CN109943666A - A kind of the multiple PCR primer group and its application of the main virus of detection tomato - Google Patents
A kind of the multiple PCR primer group and its application of the main virus of detection tomato Download PDFInfo
- Publication number
- CN109943666A CN109943666A CN201910343406.2A CN201910343406A CN109943666A CN 109943666 A CN109943666 A CN 109943666A CN 201910343406 A CN201910343406 A CN 201910343406A CN 109943666 A CN109943666 A CN 109943666A
- Authority
- CN
- China
- Prior art keywords
- primer
- virus
- seq
- tomato
- pcr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the multiple RT-PCR primer sets and its application of a kind of main virus of detection tomato.The primer sets sequence can detect that 5 kinds of tobacco mosaic virus (TMV), capsicum arteries and veins mottle virus, capsicum arteries and veins yellow virus, tomato chlorisis virus and tomato ring grain mottle virus etc. infects the main virus infection of tomato as shown in SEQ ID No.1 ~ No.10.Multiple RT-PCR detection is carried out to tomato diseased tissue using the primer sets, a variety of pathogenic virus can be quickly detected simultaneously, is had the characteristics that efficient, economical and easy.
Description
Technical field
The invention belongs to field of biological detection, further belong to plant virus detection field, and in particular to a kind of tomato master
Want the multiple PCR primer group and its application of virus.
Background technique
Tomato is that the most universal one of fruit and vegetable are cultivated in the whole world, about 8,050,000 mu of world's tomato cultivation area in 2011,
About 3 773 ten thousand t of annual output, China are one of tomato planting big country, the world, 96667 hectares of area in 2011, yield about 679
Ten thousand t, as tomato planting area constantly expands, the harm of tomato virus disease aggravates year by year.Yunnan Province's tomato virus disease is carried out
Investigation discovery, the virus causing disease for infecting tomato mainly have tobacco mosaic virus (TMV) (Tobacco mosaic virus TMV), capsicum
Arteries and veins mottle virus (Chilli veinal mottle virus, ChiVMV), capsicum arteries and veins yellow virus (Pepper vein
Yellows virus, PeVYV), tomato chlorisis virus (Tomato chlorosis virus, ToCV), tomato ring grain spot
Viral (Tomato zonate spot virus, TZSV), and these normal Combined Infections of virus are found in investigation, it directly affects
The yield and quality of tomato causes serious economic loss.And still lacks carry out multiple RT-PCR for the above virus at present
Detection technique.
Plant virus detection method common at present has electron microscopy, serological Identification and round pcr etc..
Electron microscopy is a kind of easy, sensitive viral diagnosis method.Under Electronic Speculum, the shape of virus can be intuitively observed
State, size, surface texture etc..Electronic Speculum detection has the advantages that intuitive, quick, but accuracy of detection is inadequate, can only be according to virus
Form auxiliary judgment Tobamovirus, cannot be accurate to kind.
Serological Identification principle is the association reaction based on antibody and corresponding antigens, is called immune response.Most plants
The virus protein of encoding viral has good antigenicity.As long as therefore preparing the antibody of virus, antigen-antibody can be utilized
Specific reaction judges the malicious situation of band of plant.Currently, most widely used is enzyme linked immunosorbent assay (ELISA), wherein
It is the most classical with double antibody sandwich method.Double antibody sandwich method is the antibody that antigen to be checked is added on specific supporting carrier, so
After add measuring samples, be eventually adding corresponding enzyme labelled antibody, make the sandwich complex to form antibody-antigen-antibody.Due to
The antibody (secondary antibody) being eventually adding is the antibody of enzyme label, therefore the substrate of the enzyme can be added, and makes to generate chromogenic reaction, according to aobvious
The case where colour response can judgement sample whether contain antigen.Serum detection is with easy to operate, quick, sensitivity is high, special
The features such as property is strong, the disadvantage is that sero-fast preparation time is longer；It is interfered, is easily occurred false by autoantibody, non-specific antibody etc.
It is positive；When detecting the virus of Combined Infection, sensitivity decline is identified more difficult.
PCR(Polymerase chain reaction, PCR) i.e. polymerase chain reaction is a kind of molecular biology
Technology is used for amplification in vitro specific purpose DNA sequence.With the development of science and technology, PCR technology has been widely applied
In multiple fields, which has stronger specificity, higher sensibility and accuracy.It is the virus of DNA for nucleic acid,
Directly template is done with DNA to be expanded.And for nucleic acid be the virus of RNA, then need first by mRNA reverse transcription at
CDNA, then use cDNA as template, PCR amplification is carried out, this method claims RT-PCR(Reverse transcription,
PCR).RT-PCR is a kind of quickly and effectively method for detecting RNA virus, and sensitivity and accuracy are than traditional serological side
Method is high.But the RT-PCR technology single of substance can only detect a kind of virus, when needing to carry out a variety of viral diagnosis to sample,
It needs to want a large amount of repeated work, while a large amount of detection reagent can be consumed again.
Multiple RT-PCR technology is a kind of detection technique to grow up on the basis of substance RT-PCR.Multiplex PCR be
In same reaction system, the PCR for being amplified multiple nucleic acid fragments simultaneously using one or more pairs of primers is reacted, than common substance
PCR is highly efficient, economical and easy.Not only has the advantages that substance RT-PCR technology, and multiple RT-PCR reaction once can be same
When the advantages of quickly detecting a variety of cause of diseases, be able to satisfy the requirement that production is upper while being detected to a variety of cause of diseases.
For this purpose, we research and develop the multiple PCR primer group of the main virus of tomato, the primer sets can be quickly detected from simultaneously
Infect a variety of main viruses of tomato.
Summary of the invention
The first object of the present invention is to provide a kind of multiple PCR primer group of main virus of detection tomato, and feature exists
In the main virus of the tomato includes TMV, ChiVMV, PeVYV, ToCV and TZSV, and the primer sets include:
The primer pair of TMV is detected,
Its upstream primer sequence is as shown in SEQ ID No.1: 5 '-TGAGATTTCGCTGGCGTTTG-3 ',
Primer sequence is as shown in SEQ ID No.2 downstream: 5 '-TTCGCTGCTGTCATTGGGTC-3 '；
The primer pair of ChiVMV is detected,
Its upstream primer sequence is as shown in SEQ ID No.3: 5 '-TGTGGCGTCCATTCGTCTGA-3 ',
Primer sequence is as shown in SEQ ID No.4 downstream: 5 '-ATGCCCAATCGGCGTAGTGA-3 '；
The primer pair of PeVYV is detected,
Its upstream primer sequence is as shown in SEQ ID No.5: 5 '-GCGTTGCGGAATGGATGCTA-3 ',
Primer sequence is as shown in SEQ ID No.6 downstream: 5 '-GACCCTTTGGGCGAGGAGTG-3 '；
The primer pair of ToCV is detected,
Its upstream primer sequence is as shown in SEQ ID No.7: 5 '-TGTCGCAGCCAATCACTACCT-3 ',
Primer sequence is as shown in SEQ ID No.8 downstream: 5 '-TCCTTTCTTTCTCGCCAACC-3 '；
The primer pair of TZSV is detected,
Its upstream primer sequence is as shown in SEQ ID No.9: 5 '-GGAGTCAGAGATGGATGAAAATGAT-3 ',
Primer sequence is as shown in SEQ ID No.10 downstream: 5 '-TATTGAATGGTAGCACCAGAATGGT-3 '.
The second object of the present invention is to provide a kind of method for detecting and infecting tomato virus, the method be utilize described in
Primer sets carry out multiple RT-PCR detection.
The third object of the present invention is to provide a kind of containing primer pairs one or more kinds of in the primer sets of having the right
Detection kit, the kit can be used for detecting the corresponding tomato virus of one or more kinds of primer pairs institute.
Detailed description of the invention
Fig. 1 uses individually the virus causing disease of 5 groups of primer detection diseased plants；
In figure, M. DL2000; 1. 17YV1381; 2. 17YV1365-H; 3. 17YV1385; 4. 17YV1367; 5.
17YV1372-T; 6. 17YV1377; 7. 17YV1383; 8. 17YV1370-T; 9. 17YV1382; 10.
17YV1366; 11. 17YV1372; 12 17YV1368。
The optimization of Fig. 2 Multiple RT-PCR detec-tion system；
In figure, M. DL2000; 1. 17YV1365-H; 2. 17YV1366 ;3. 17YV1367; 4. 17YV1368; 5.
17YV1377; 6.17YV1381; 7.17YV1385。
Fig. 3 detects the virus causing disease of diseased plant using multiple PCR primer group；
In figure, 1. negative controls；2. TZSV is positive；3. ToCV is positive；4. TMV is positive；5. ChiVMV is positive； 6.
PeVYV is positive；7. 18YV672；8. 18YV673； 9. 18YV674；10. 18YV675；11. 18YV676；12.
18YV677；13. 18YV680；14. 18YV681.
Specific embodiment
The present invention will be further described below with reference to the drawings, but the present invention is limited in any way, base
In present invention teach that done it is any transform or replace, all belong to the scope of protection of the present invention.
The present invention provides a kind of multiple PCR primer groups of main virus of detection tomato, wherein the tomato is mainly viral
Including TMV, ChiVMV, PeVYV, ToCV and TZSV, the primer sets include:
The primer pair of TMV is detected,
Its upstream primer sequence is as shown in SEQ ID No.1: 5 '-TGAGATTTCGCTGGCGTTTG-3 ',
Primer sequence is as shown in SEQ ID No.2 downstream: 5 '-TTCGCTGCTGTCATTGGGTC-3 '；
The primer pair of ChiVMV is detected,
Its upstream primer sequence is as shown in SEQ ID No.3: 5 '-TGTGGCGTCCATTCGTCTGA-3 ',
Primer sequence is as shown in SEQ ID No.4 downstream: 5 '-ATGCCCAATCGGCGTAGTGA-3 '；
The primer pair of PeVYV is detected,
Its upstream primer sequence is as shown in SEQ ID No.5: 5 '-GCGTTGCGGAATGGATGCTA-3 ',
Primer sequence is as shown in SEQ ID No.6 downstream: 5 '-GACCCTTTGGGCGAGGAGTG-3 '；
The primer pair of ToCV is detected,
Its upstream primer sequence is as shown in SEQ ID No.7: 5 '-TGTCGCAGCCAATCACTACCT-3 ',
Primer sequence is as shown in SEQ ID No.8 downstream: 5 '-TCCTTTCTTTCTCGCCAACC-3 '；
The primer pair of TZSV is detected,
Its upstream primer sequence is as shown in SEQ ID No.9: 5 '-GGAGTCAGAGATGGATGAAAATGAT-3 ',
Primer sequence is as shown in SEQ ID No.10 downstream: 5 '-TATTGAATGGTAGCACCAGAATGGT-3 '.
Multiple RT-PCR detection is carried out to tomato tissue of catching an illness using the primer sets, simultaneously and can quickly detect and infect
A variety of pathogenic virus of tomato.
Preferably as one kind of the invention, the reaction system and condition of reverse transcription (RT) of the present invention are as follows: with power traction
2 μ L of object (oligo (dT) Primer) 5 μ L, plant total serum IgE 4 μ L, 10 mmol/L dNTP Mixture, 65 DEG C of denaturation 5
Min, rapid 5 min of ice bath;Add 5 × PrimeScriptTM8 μ L, 40U/ μ L RNase Inhibitor of Buffer
1 μ L, PrimeScriptTM RTase (TaKaRa Takara-PrimeScriptTMRT reagent Kit) 1 μ L,
RNase Free H2O 4 μ L, 42 DEG C of 60 min, 95 DEG C of 5 min are to terminating.
Preferably as one kind of the invention, the reaction system of PCR of the present invention is 30 μ L:Premix Taq
(TaKaRaTaq2.0 plus dye of Version) each each 0.8 μ L of 0.8 μ L, ToCV or more primer of 15 μ L, TZSV or more primers,
Each each 1 μ L of 0.4 μ L, PeVYV or more primer of each 0.4 μ L, ChiVMV or more primer of TMV or more primer；CDNA 5 μ L, ddH2O
It mends to 30 μ L of total system.The response procedures of the PCR are as follows: 94 DEG C of 5min;94 DEG C of 30s, 56 DEG C of 2 min, 42 circulations;
72℃ 10min。
Primer pair in primer sets of the present invention has high specificity and sensitivity, can be used for corresponding to individually detection
Virus, can also combine and detect a variety of viruses.Therefore, it can be made using the primer sets containing in the primer sets
The detection kit of one or more kinds of primer pairs, for detecting the corresponding tomato disease of one or more kinds of primer pair institutes
Poison.
The present invention is based on multiplex PCR principle, multiple PCR primer group is designed and obtains, it can be to dye using the primer sets
Sick tomato carries out multiple RT-PCR detection, can detect a variety of pathogenic virus simultaneously, more highly efficient than regular-PCR, economical and simple
Just, it is able to satisfy the requirement that production is upper while being detected to a variety of cause of diseases.
Below in conjunction with specific embodiment, the present invention is further detailed.
The design of embodiment 1:RT-PCR specific detection primer
Virus sequence, TMV accession number: HE818416 are downloaded from ncbi database；ChiVMV accession number: KC711055；PeVYV
Accession number: KP326573；ToCV accession number: KY618797；TZSV accession number: NC_010490.Using primer5.0 software into
Row design of primers, primer sequence and amplification length are shown in Table 1.
Embodiment 2: substance RT-PCR verifying.
1. sample to be tested information.
Totally 12, the virosis symptom tomato sample of selection Yuanmou of Yunnan Province acquisition, respectively 17YV1381,17YV1365-H,
17YV1385、17YV1367、17YV1372-T、17YV1377、17YV1383、17YV1370-T、17YV1382、17YV1366、
17YV1372 and 17YV1368.Macro gene order-checking these samples the phenomenon that there are a variety of viral Combined Infections as the result is shown, because
This design specific primer carries out RT-PCR verifying analysis.
2. reverse transcription.
1 μ L random primer (oligo (dT) Primer), 5 μ L are added in 0.2 mL RNase Free centrifuge tube
RNA, 70 DEG C of denaturation 10 min, rapid 5 min of ice bath;Add 4 μ 5 × M-MLV of L Buffer, 1 μ L
10mMdNTP, 0.5 μ L RNase Inhibitor (TaKaRa), 1 μ L RTase-MLV (TaKaRa), 3.5 μ L
RNase Free H2O, 42 DEG C of 60min, 70 DEG C of 15 min.
3. PCR condition system.
PCR system is 30 μ L:Premix Taq(TaKaRa Taq 2.0 plus dye of Version) 15 μ L, upper,
Downstream primer (10 μm of ol/L) each 0.75 μ L, cDNA 5 μ L, ddH2O 8.5 μL.PCR program are as follows: 94 DEG C 5
min;94 DEG C of 30s, 56 DEG C of 45s, 30 circulations; 72℃ 10min.Take 6 2% Ago-Gels of μ L PCR product
Electrophoresis detection is carried out, is observed in gel imager.As a result this 5 pairs of primer pairs can be in tested sample as shown in Figure 1:
In amplify target fragment of corresponding size, and without non-specific background's band.
4. being detected sample virus sequence verification.
It after the target fragment that RT-PCR is amplified is tapped and recovered, is connected into PMD18-T cloning vector (TaKaRa), conversion is big
Positive colony, which is selected, using the method for bacterium colony PCR after enterobacteria carries out sequencing analysis (holding up section's biology).Sequencing result shows substance
The target fragment that PCR amplification goes out is correlated virus sequence.Confirm that this 5 pairs of primers can be used for the RT-PCR inspection of tomato correlated virus
It surveys, testing result is shown in Table 2 statistics.
Embodiment 3: multiple RT-PCR detection architecture optimization.
1. sample to be tested information.
It chooses in embodiment 2 and is identified through substance RT-PCR, and determine 7 samples of virus kind through sequencing analysis
17YV1365-H, 17YV1366,17YV1367,17YV1368,17YV1377,17YV1381 and 17YV1385 are as multiple RT-
The sample of PCR detection.
2. reverse transcription system optimizes.
The synthesis of cDNA used in multiplex PCR: 5 μ L random primer (oligo dT are added in 0.2 mL centrifuge tube
Primer), 4 μ L, 2 μ L dNTP Mixture (10 mmol/L each), 65 DEG C of 5 min of denaturation of total serum IgE, rapidly
5 min of ice bath;Add 8 μ 5 × Prime of L ScriptTMBuffer, 1 μ L RNase Inhibitor (40U/ μ
L), 1 μ L Prime ScriptTMRTase, 4 μ L RNase Free H2O, 42 DEG C of 60 min, 95 DEG C of 5 min.
3.PCR system optimization.
According to the band brightness adjustment primer concentration of each virus, the concentration of each primer pair is respectively as follows: TZSV 0.8 after optimization
0.8 0.4 0.4 1 μ L of μ L, PeVYV of μ L, ChiVMV of μ L, TMV of μ L, ToCV.
The final system of multiplex PCR is 30 μ L:Premix Taq(TaKaRa Taq Version, 2.0 plus dye)
0.8 0.8 0.4 0.4 1 μ L of μ L, PeVYV of μ L, ChiVMV of μ L, TMV of μ L, ToCV of 15 μ L, upstream and downstream primer TZSV；cDNA
5 μ L, ddH2O mends 20 μ L of total system.PCR program are as follows: 94 DEG C of 5min;94 DEG C of 30s, 56 DEG C of 2 min, 42 circulations;
72℃ 10min.6 μ L PCR products are taken to carry out electrophoresis detection with 2% Ago-Gel, testing result is shown in Fig. 2, as a result counts
It is shown in Table 3.
Multiplex PCR sample 17YV1365-H, 17YV1366 detected, 17YV1367,17YV1368,17YV1377,
17YV1381 and 17YV1385 is consistent with substance PCR test sample, can find from the result of table 1,2 statistics: multiplex PCR detection knot
Fruit is consistent with substance PCR testing result, illustrates that multi-PCR detection method is reliable, can be used for the detection of tomato virus.
Embodiment 4: multiple RT-PCR detects the main virus applications example of tomato.
1. plant sample Total RNAs extraction.
Choose tool virosis symptom 8 tomato samples: 18YV672,18YV673,18YV674,18YV675,
18YV676,18YV677,18YV680 and 18YV681 press the full formula gene of Tripure() reagent specification is from tomato leaf or fruit
Total serum IgE is extracted in reality, precipitating is dissolved in 30 μ L without in RNase water, and -80 DEG C save backup.
2. reverse transcription.
It is added in 0.2 mL centrifuge tube 5 μ L random primers (oligo (dT) Primer), 4 μ L of plant total serum IgE,
2 μ L dNTP Mixture (10 mmol/L each), 65 DEG C of denaturation 5 min, rapid 5 min of ice bath;Add 8 μ
L 5 × PrimeScriptTM Buffer, 1 μ L RNase Inhibitor (40U/ μ L), 1 μ L PrimeScriptTM
RTase, 4 μ L RNase Free H2O, 42 DEG C of 60 min, 95 DEG C of 5 min.
3. multiplex PCR.
30 μ L:Premix Taq(TaKaRa Taq Version, 2.0 plus dye) 15 μ L, upstream and downstream primer
0.8 0.8 0.4 0.4 1 μ L of μ L, PeVYV of μ L, ChiVMV of μ L, TMV of μ L, ToCV of TZSV；CDNA 5 μ L, ddH2O are mended to total
30 μ L of system.PCR program are as follows: 94 DEG C of 5min;94 DEG C of 30s, 56 DEG C of 2 min, 42 circulations; 72℃ 10min.
4. positive control and negative control.
There is the plasmid of viral target fragment, feminine gender is healthy tomato sample.Positive and negative RT-PCR amplification method
It is consistent with the above method.
5. Ago-Gel detects.
6 μ L PCR products are taken to carry out electrophoresis detection with 2% Ago-Gel, loading Marker is 500bp.In gel
It is observed on imager.
6. Analysis of test results.
Testing result is as shown in Figure 3: negative control can amplify the item of purpose size without purpose band, positive control
Band, and measuring samples can also amplify the purpose band of one or more Yu positive control same size.
In conclusion using the primer sets multiple RT-PCR detection can be carried out to the tomato that catches an illness, can detect simultaneously
A variety of pathogenic virus, it is more highly efficient than regular-PCR, economical and easy, it is able to satisfy production above while a variety of cause of diseases is detected
Requirement.
SEQUENCE LISTING
<110>KUNMING INST OF BOTANY CAS
<120>a kind of the multiple PCR primer group and its application of the main virus of detection tomato
<130> 2019
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>artificial synthesized
<400> 1
tgagatttcg ctggcgtttg 20
<210> 2
<211> 20
<212> DNA
<213>artificial synthesized
<400> 2
ttcgctgctg tcattgggtc 20
<210> 3
<211> 20
<212> DNA
<213>artificial synthesized
<400> 3
tgtggcgtcc attcgtctga 20
<210> 4
<211> 20
<212> DNA
<213>artificial synthesized
<400> 4
atgcccaatc ggcgtagtga 20
<210> 5
<211> 20
<212> DNA
<213>artificial synthesized
<400> 5
gcgttgcgga atggatgcta 20
<210> 6
<211> 20
<212> DNA
<213>artificial synthesized
<400> 6
gaccctttgg gcgaggagtg 20
<210> 7
<211> 21
<212> DNA
<213>artificial synthesized
<400> 7
tgtcgcagcc aatcactacc t 21
<210> 8
<211> 20
<212> DNA
<213>artificial synthesized
<400> 8
tcctttcttt ctcgccaacc 20
<210> 9
<211> 25
<212> DNA
<213>artificial synthesized
<400> 9
ggagtcagag atggatgaaa atgat 25
<210> 10
<211> 25
<212> DNA
<213>artificial synthesized
<400> 10
tattgaatgg tagcaccaga atggt 25
Claims (6)
1. a kind of multiple RT-PCR primer sets of the main virus of detection tomato, which is characterized in that the main virus packet of the tomato
TMV, ChiVMV, PeVYV, ToCV and TZSV are included, the primer sets include:
The primer pair of TMV is detected,
Its upstream primer sequence is as shown in SEQ ID No.1: 5 '-TGAGATTTCGCTGGCGTTTG-3 ',
Primer sequence is as shown in SEQ ID No.2 downstream: 5 '-TTCGCTGCTGTCATTGGGTC-3 '；
The primer pair of ChiVMV is detected,
Its upstream primer sequence is as shown in SEQ ID No.3: 5 '-TGTGGCGTCCATTCGTCTGA-3 ',
Primer sequence is as shown in SEQ ID No.4 downstream: 5 '-ATGCCCAATCGGCGTAGTGA-3 '；
The primer pair of PeVYV is detected,
Its upstream primer sequence is as shown in SEQ ID No.5: 5 '-GCGTTGCGGAATGGATGCTA-3 ',
Primer sequence is as shown in SEQ ID No.6 downstream: 5 '-GACCCTTTGGGCGAGGAGTG-3 '；
The primer pair of ToCV is detected,
Its upstream primer sequence is as shown in SEQ ID No.7: 5 '-TGTCGCAGCCAATCACTACCT-3 ',
Primer sequence is as shown in SEQ ID No.8 downstream: 5 '-TCCTTTCTTTCTCGCCAACC-3 '；
The primer pair of TZSV is detected,
Its upstream primer sequence is as shown in SEQ ID No.9: 5 '-GGAGTCAGAGATGGATGAAAATGAT-3 ',
Primer sequence is as shown in SEQ ID No.10 downstream: 5 '-TATTGAATGGTAGCACCAGAATGGT-3 '.
2. a kind of detect the method for infecting tomato virus, which is characterized in that carried out using primer sets described in claim 1 multiple
RT-PCR detection.
3. according to claim 2 detect infects the method for tomato virus, which is characterized in that the reaction system of the RT and
Condition are as follows: random primer [oligo (dT) Primer] 5 μ L, plant total serum IgE 4 μ L, 10 mmol/L dNTP Mixture
2 μ L, 65 DEG C of denaturation 5 min, rapid 5 min of ice bath;Add 5 × PrimeScriptTM8 μ L, 40U/ μ L of Buffer
RNase Inhibitor 1 μ L, PrimeScriptTM1 μ L, RNase Free H of RTase2O 4 μ L, 42 DEG C of 60 min,
95 DEG C of 5 min is to terminating.
4. according to claim 2 detect the method for infecting tomato virus, which is characterized in that the reaction system of PCR is 30
μ L:Premix Taq 15 each each 0.4 μ of 0.8 μ L, TMV or more primer of each 0.8 μ L, ToCV or more primer of μ L, TZSV or more primer
Each each 1 μ L of 0.4 μ L, PeVYV or more primer of primer above or below L, ChiVMV；CDNA 5 μ L, ddH2O is mended to 30 μ L of total system.
5. according to claim 2 detect the method for infecting tomato virus, which is characterized in that the response procedures of the PCR
Are as follows: 94 DEG C of 5min;94 DEG C of 30s, 56 DEG C of 2 min, 42 circulations; 72℃ 10min.
6. a kind of detection kit containing primer pairs one or more kinds of in primer sets described in claim 1, feature exist
In the kit can be used for detecting the corresponding tomato virus of one or more kinds of primer pair institutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910343406.2A CN109943666B (en) | 2019-04-26 | 2019-04-26 | Multiplex PCR primer group for detecting main tomato viruses and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910343406.2A CN109943666B (en) | 2019-04-26 | 2019-04-26 | Multiplex PCR primer group for detecting main tomato viruses and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109943666A true CN109943666A (en) | 2019-06-28 |
| CN109943666B CN109943666B (en) | 2022-07-12 |
Family
ID=67016374
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910343406.2A Active CN109943666B (en) | 2019-04-26 | 2019-04-26 | Multiplex PCR primer group for detecting main tomato viruses and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109943666B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363856A (en) * | 2020-05-07 | 2020-07-03 | 山东农业大学 | Method for simultaneously detecting four tomato viruses by multiple RT-PCR |
| CN113481329A (en) * | 2021-07-23 | 2021-10-08 | 云南省农业科学院生物技术与种质资源研究所 | Method for synchronously detecting five important virus pathogens in tomato |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484570A (en) * | 2013-09-28 | 2014-01-01 | 甘肃出入境检验检疫局检验检疫综合技术中心 | Tomato seed-borne pathogen multiple PCR detection kit and tomato seed-borne pathogen multiple PCR detection method |
| CN105087567A (en) * | 2015-08-28 | 2015-11-25 | 青岛农业大学 | Duplex PCR (polymerase chain reaction) primer and method for identifying tomato chlorosis viruses and tomato yellow leaf curl viruses |
-
2019
- 2019-04-26 CN CN201910343406.2A patent/CN109943666B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484570A (en) * | 2013-09-28 | 2014-01-01 | 甘肃出入境检验检疫局检验检疫综合技术中心 | Tomato seed-borne pathogen multiple PCR detection kit and tomato seed-borne pathogen multiple PCR detection method |
| CN105087567A (en) * | 2015-08-28 | 2015-11-25 | 青岛农业大学 | Duplex PCR (polymerase chain reaction) primer and method for identifying tomato chlorosis viruses and tomato yellow leaf curl viruses |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363856A (en) * | 2020-05-07 | 2020-07-03 | 山东农业大学 | Method for simultaneously detecting four tomato viruses by multiple RT-PCR |
| CN111363856B (en) * | 2020-05-07 | 2021-11-23 | 山东农业大学 | Method for simultaneously detecting four tomato viruses by multiple RT-PCR |
| CN113481329A (en) * | 2021-07-23 | 2021-10-08 | 云南省农业科学院生物技术与种质资源研究所 | Method for synchronously detecting five important virus pathogens in tomato |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109943666B (en) | 2022-07-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103667525B (en) | Fast detection kit and method of strawberry mottle virus | |
| CN109913565B (en) | Kit, primer pair, probe and method for detecting vibrio parahaemolyticus | |
| CN110863058A (en) | RPA primer for identifying potato rot stem nematode and application thereof | |
| Asano et al. | Simultaneous detection of Tomato spotted wilt virus, Dahlia mosaic virus and Chrysanthemum stunt viroid by multiplex RT‐PCR in dahlias and their distribution in Japanese dahlias | |
| Nabi et al. | Association of Apple necrotic mosaic virus (ApNMV) with mosaic disease in commercially grown cultivars of apple (Malus domestica Borkh) in India | |
| Watpade et al. | Simultaneous detection of Apple Chlorotic Leaf Spot Virus and Apple mosaic virus in crab apples and apple rootstocks by duplex RT-PCR | |
| CN109943666A (en) | A kind of the multiple PCR primer group and its application of the main virus of detection tomato | |
| CN107142334A (en) | The viruliferous RT LAMP detection methods of mulberry arteries and veins and its primer sets, kit and application | |
| KR101301865B1 (en) | Primer set for detecting pathogenic bacteria and pathogenic virus in Solanaceae, and method for detecting pathogens thereof using the same | |
| CN107805675A (en) | A kind of Potato viruses detection kit | |
| Fidan et al. | Occurrence of Alfalfa Mosaic Virus (AMV) diseases on potato crops in northern Cyprus | |
| CN104195269B (en) | A kind of method detecting tomato spotted wilf virus | |
| Yu et al. | A one‐step multiplex RT‐PCR assay for simultaneous detection of four viruses that infect peach | |
| Guček et al. | One-step multiplex RT-PCR for simultaneous detection of four viroids from hop (Humulus lupulus L.) | |
| CN103498010B (en) | Primer and kit for rapidly detecting tomato chlorosis virus (ToCV) and application thereof | |
| CN103710463B (en) | Rapid detection kit and method of strawberry mild yellow edge virus | |
| Khurana et al. | Recent developments towards detection and diagnosis for management of plant viruses | |
| CN102134603B (en) | Primer, probe, test kit and method for testing genetically modified rice or products thereof | |
| CN116377118A (en) | Multiplex PCR detection method for three pathogenic bacteria of Solanaceae and application thereof | |
| CN102796825A (en) | Specificity polymerase chain reaction (PCR) method for detecting heterodera elachista ohshima | |
| CN103194544A (en) | Reagent assisting to identify tobacco ringspot virus and application thereof | |
| CN116083654A (en) | Double PCR detection method for type 2 and type 3 herpesviruses of carp | |
| CN106521038B (en) | A kind of real-time fluorescence quantitative PCR detection methods of highly sensitive BHV 2 and kit | |
| Alvarez-Díaz et al. | Duplex RT-PCR assay for simultaneous detection of TSWV and CSVd in chrysanthemum | |
| CN111662991A (en) | Primer, probe and method for detecting soybean nematodes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zheng Kuanyu Inventor after: Zhang Zhongkai Inventor after: Wei Jianli Inventor after: Zhang Jie Inventor after: Zheng Xue Inventor after: Zhao Lihua Inventor after: Zhao Liling Inventor before: Zhang Zhongkai Inventor before: Zheng Kuanyu Inventor before: Wei Jianli Inventor before: Zhang Jie Inventor before: Zheng Xue Inventor before: Zhao Lihua Inventor before: Zhao Liling |
|
| CB03 | Change of inventor or designer information | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230927 Address after: 651300 Modern Seed Industry Science and Technology Park on the Extension Line of Faxiang North Road, Yuanma Town, Yuanmou County, Chuxiong Yi Autonomous Prefecture, Yunnan Province Patentee after: YUNNAN SINONG VEGETABLES SEED Co.,Ltd. Address before: No.9 Xueyun Road, Wuhua District, Kunming, Yunnan, 650223 Patentee before: BIOTECHNOLOGY AND GERMPLASM RESOURCE INSTITUTE, YUNNAN AGRICULTURAL ACADEMY |
|
| TR01 | Transfer of patent right |