CN117512220A - Detection method for strawberry whitening related viruses and application thereof - Google Patents
Detection method for strawberry whitening related viruses and application thereof Download PDFInfo
- Publication number
- CN117512220A CN117512220A CN202311664654.XA CN202311664654A CN117512220A CN 117512220 A CN117512220 A CN 117512220A CN 202311664654 A CN202311664654 A CN 202311664654A CN 117512220 A CN117512220 A CN 117512220A
- Authority
- CN
- China
- Prior art keywords
- spav
- strawberry
- whitening
- primer
- reaction
- 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.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 59
- 241000700605 Viruses Species 0.000 title claims abstract description 49
- 235000016623 Fragaria vesca Nutrition 0.000 title claims abstract description 35
- 235000011363 Fragaria x ananassa Nutrition 0.000 title claims abstract description 35
- 230000002087 whitening effect Effects 0.000 title claims abstract description 24
- 240000009088 Fragaria x ananassa Species 0.000 title abstract 2
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000007397 LAMP assay Methods 0.000 claims abstract description 22
- 238000010839 reverse transcription Methods 0.000 claims abstract description 16
- 238000006243 chemical reaction Methods 0.000 claims description 50
- 241000220223 Fragaria Species 0.000 claims description 36
- 230000003321 amplification Effects 0.000 claims description 25
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 25
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 claims description 16
- 102100034343 Integrase Human genes 0.000 claims description 9
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 9
- 239000000523 sample Substances 0.000 claims description 8
- 238000011901 isothermal amplification Methods 0.000 claims description 6
- 230000003612 virological effect Effects 0.000 claims description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 4
- 239000012472 biological sample Substances 0.000 claims description 4
- 239000007850 fluorescent dye Substances 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 238000004040 coloring Methods 0.000 claims description 2
- 238000003384 imaging method Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims 1
- 201000010099 disease Diseases 0.000 abstract description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- 238000011835 investigation Methods 0.000 abstract 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 22
- 239000000047 product Substances 0.000 description 12
- 238000003757 reverse transcription PCR Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 9
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 8
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 description 8
- 229960003237 betaine Drugs 0.000 description 8
- 239000007795 chemical reaction product Substances 0.000 description 8
- 238000011282 treatment Methods 0.000 description 8
- 238000001962 electrophoresis Methods 0.000 description 7
- 230000035484 reaction time Effects 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 230000000007 visual effect Effects 0.000 description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 241000710036 Strawberry mild yellow edge virus Species 0.000 description 3
- 241000413494 Strawberry mottle virus Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000011895 specific detection Methods 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 239000013614 RNA sample Substances 0.000 description 2
- 241000218632 Strawberry vein banding virus Species 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000002480 mineral oil Substances 0.000 description 2
- 235000010446 mineral oil Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 235000021012 strawberries Nutrition 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 206010001557 Albinism Diseases 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 241000948223 Octomys Species 0.000 description 1
- 108020005089 Plant RNA Proteins 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 101150023114 RNA1 gene Proteins 0.000 description 1
- 101150084101 RNA2 gene Proteins 0.000 description 1
- 101100353432 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) PRP2 gene Proteins 0.000 description 1
- 241000191471 Strawberry crinkle cytorhabdovirus Species 0.000 description 1
- 241000317669 Strawberry pallidosis-associated virus Species 0.000 description 1
- 241000018137 Trialeurodes vaporariorum Species 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000008094 contradictory effect Effects 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a detection method of strawberry whitening related viruses and application thereof. The reverse transcription loop-mediated isothermal amplification (reverse transcription loop-mediated isothermal amplification, RT-LAMP) detection system constructed in the invention can simply, conveniently, rapidly and sensitively detect SPaV without other expensive instruments in the detection process, and is particularly suitable for basic-level personnel to use for identification of detoxified seedlings and investigation of field virus diseases.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a detection method of strawberry whitening related viruses and application thereof.
Background
The strawberry albino related virus (Strawberry pallidosis-associated virus, SPaV) belongs to the genus Maofaciviridae, and can cause strawberry albinism, especially after complex infection with strawberry mild yellow edge virus and strawberry mottle virus, the symptoms of viral diseases can be aggravated. SPaV has a double-segmented positive single-stranded RNA genome in which RNA1 and RNA2 are 8067nt and 7979nt long, respectively. Grafting and insect mediator the greenhouse trialeurodes vaporariorum can transmit SPaV. In 2017, the existence of the virus SPaV is reported for the first time in the domestic Fujian area, and the virus SPaV is also found in strawberries in the south of the river in 2018. It can be seen that the virus SPaV presents an expanding situation in China, which threatens the healthy growth of strawberries.
Japanese scholars Notomi proposed loop-mediated isothermal amplification (LAMP) technology in 2000, and has the advantages of strong specificity, simple operation, high amplification efficiency and the like. Reverse transcription loop-mediated isothermal amplification (reverse transcription loop-mediated isothermal amplification, RT-LAMP) based on LAMP technology is a one-step amplification of RNA templates by adding reverse transcriptase to the reaction system. The technology is widely applied to detection of other viruses, and at present, no report of SPaV detection by RT-LAMP technology is found. The detection means of the virus SPaV are mainly a traditional RT-PCR method, a high-throughput sequencing method and a PCR-based improved method. These methods are time consuming, expensive instruments and often cannot be visually determined by the naked eye. Therefore, a detection method which is simple and convenient to develop and operate, short in time consumption and capable of being intuitively judged by naked eyes is necessary.
Disclosure of Invention
The invention mainly aims to provide a detection method and application of strawberry whitening related viruses, and aims to provide an RT-LAMP detection method of strawberry whitening related viruses, which has the advantages of strong specificity, high sensitivity, simple operation, low instrument cost and visual identification result.
In order to achieve the above purpose, the invention provides a detection method of strawberry whitening-related viruses, which uses a coat protein coding gene of the strawberry whitening-related viruses as a target sequence to design a primer, and uses the primer to carry out reverse transcription loop-mediated isothermal amplification to identify the strawberry whitening-related viruses.
Optionally, the primers include SPaV-F3 primer, SPaV-B3 primer, SPaV-FIP primer, SPaV-BIP primer and SPaV-LB primer, and the specific sequences are as follows:
SPaV-F3：AGGTTTTTCAGGTTTAGGGA；
SPaV-B3：GGTCCGGAGATAAAATCAGT；
SPaV-FIP：ATGTTCAAAACTTGCCCGCACAGTGACCTTCTTGGCTTT A；
SPaV-BIP：GTTTCCGAGTCTCAAAAACCGCAAAGACCTTCCAACTAT GGT；
SPaV-LB：AACCCCATCTATGAAGATAGACACA。
optionally, the detection method includes the steps of:
s1, extracting total RNA of a biological sample to be detected;
s2, using the total RNA as a template, and carrying out reverse transcription loop-mediated isothermal amplification by using the primer as claimed in claim 2 to obtain an amplification product;
and S3, identifying whether the biological sample to be detected contains the strawberry whitening related virus or is infected with the strawberry whitening related virus according to the amplification product.
Optionally, in step S3, the authentication is performed by the method described in 1) or 2) below:
1) Adding 0.3 mu L of fluorescent dye SYBR Green I into the amplification product, directly observing color change by naked eyes, and coloring a sample containing the virus SPaV into Green, wherein the sample without the virus SPaV is orange;
2) Conventional agarose gel electrophoresis and ultraviolet imaging observations, samples containing viral SPaV can form waterfall-type bands.
Alternatively, the reverse transcription loop-mediated isothermal amplification employs a reaction system of 2.5. Mu.L 10× Isothermal Amplification Buffer, 6mmol/L Mg 2+ 1mmol/L dNTPs,1. Mu. Mol/L SPaV-FIP and SPaV-BIP, 0.1. Mu. Mol/L SPaV-F3 and SPaV-B3, 0.3. Mu. Mol/L SPaV-LB,0.4mmol/L Betaine, 0.256U/. Mu.L WarmStart Bst2.0 DNAPolymerase, 0.12U/. Mu. L WarmStart RTx Reverse Transcriptase, 1. Mu.L template RNA, ddH was added 2 O makes up 25. Mu.L.
Optionally, the reverse transcription loop-mediated isothermal amplification adopts a reaction program of 61 ℃ for 40min;80 ℃ for 20min.
The invention also provides application of the detection method of the strawberry whitening-associated virus in preparation of a kit for detecting the strawberry whitening-associated virus.
The invention has the beneficial effects that:
(1) According to the technical scheme provided by the invention, the detection method for the strawberry whitening related virus shortens the detection time, and has the advantages of simplicity in operation, strong specificity, high sensitivity, low instrument cost and visual identification result.
(2) The invention provides a visual rapid detection RT-LAMP amplification method for virus SPaV, which can finish amplification detection within 1h, is simple to operate, can directly observe an identification result with naked eyes after being dyed by fluorescent dye SYBR Green I, and can be applied to a strawberry whitening related virus kit in view of the intuitiveness of the detection method, thereby being convenient and rapid.
(3) According to the invention, the reaction conditions of the conventional RT-LAMP are optimized, and the amplification reaction is carried out under the optimized conditions, so that the reaction result can be obtained rapidly, and the raw material cost is saved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the detection of amplified products at each reaction temperature of SPaV RT-LAMP amplification in example 2 of the present invention (M: DNA Marker TL-2000, NTC is a negative control without addition of RNA template);
FIG. 2 is a graph showing the detection of different reaction times of SPaV RT-LAMP amplification in example 2 of the present invention;
FIG. 3 is an amplification plot of primers SPaV-FIP/BIP at different concentrations in SPaV RT-LAMP in example 2 of the present invention;
FIG. 4 is an amplification plot of primers SPaV-F3/B3 at different concentrations in SPaV RT-LAMP in example 2 of the present invention;
FIG. 5 is an amplification plot of primers SPaV-LB in SPaV RT-LAMP in example 2 of the present invention at different concentrations;
FIG. 6 is an amplification plot of the SPaV RT-LAMP of example 2 of the present invention at different final concentrations of Mg2+;
FIG. 7 is an amplification plot of different final concentrations of dNTPs in SPaV RT-LAMP in example 2 of the present invention;
FIG. 8 is an amplification plot of the different final concentrations of Betaine in SPaV RT-LAMP in example 2 of the invention;
FIG. 9 is an amplification plot of reverse transcriptase at various final concentrations in SPaV RT-LAMP in example 2 of the present invention;
FIG. 10 is an amplification plot of the SPaV RT-LAMP of example 2 of the present invention at different final concentrations of Bst 2.0;
FIG. 11 is a diagram showing the specific detection of the SPaV RT-LAMP optimization system in example 3 of the present invention (M: DNA Marker TL-2000;1: no viral RNA sample; 2-6: plant RNA samples infected with viruses SPaV, SVBV, SMoV, SMYEV, SCrV3 and SCrV4 alone or in combination, wherein only sample 3 contains SPaV virus);
FIG. 12 is a sensitivity detection chart of the RT-PCR and RT-LAMP detection methods of SPaV in example 4 of the present invention;
FIG. 13 is a test chart of SPaV RT-LAMP field application in example 5 of the present invention.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention. The techniques and methods in the following examples are conventional unless otherwise specified. The reagents and apparatus are conventional products commercially available unless otherwise specified.
The main reagent comprises: the plant total RNA small extraction kit and the agarose gel DNA recovery kit are purchased from Guangzhou Meiyi Biotechnology Co., ltd; cDNA synthesis kit, DNA Marker, DH 5. Alpha. E.coli competence were purchased from Tu Lu Kong biotechnology Co., ltd; 2 XEs Taq MasterMix for PCR amplification was purchased from Kangji Biotechnology Co., ltd; 10× Isothermal Amplification Buffer, bst2.0 WarmStart DNA Polymerase, warmStart RTx Reverse Transcriptase, mgSO4 from New England BioLabs (NEB, beijing); betaine is available from Shanghai Seiyaka Biotechnology Co., ltd; dNTPs and SYBR Green I dyes were purchased from Beijing Soy Bao technology Co., ltd; the TA cloning vector pMD19 and DNA ligation kit were purchased from Takara Bio-engineering (Dalian) Inc.
Example 1 acquisition of primers for detection of viral SPaV
The virus SPaV detected by high-throughput sequencing in the early stage of a laboratory and SPaV sequences (accession numbers are OK04297.1, MZ328109.1, OP839188.1, NC_005896.2 and MN747002.1 respectively) published by GenBank are subjected to homology analysis by DNAMAN, a conserved region in the sequence is used as a template, 5 specific primers are designed for RT-LAMP detection by using NEB company on-line LAMP primer design software (https:// LAMP. NEB. Com/# | /), and the primer sequences are synthesized by Wuhan Octomy biotechnology Co., ltd.
TABLE 1 viral SPaV detection primer set
Example 2 optimization of the detection method
The invention optimizes each condition of the RT-LAMP detection method, and the used samples are taken from the strawberry planting base in the southern lake of Wuhan city. Taking 0.1g of fresh strawberry leaves, extracting total RNA of a sample by adopting a plant total RNA small extraction kit, carrying out reverse transcription, carrying out PCR amplification detection by using specific detection primers SPaVF (TTCGCCCT CGGATAAACTCACT) and SPaVR (CTTTGGTTCGGAAATGATGGTG), and preserving the total RNA of the sample containing SPaV at-80 ℃ for later use.
The specific method for optimizing the RT-LAMP reaction conditions is as follows:
(1) Initial reaction system
The basic detection system was 2.5. Mu.L 10X Isothermal Amplification Buffer,8mM MgSO 4 1.4mmol/L dNTPs, 1.6. Mu. Mol/L SPaV-FIP and SPaV-BIP, 0.2. Mu. Mol/L SPaV-F3 and SPaV-B3, 0.4. Mu. Mol/L SPaV-LB,0.4mol/L betaine, 1. Mu.L Bst2.0 WarmStart DNA Polymerase (8U/. Mu.L), 0.2. Mu. L WarmStart RTx Reverse Transcriptase (15U/. Mu.L), 1. Mu.L template RNA, ddH added 2 O was made up to 25. Mu.L and finally 20. Mu.L of sterile mineral oil was added to the reaction mixture to cover it to prevent aerosol contamination during the reaction and open-cover detection. In addition, 0.3. Mu.L of SYBR Green I dye was added to the inner wall of the tube cap prior to the reaction for post-reaction staining observation. The reaction procedure was followed by incubation at 63℃for 60min and treatment at 80℃for 20min.
(2) Selection of optimum temperature
RNA containing SPaV is used as a template, and the reaction temperature is optimized according to the reaction system. A total of 7 reaction temperatures of 60, 61, 62, 63, 64, 65, 66℃were set. The reaction is that after incubation for 60min at different temperatures, the reaction is treated for 20min at 80 ℃. The same reaction temperature is set with ddH 2 O replaces the negative control of the template RNA.
After the reaction, the fluorescent dye SYBR Green I is mixed into the reaction liquid by centrifugation, and the color change is observed; meanwhile, 0.5. Mu.L of the reaction solution was taken and subjected to agarose gel electrophoresis. The results show that waterfall type bands can be formed at 61-65 ℃, and the corresponding reaction products become Green after being dyed by SYBR Green I, and have no obvious difference (see figure 1), and the 61 ℃ is determined to be the isothermal amplification temperature.
(3) Selection of optimal reaction time
And (3) selecting 61 ℃ as a reaction temperature, setting 10, 20, 30, 40, 50 and 60min for 6 different reaction times, and otherwise, performing the same steps (2). The detection result shows that the products have waterfall-shaped strips within 30-60min, when the reaction time is 40min, the electrophoresis strips of the products are clear and obvious Green (see figure 2) is formed after SYBR Green I is dyed, and the optimal reaction time is determined to be 40min.
(4) Optimal primer concentration selection
The primer SPaV-FIP/BIP was subjected to 5 different treatments such as 1.0, 1.2, 1.4, 1.6 and 1.8. Mu. Mol/L, and the reaction was carried out at 61℃for 40min and then at 80℃for 20min. The detection results show that the reaction products can generate waterfall type strips at different concentrations, are obvious Green after being dyed by SYBR Green I, and have no obvious difference (see figure 3), so that the reaction concentration of SPaV-FIP/BIP is 1.0 mu mol/L.
The primer SPaV-F3/B3 is provided with 5 reaction concentrations of 0.1, 0.15, 0.2, 0.25, 0.3 mu mol/L and the like, after the reaction, the detection result shows that the reaction products can generate waterfall type strips at different concentrations, and the reaction products are obvious Green after being dyed by SYBR Green I without obvious difference (see figure 4), so that the reaction concentration of SPaV-F3/B3 is selected to be 0.1 mu mol/L.
The loop primer SPaV-LB is provided with 6 reaction concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5 mu mol/L and the like, after the reaction is finished, the detection result shows that the reaction products can be dyed into Green by SYBR Green I at different concentrations, the electrophoresis result has waterfall type bands, but the bands at the concentration of 0.3-0.5 mu mol/L are clearer and brighter (see FIG. 5), so that the reaction concentration of the selected primer SPaV-LB is 0.3 mu mol/L.
(5)Mg 2+ Concentration selection
For Mg 2+ Setting the final concentration of the reaction to 2, 4, 6, 8, 10mmol/L and other 5 different treatments, after the reaction is finished, the detection result shows that the reaction product has a waterfall type strip at two final concentrations of 6mmol/L and 8mmol/L, and the strip is brighter when the final concentration is 6mmol/L after being dyed by SYBR Green I (see figure 6), thus Mg is selected 2+ The final concentration of the reaction was 6mmol/L.
(6) dNTPs concentration selection
The final concentration of dNTPs in the reaction system is set to be 0, 0.5, 1.0, 1.4, 2.0mmol/L and other 5 different treatments, and after the reaction is finished, detection of the reaction product shows that when the final concentration of dNTPs is 1.0mmol/L, the electrophoresis band of the product is brightest, and the product is most obvious in green after dyeing (see FIG. 7), so that the final concentration of dNTPs is determined to be 1.0mmol/L.
(7) Betaine concentration determination
5 treatments of 0.4, 0.8, 1.0, 1.2, 1.4mol/L and the like are set for the final concentration of the Betaine in the reaction system, and after the reaction is finished, the detection result shows that the band of the reaction product is brightest and is most obvious when being dyed into Green by SYBR Green I (see figure 8), so that the final concentration of the Betaine added into the reaction system is determined to be 0.4mol/L.
(8) Determination of enzyme concentration in reaction System
The final concentrations of reverse transcriptase in the reaction system were set to 6 different treatments of 0, 0.06, 0.12, 0.18, 0.24, 0.30U/. Mu.L, etc., and after the reaction was completed, the results showed that the electrophoresis detection band was brightest at the final concentrations of 0.12U/. Mu.L and 0.18U/. Mu.L, and the staining result was clear green (see FIG. 9), and the final concentration of reverse transcriptase was selected to be 0.12U/. Mu.L from the viewpoint of cost saving.
The final concentration of Bst2.0 in the reaction system was set to 6 different treatments of 0, 0.064, 0.128, 0.192, 0.256 and 0.32U/. Mu.L, and after the reaction was completed, the detection result showed that the electrophoresis band was brightest and clear and the staining was significantly green at the final concentration of 0.256U/. Mu.L (see FIG. 10), and therefore, the final concentration of Bst2.0 was selected to be 0.256U/. Mu.L.
Through the screening test, the finally determined optimized reaction system is 2.5μL 10×Isothermal Amplification Buffer、6mmol/L Mg 2+ 1mmol/L dNTPs,1. Mu. Mol/L SPaV-FIP and SPaV-BIP, 0.1. Mu. Mol/L SPaV-F3 and SPaV-B3, 0.3. Mu. Mol/L SPaV-LB,0.4mmol/L Betaine, 0.256U/. Mu.L WarmStart Bst 2.0DNA Polymerase,0.12U/. Mu. L WarmStart RTx Reverse Transcriptase, 1. Mu.L RNA, ddH added 2 O was added in 25. Mu.L, and the reaction was carried out at 61℃for 40min.
Example 3RT-LAMP method specific detection
RT-LAMP specificity was determined according to the optimal system determined in example 2, using RNA containing one or more of SPaV, SVBV, SMoV, SMYEV, SCrV and SCrV4 viruses and RNA from healthy strawberry leaves without these viruses as templates. And (3) after the detection result is determined through electrophoresis and staining, carrying out TA (time-series) clone sequencing identification on the positive amplification product. The detection result showed that only the sample carrying the virus SPaV appeared in a waterfall type band, and the color of the product turned green (see FIG. 11). A band with the size of 225bp is cut, 4 different monoclonal sequencing is selected through TA cloning, and the result is the virus SPaV through BLASTN comparison. The detection system has good specificity.
Example 4 sensitivity detection by RT-LAMP method
Subjecting RNA containing SPaV to 10-time gradient stepwise dilution to obtain RNA stock solution and 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 And 10 -7 And (3) carrying out RT-LAMP and RT-PCR amplification on RNA molds with different dilution multiples, and comparing the sensitivity of the two methods. Wherein the RT-LAMP amplification system was the optimized system of example 2. RT-PCR comprises two steps, wherein the reverse transcription step is: the reverse transcription system was 4. Mu.L of 5 Xall-in-one RT buffer, 1. Mu.L of All-in-one Enzyme Mix, 1. Mu.L of total RNA, and ddH was added 2 After the O was made up to 20. Mu.L, the reaction was carried out at 25℃for 5min and at 60℃for 15min, followed by a final treatment at 85℃for 5s. The synthesized cDNA is preserved at-20 ℃ for standby. PCR system was 5. Mu.L of 2 XEs Taq Master mix, 0.4. Mu. Mol/L primers SPaVF and SPaVR, 1. Mu.L template cDNA, and ddH was added 2 O was made up to 10. Mu.L, and finally 20. Mu.L of mineral oil was added to the reaction mixture to prevent the amplification reaction system from evaporating to dryness. The reaction procedure was 95℃pre-denaturation3min; denaturation at 94℃for 30s, annealing at 62℃for 30s, extension at 72℃for 40s, and cycling 34 times; extending at 72℃for 5min.
The results showed that SPaV virus could be detected only in the stock solution in RT-PCR detection (see FIG. 12A), while the results of RT-LAMP detection showed that waterfall bands appeared in the electrophoresis detection when the stock solution and RNA diluted 10 times were used as templates, and the corresponding SYBR Green I was also clearly Green (see FIG. 12B). Thus, RT-LAMP detects SPaV in strawberry leaf samples 10 times more sensitive than RT-PCR.
EXAMPLE 5 applicability of RT-LAMP detection method
Leaf tissues of 14 strawberry plants of a production base are randomly collected, total RNA of the leaf tissues is extracted, and then a RT-PCR method and a RT-LAMP method are respectively used for detecting virus SPaV. The RT-PCR detection result is detected by agarose gel electrophoresis, and the RT-LAMP detection result is identified by SYBR Green I staining. The results showed that 4 samples were amplified to the target size band by RT-PCR (see FIG. 13A), and the corresponding 4 samples were stained by SYBR Green I in RT-LAMP detection as clear Green (see FIG. 13B). Therefore, the RT-LAMP detection result is consistent with the RT-PCR detection result, and the constructed SPaV RT-LAMP visual detection method is applicable to quick detection of strawberry field samples.
In conclusion, the reverse transcription loop-mediated isothermal amplification detection method based on the LAMP technology is suitable for detecting the strawberry albino virus, is more sensitive in reaction, shorter in reaction time and stronger in specificity compared with a conventional RT-PCR method, and can be used for directly observing identification results with naked eyes.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (7)
1. A detection method of strawberry albino related virus is characterized in that a primer is designed by taking an outer shell protein coding gene of the strawberry albino related virus as a target sequence, and reverse transcription loop-mediated isothermal amplification is carried out by using the primer to identify the strawberry albino related virus.
2. The method for detecting strawberry whitening associated virus according to claim 1, wherein the primer comprises a SPaV-F3 primer, a SPaV-B3 primer, a SPaV-FIP primer, a SPaV-BIP primer and a SPaV-LB primer, and the specific sequences are as follows:
SPaV-F3：AGGTTTTTCAGGTTTAGGGA；
SPaV-B3：GGTCCGGAGATAAAATCAGT；
SPaV-FIP：ATGTTCAAAACTTGCCCGCACAGTGACCTTCTTGGCTTT A；
SPaV-BIP：GTTTCCGAGTCTCAAAAACCGCAAAGACCTTCCAACTAT GGT；
SPaV-LB：AACCCCATCTATGAAGATAGACACA。
3. the method for detecting a strawberry whitening-associated virus according to claim 1, comprising the steps of:
s1, extracting total RNA of a biological sample to be detected;
s2, using the total RNA as a template, and carrying out reverse transcription loop-mediated isothermal amplification by using the primer as claimed in claim 2 to obtain an amplification product;
and S3, identifying whether the biological sample to be detected contains the strawberry whitening related virus or is infected with the strawberry whitening related virus according to the amplification product.
4. A method for detecting a strawberry whitening-associated virus as in claim 3, wherein in step S3, the identification is performed by the method of 1) or 2) as follows:
1) Adding 0.3 mu L of fluorescent dye SYBR Green I into the amplification product, directly observing color change by naked eyes, and coloring a sample containing the virus SPaV into Green, wherein the sample without the virus SPaV is orange;
2) Conventional agarose gel electrophoresis and ultraviolet imaging observations, samples containing viral SPaV can form waterfall-type bands.
5. The method for detecting strawberry whitening associated virus according to claim 1, wherein the reaction system for reverse transcription loop-mediated isothermal amplification is 2.5. Mu.L 10X Isothermal Amplification Buffer, 6mmol/L Mg 2 + 1mmol/L dNTPs,1. Mu. Mol/L SPaV-FIP and SPaV-BIP, 0.1. Mu. Mol/L SPaV-F3 and SPaV-B3, 0.3. Mu. Mol/L SPaV-LB,0.4mmol/LBetaine, 0.256U/. Mu.L WarmStart Bst 2.0DNA Polymerase,0.12U/. Mu. L WarmStart RTx Reverse Transcriptase, 1. Mu.L template RNA, ddH was added 2 O makes up 25. Mu.L.
6. The method for detecting strawberry whitening associated virus according to claim 1, wherein the reverse transcription loop-mediated isothermal amplification adopts a reaction procedure of 61 ℃ for 40min and 80 ℃ for 20min.
7. The use of the method for detecting a strawberry whitening-associated virus according to claim 1 in the preparation of a kit for detecting a strawberry whitening-associated virus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311664654.XA CN117512220A (en) | 2023-12-06 | 2023-12-06 | Detection method for strawberry whitening related viruses and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311664654.XA CN117512220A (en) | 2023-12-06 | 2023-12-06 | Detection method for strawberry whitening related viruses and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117512220A true CN117512220A (en) | 2024-02-06 |
Family
ID=89756733
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311664654.XA Pending CN117512220A (en) | 2023-12-06 | 2023-12-06 | Detection method for strawberry whitening related viruses and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117512220A (en) |
-
2023
- 2023-12-06 CN CN202311664654.XA patent/CN117512220A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108060257B (en) | Primer composition for detecting pythium androsaceus based on loop-mediated isothermal amplification technology and detection method thereof | |
| CN110951838A (en) | Primer, probe, kit and application for detecting meloidogyne hapla based on RPA-LFD technology | |
| CN107022624A (en) | The LAMP methods and kit of quick detection bakanae disease of rice germ from seed rice | |
| CN107142334A (en) | The viruliferous RT LAMP detection methods of mulberry arteries and veins and its primer sets, kit and application | |
| CN112280879A (en) | RPA primer and kit for rapidly detecting citrus yellow shoot Asian species, detection method and application thereof | |
| CN102010910A (en) | Loop-mediated isothermal amplification technology-based plasmodium genus and species nucleic acid screening method | |
| CN111690759B (en) | Specific primer, kit and method for detecting RPA of citrus canker pathogen | |
| CN109371110B (en) | LAMP (loop-mediated isothermal amplification) detection kit for bacterial canker pathogen of poplar | |
| CN111534626A (en) | LAMP (loop-mediated isothermal amplification) detection primer composition for pythium bellatus, detection kit and visual detection method of LAMP detection primer composition | |
| CN112646914A (en) | LAMP primer group for rapidly detecting amycolatopsis persicae and rapid detection method and kit thereof | |
| CN114015799B (en) | LAMP (loop-mediated isothermal amplification) detection primer group, kit and LAMP detection method for Helminthosporium putrescens | |
| CN117512220A (en) | Detection method for strawberry whitening related viruses and application thereof | |
| CN116287475A (en) | Nucleic acid sequence combination, kit and application of brassica yellow virus RT-LAMP-CRISPR isothermal detection | |
| CN108315493A (en) | A kind of LAMP primer group and detection method for detecting barley yellow mosaic virus | |
| CN108411017A (en) | Detect the LAMP primer and method of pseudomonas syringae tomato pvs oryzae and oryzicola | |
| CN112048573B (en) | RPA primer and kit for detecting cotton leaf curl virus, and detection method and application thereof | |
| CN110904242B (en) | Primer composition and application thereof in identification of Hirudinaria manillensis | |
| CN114032336A (en) | Method and kit for detecting cucumber mosaic virus | |
| CN118064642A (en) | Rapid detection method for strawberry potato leaf curl virus 1 and application thereof | |
| CN111304357A (en) | Fusarium oxysporum bitter gourd specialized LAMP visual detection method and kit thereof | |
| CN112210624A (en) | LAMP primer and kit for efficient triple detection of three pathogens of Chinese softshell turtles | |
| CN107828918B (en) | Dual RT-PCR (reverse transcription-polymerase chain reaction) detection kit for Nihonin latent virus and narcissus common latent virus and detection method thereof | |
| CN117721254A (en) | Rapid detection method for strawberry hairy virus 4 | |
| CN114592079B (en) | LAMP detection primer group, reagent and detection method for detecting pathogenic bacteria of potato scab and application of LAMP detection primer group | |
| CN113322353B (en) | RPA kit for detecting sweet potato pinnate mottle virus and sweet potato chlorotic stunt virus |
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 |