4 DEG C terminate reaction.
The beneficial effects of the present invention is:
For the light mosaic virus of Rhizoma Iridis Tectori, narcissus cryptovirus and three kinds of viral shells of bean virus 2
The nucleotide sequence of protein gene, separately designs and has synthesized three to corresponding specific primer, and utilize should
The three pairs of specific primer mosaic viruss light to the Rhizoma Iridis Tectori in napiform root Iris Leaves simultaneously, narcissus cryptovirus and
Bean virus 2 carries out mRT-PCR reaction, establishes a kind of primary first-order equation of passing through and just can detect simultaneously
Go out 3 kinds of napiform root Rhizoma Iridis Tectori virus light mosaic viruss of i.e. Rhizoma Iridis Tectori, narcissus cryptovirus and bean virus 2
Detection method, has that detection efficiency is high, the feature of required low cost, though and 3 kinds of napiform root kites in sample
The rna content of tail virus is the lowest, and the present invention also can detect, thus the present invention has the highest detection
Sensitivity.
[accompanying drawing explanation]
The invention will be further described the most in conjunction with the embodiments.
Fig. 1 is 1% agarose gel electrophoresis figure of embodiment one in the present invention.
Fig. 2 is 1% agarose gel electrophoresis figure of embodiment two in the present invention.
Fig. 3 is 1% agarose gel electrophoresis figure of embodiment three in the present invention.
[detailed description of the invention]
The present invention lists following representative embodiment, and these embodiments are only exemplary, and not
For limiting the scope of the present invention, these embodiments are merely to illustrate the implementation of the present invention.
Embodiment one
The detection method of the present invention a kind of napiform root Rhizoma Iridis Tectori virus, hides according to the light mosaic virus of Rhizoma Iridis Tectori, narcissus
The conserved nucleotide sequence of virus and three kinds of viral coat protein genes of bean virus 2 separately designs
Synthesized Rhizoma Iridis Tectori light mosaic virus primer to, narcissus cryptovirus primer to and bean virus 2 primer
Right, then it is utilized respectively Rhizoma Iridis Tectori light mosaic virus primer to mosaic disease light to the Rhizoma Iridis Tectori in napiform root Iris Leaves
Poison, narcissus cryptovirus primer is to the narcissus cryptovirus in napiform root Iris Leaves, Kidney bean yellow mosaic
Poison primer, finally will be anti-to the bean virus 2 in napiform root Iris Leaves carries out mRT-PCR reaction
Product is answered to carry out 1% agarose gel electrophoresis experiment and cloning and sequencing comparison.
Wherein, each primer to particularly as follows: (1) Rhizoma Iridis Tectori light mosaic virus primer to (as SEQ ID NO:1,
Shown in 2): forward primer 5'-GAAGGTAAAGCACCATACAT-3', reverse primer 5'-
CGTGCTAGTGACATATCGGTAA-3'；(2) narcissus cryptovirus primer is to (such as SEQ ID
NO:3, shown in 4): forward primer 5'-AATTGGTGCATTTGGTGC-3', reverse primer
5'-TCAAAAGCGTAGGGGATCAT-3'；(3) bean virus 2 primer is to (such as SEQ ID
NO:5, shown in 6): forward primer 5'-AGACACACAGCAGGAGATGT-3', reverse primer
5'-GCTTACCCTGTCAGAGTAGA-3'。
It addition, applicant is for convenience of describing, below light for Rhizoma Iridis Tectori mosaic virus is referred to as IMMV, narcissus
Cryptovirus is referred to as NLV, and bean virus 2 is referred to as BYMV.The tool of detection method
Body operating procedure is as follows:
Step 100: according to the light mosaic virus of Rhizoma Iridis Tectori, narcissus cryptovirus and bean virus 2 three kinds
The coat protein gene nucleotide sequence of virus, utilizes Primer5.0 software design three to specific primer
(the most described Rhizoma Iridis Tectori light mosaic virus primer to, narcissus cryptovirus primer to and bean virus 2 draw
Thing to), then with DNAman software, three pairs of designed specific primers are corrected, it was demonstrated that three is right
Self coupling and competitive amplification is there is not between primer, bright to guarantee that above-mentioned three kinds of viruses can amplify difference
Aobvious specific fragment, and synthesized above-mentioned three pairs of specific primers by Shanghai Sheng Gong biological engineering company limited.
Step 200:RNA is extracted: will be combined by tri-kinds of viruses of IMMV, NLV and BYMV simultaneously
Napiform root Iris Leaves 0.2g, the healthy leaves 0.2g infected carries out RNA extraction respectively, and with healthy leaf
Sheet is negative control.RNA extraction employing Trizol method: 0.2g napiform root Rhizoma Iridis Tectori band virus blade or healthy leaf
Sheet is put in mortar, adds liquid nitrogen grinding powdering, is then placed in the first centrifuge tube by powdery blade,
Add 1mL Trizol acutely concussion 20～30S, then the first centrifuge tube is placed in left at room temperature 5
Min is so that blade fully cracks, then by the first centrifuge tube centrifugal 5min under 10000rpm, by upper
Move to clearly in the second centrifuge tube, in the second centrifuge tube, add 200uL chloroform, and mixing of vibrating, in room
Place 3min under temperature, then at 4 DEG C, centrifugal 15min under 10000rpm, then draw upper strata aqueous phase
To the 3rd centrifuge tube, in the 3rd centrifuge tube, add the mixing of 0.5mL isopropanol, and place at room temperature
10min, then by the 3rd centrifuge tube in 4 DEG C, centrifugal 10min under the conditions of 10000rpm, and discards
Clear liquid, then at the bottom of RNA is sunken to pipe；Add 1mL75% ethanol (DEPC process water preparation) afterwards in the
In three centrifuge tubes, the gentleest vibration the 3rd centrifuge tube, obtains suspension, then at 4 DEG C, 10000rpm condition
Under be centrifuged 5min, and abandoning supernatant, then the 3rd centrifuge tube is placed in and dries 10min on ice, finally
Water dissolution RNA sample is processed with 30uL DEPC.
Step 300: reverse transcription cDNA: the RNA reverse transcription processing gained through step 200 is become cDNA,
Particular content is: be placed in by reaction tube on ice, adds 1uL Primer Oligo (dt), 1uL dNTP mixing
Thing (10mmol/L), 4uL DEPC process in water, 4uL RNA sample to reaction tube, mix gently again
Somewhat it is centrifuged, after then reaction tube is placed in 65 DEG C of water-baths heating 5min, immediately by reaction tube
It is placed in 5min on ice, then again reaction tube is placed on ice after somewhat centrifugal for reaction tube, and adds 4uL
5 × reverse transcription buffer, the RNAase inhibitor of 0.5uL20U/uL, 1uL200U/uL reverse transcription
Enzyme, 4.5uL DEPC process water in reaction tube, and then reaction tube is placed in 42 DEG C of water-baths heating 60
Min, then reaction tube is placed in thermal shock 10min at 70 DEG C, finally terminate reaction.
Step 400:PCR expands: to process the cDNA of gained through step 300 as template, and with step
In rapid 100 obtain three pairs of primers be primer to carrying out pcr amplification reaction simultaneously, obtain PCR amplification
Product.
In order to contrast, the present embodiment has carried out substance pcr amplification reaction simultaneously and multiplex PCR expands
Increase reaction, i.e. the reaction system of pcr amplification reaction includes that substance PCR reaction system and multiplex PCR are anti-
Answer system, wherein, substance pcr amplification reaction consistent with the response procedures that multiplexed PCR amplification reacts and
Reaction system is different.Reaction system and the response procedures particular content of pcr amplification reaction are as follows:
Substance PCR reaction system: the cumulative volume of reaction system is 50 μ L, including 0.3 μ L5U/ μ L's
Taq enzyme, 10 × Taq enzyme buffer of 5 μ L, the dNTPs of 4 μ L2.5mmol/L, 1 μ L template, 1 μ L
The forward primer of 10 μm ol/L IMMV or NLV or BYMV, 1 μ L10 μm ol/L IMMV or NLV
Or the reverse primer of BYMV, remaining composition is sterilizing distilled water.
Multi-PRC reaction system: the cumulative volume of reaction system is 50 μ L, containing 0.3 μ L5U/ μ L's
Taq enzyme, 10 × Taq enzyme buffer of 5 μ L, the dNTPs of 4 μ L2.5mmol/L, 1 μ L template, 0.16 μm ol/L
IMMV forward primer, the IMMV reverse primer of 0.16 μm ol/L, the NLV of 0.12 μm ol/L
Forward primer, the NLV reverse primer of 0.12 μm ol/L, the BYMV forward primer of 0.12 μm ol/L,
The BYMV reverse primer of 0.12 μm ol/L, remaining composition is sterilizing distilled water；Wherein, described IMMV
Forward primer and IMMV reverse primer, NLV forward primer and NLV reverse primer, BYMV forward
The concentration of primer and BYMV reverse primer is all on the basis of the cumulative volume 50 μ L of reaction system.
PCR response procedures: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 30s, 54 DEG C of annealing 30s, 72 DEG C
Extend 1min, circulate 35 times；72 DEG C extend 10min；4 DEG C terminate reaction.
Step 500: taking 5 μ L PCR primer with 1% agarose gel (fluorescent dye) is that electrophoresis support is situated between
Matter, electrophoresis 30min under 0.5 × TBE and 120V voltage, then at 0.5 μ g/mL ethidium bromide staining
Dye in liquid 15min, observes with ultraviolet transilluminator, and specific experiment result is shown in Fig. 1.
The present embodiment 1% agarose gel electrophoresis experimental result is as it is shown in figure 1, the substance of IMMV primer pair
Pcr amplification reaction result is as shown in the mark 1 in Fig. 1, and the substance PCR amplification of NLV primer pair is anti-
Should result as shown in the mark 2 in Fig. 1, the substance pcr amplification reaction result of BYMV primer pair as figure
Shown in mark 3 in 1, three couple of BYMV primer pair is drawn by, NLV primer IMMV primer
Thing participate in multiplexed PCR amplification reaction result as shown in the mark 4 in Fig. 1, the substance PCR of healthy leaves
Amplified reaction result is as shown in the mark 5 in Fig. 1.As shown in Figure 1, the substance PCR of IMMV primer pair
Amplified reaction obtains the band that expanding fragment length is 770bp, and the substance PCR amplification of NLV primer pair is anti-
Should obtain the band that expanding fragment length is 266bp, the substance pcr amplification reaction of BYMV primer pair obtains
It is the band of 186bp to expanding fragment length, by IMMV primer to, NLV primer to, BYMV
The reaction of multiplexed PCR amplification that three pairs of primers are participated in by primer, can obtain simultaneously expanding fragment length 770bp,
Three bands limpid in sight of 266bp and 186bp, it is seen then that the present invention can detect the most simultaneously
Go out tri-kinds of viruses of IMMV, NLV, BYMV of napiform root Rhizoma Iridis Tectori, and specificity is good, detection efficiency is high.
Embodiment two
Pcr amplification product multi-PRC reaction system in embodiment one step 400 obtained is through cutting
Glue reclaims, and carries out Ligation in vitro, then picking positive colony with pMD18-T simple vector the most respectively,
Finally extract plasmid order-checking, to check the light mosaic virus of Rhizoma Iridis Tectori, narcissus cryptovirus and Kidney bean yellow mosaic
Three kinds of viral amplifications of poison.
And sequencing result is: the amplified production of IMMV, NLV and BYMV is by 770,266 respectively
The sequence formed with 186 nucleotide, wherein said IMMV amplified production sequence such as SEQ ID NO:
Shown in 7, described NLV amplified production sequence is as shown in SEQ ID NO:8, and described BYMV expands product
Thing sequence is as shown in SEQ ID NO:9.Analyze homology result with NCBI to show, the amplification of gained
Product Sequence SEQ ID NO:7 and the homology of the reference sequences i.e. Coat protein gene sequence of IMMV
Reach 99%, the homology of the Coat protein gene sequence of sequence SEQ ID NO:8 and reference sequences NLV
Rate reaches 98%, the Coat protein gene sequence of sequence SEQ ID NO:9 and reference sequences BYMV
Homology reaches 100%.As can be seen here, amplified production SEQ ID NO:7, the SEQ ID NO of gained:
8 and SEQ ID NO:9 sequences are respectively the portion of the coat protein gene of IMMV, NLV and BYMV
Sub-sequence, thus demonstrate the reliability of testing result of the present invention.
Additionally, the sensitivity of the detection method in order to investigate napiform root Rhizoma Iridis Tectori of the present invention virus, applicant is to reality
The RNA sample executed in example one step 200 carries out 10-1、10-2、10-3、10-4、10-5A series of gradient
Dilution, is then passed through multiplex RT-PCR amplification reaction, then carries out 1% agarose gel electrophoresis experiment, tool
Body result is as shown in Figure 2.As shown in Figure 2, tri-kinds of viruses of IMMV, NLV and BYMV are dense at sample
All remain to amplify specific band after degree dilution 100 times, it can be seen that the present invention has the highest sensitivity.
Embodiment three
Multiple RT-PCR detects the application of IMMV, NLV and BYMV simultaneously:
Step 1: sampling spot is Fujian Academy flowers research center Germplasm Resources.Randomly select 6
Individual big Tanaka has the napiform root Iris Leaves sample of Combined Infection symptom, and 6 napiform root Iris Leaves samples are equal
For 0.2g；Then each sample is carried out RNA extraction (operating the step 200 with embodiment one), and will
RNA reverse transcription becomes cDNA (operating the step 300 with embodiment one).
Step 2: with process gained cDNA as template, and with three pairs of primers of the present invention for drawing
Thing, to carrying out pcr amplification reaction simultaneously, obtains pcr amplification product.The reactant of pcr amplification reaction
System and response procedures particular content are as follows:
PCR reaction system: the cumulative volume of reaction system is 50 μ L, containing the Taq of 0.3 μ L5U/ μ L
Enzyme, 10 × Taq enzyme buffer of 5 μ L, the dNTPs of 4 μ L2.5mmol/L, 1 μ L template, 0.20 μm ol/L
IMMV forward primer, the IMMV reverse primer of 0.20 μm ol/L, 0.16 μm ol/L NLV just
To primer, the NLV reverse primer of 0.16 μm ol/L, the BYMV forward primer of 0.16 μm ol/L,
The BYMV reverse primer of 0.16 μm ol/L, remaining composition is sterilizing distilled water；PCR response procedures is
94 DEG C of denaturations 5min, 94 DEG C of degeneration 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 1min, circulate 35
Secondary；72 DEG C extend 10min；4 DEG C terminate reaction.
Step 3: taking 5 μ L PCR primer is electrophoresis supporting dielectric with 1% agarose gel (fluorescent dye),
Electrophoresis 30min under 0.5 × TBE and 120V voltage, then in 0.5 μ g/mL ethidium bromide staining liquid
Dyeing 15min, observes with ultraviolet transilluminator, and specific experiment result is shown in Fig. 3.
1% agarose gel electrophoresis of the present embodiment is tested as it is shown on figure 3, utilize multiple RT-PCR system
When detecting 6 napiform root Iris Leaves samples, each described sample all can amplify three and become clear clear
Clear specific band, illustrates that gathered sample is combined by tri-kinds of viruses of IMMV, NLV and BYMV
Infect, and specific band fragment length 770bp, 226bp and 186bp respectively that amplification obtains, with reality
Execute substance PCR testing result in example one consistent.Visible detection method can detect the most simultaneously
Go out tri-kinds of viruses of IMMV, NLV, BYMV of Rhizoma Dioscoreae esculentae, specificity is good, detection efficiency high specific is good,
Efficiency is high.
The present invention is directed to the nucleotide of tri-kinds of viral coat protein genes of IMMV, NLV and BYMV
Sequence, separately designs and has synthesized three to corresponding specific primer, and utilize these three pairs of specific primers with
Time IMMV, NLV and the BYMV in napiform root Iris Leaves is carried out mRT-PCR reaction, establish
A kind of new PCR reaction system and response procedures, make mRT-PCR reaction obtain difference in size obvious,
Length is respectively the amplification sheet of three IMMV, NLV and BYMV of 770bp, 226bp and 186bp
Duan Xulie；Present invention achieves primary first-order equation just can detect simultaneously 3 kinds of napiform root i.e. IMMV of Rhizoma Iridis Tectori virus,
The target of NLV and BYMV, thus detection efficiency of the present invention is high, agents useful for same of the present invention is that molecule is raw
Thing common agents, required low cost, even and if in sample the rna content of 3 kinds of napiform root Rhizoma Iridis Tectoris virus is very
Low, the present invention also can detect, thus the present invention has the highest detection sensitivity.
Claims (4)
1. a napiform root Rhizoma Iridis Tectori Viral diagnosis primer, it is characterised in that: specifically include following three primer pair:
Rhizoma Iridis Tectori light mosaic virus primer pair: forward primer 5'-GAAGGTAAAGCACCATACAT-3',
Reverse primer 5'-CGTGCTAGTGACATATCGGTAA-3'；
Narcissus cryptovirus primer pair: forward primer 5'-AATTGGTGCATTTGGTGC-3', reversely
Primer 5'-TCAAAAGCGTAGGGGATCAT-3'；
Bean virus 2 primer pair: forward primer 5'-AGACACACAGCAGGAGATGT-3',
Reverse primer 5'-GCTTACCCTGTCAGAGTAGA-3'.
A kind of napiform root Rhizoma Iridis Tectori Viral diagnosis primer, it is characterised in that: described
Rhizoma Iridis Tectori light mosaic virus primer amplifies the product of 770bp to mosaic virus PCR light to Rhizoma Iridis Tectori；Described water
The celestial cryptovirus primer product to narcissus cryptovirus PCR being amplified 266bp；Described Kidney bean Hemerocallis citrina Baroni
The mosaic virus primer product to bean virus 2 PCR being amplified 186bp.
3. a napiform root Rhizoma Iridis Tectori method for detecting virus, it is characterised in that: according to the light mosaic virus of Rhizoma Iridis Tectori, water
The nucleotide sequence of celestial cryptovirus and three kinds of viral coat protein genes of bean virus 2 sets respectively
Meter synthesizes the light mosaic virus primer of Rhizoma Iridis Tectori as described in the appended claim 1 to, narcissus cryptovirus primer pair
And bean virus 2 primer pair；Then Rhizoma Iridis Tectori light mosaic virus primer it is utilized respectively to napiform root Rhizoma Iridis Tectori
The light mosaic virus of Rhizoma Iridis Tectori, narcissus cryptovirus primer in blade are dived to the narcissus in napiform root Iris Leaves
Cryptovirus, bean virus 2 primer are carried out to the bean virus 2 in napiform root Iris Leaves
MRT-PCR reacts, and product finally carries out 1% agarose gel electrophoresis analysis and cloning and sequencing ratio
Right.
A kind of napiform root Rhizoma Iridis Tectori method for detecting virus, it is characterised in that: described
Reaction system and the response procedures of mRT-PCR reaction are as follows:
Reaction system: the cumulative volume of reaction system is 50 μ L, the Taq enzyme containing 0.3 μ L 5U/ μ L, 5 μ L
10 × Taq enzyme buffer, the dNTPs of 4 μ L 2.5mmol/L, 1 μ L template, 0.12-0.32 μm ol/L
Rhizoma Iridis Tectori light mosaic virus forward primer, the Rhizoma Iridis Tectori light mosaic virus reverse primer of 0.12-0.32 μm ol/L,
The narcissus cryptovirus forward primer of 0.08-0.24 μm ol/L, the narcissus cryptovirus of 0.08-0.24 μm ol/L
Reverse primer, the bean virus 2 forward primer of 0.04-0.24 μm ol/L, 0.04-0.24 μm ol/L
Bean virus 2 reverse primer, remaining composition is sterilizing distilled water；Wherein, every described primer centering
Forward primer and the concentration of reverse primer the most equal, described Rhizoma Iridis Tectori light mosaic virus forward primer, Rhizoma Iridis Tectori
Light mosaic virus reverse primer, narcissus cryptovirus forward primer, narcissus cryptovirus reverse primer, dish
Glycine max (L.) Merr. mosaic virus forward primer, bean virus 2 reverse primer concentration all with reaction system
On the basis of cumulative volume 50 μ L；
Response procedures: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 48-58 DEG C of annealing 30s, 72 DEG C
Extend 1min, circulate 35 times；72 DEG C extend 10min；4 DEG C terminate reaction.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410325043.7A CN104059999B (en) | 2014-07-09 | 2014-07-09 | A kind of napiform root Rhizoma Iridis Tectori Viral diagnosis primer and method |
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| CN102732642A (en) * | 2012-05-14 | 2012-10-17 | 福建出入境检验检疫局检验检疫技术中心 | Narcissus latent virus detection kit and detection method thereof |
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| CN102031313A (en) * | 2010-08-16 | 2011-04-27 | 深圳出入境检验检疫局动植物检验检疫技术中心 | RT-PCR (reverse transcription-polymerase chain reaction) method for detecting the infection reality of host by arabis mosaic virus, bean yellow mosaic virus and lily symptomless virus |
| CN102732642A (en) * | 2012-05-14 | 2012-10-17 | 福建出入境检验检疫局检验检疫技术中心 | Narcissus latent virus detection kit and detection method thereof |
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