CN114250313A - Composition and method for detecting drug resistance of botrytis cinerea benzimidazole bactericide - Google Patents
Composition and method for detecting drug resistance of botrytis cinerea benzimidazole bactericide Download PDFInfo
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- 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
Abstract
The invention discloses a composition and a method for detecting the drug resistance of botrytis cinerea benzimidazole, belonging to the technical field of drug resistance detection of plant pathogenic bacteria, wherein the composition comprises a primer pair for detecting botrytis cinerea, and the sequence of the primer pair is as follows: an upstream primer: 5'-GTCCATCAATTGGTTGAGAACTCTGAAAC-3', respectively; a downstream primer: 5'-GCAAATCCAACCATGAAGAAATGGAGACG-3' are provided. The composition and the method can be used for quickly detecting MBC resistant botrytis cinerea in the bacterium-carrying tissues, can complete the detection process in less than 20min, does not need to separate, purify and culture the pathogenic bacteria, saves time, and is very suitable for the quick detection requirement of a base layer on the pathogenic bacteria.
Description
Technical Field
The invention belongs to the technical field of drug resistance detection of plant pathogenic bacteria, and particularly relates to a composition and a method for detecting drug resistance of botrytis cinerea benzimidazole, which are used for rapidly detecting field resistant strains.
Background
Botrytis cinerea has a very broad host range, can cause gray mold of up to 200 plants, is listed as ten important plant pathogenic bacteria in the world, and is ranked second (Dean et al.2012). Botrytis cinerea occurs during the growth of plants, during post-harvest storage and during transportation. In recent years, the grape industry in China is rapidly developed along with the popularization of rain sheltering cultivation technology, but the grape gray mold is gradually increased to be one of the main diseases of grapes along with the increase of facility cultivation area. The disease can cause decay of the grape plant from sprouts, young shoots, inflorescences, young leaves, to fruits; in the mature period, a gray mildew layer can be formed; during the storage period, the wet rot of the cob part of the grapes can be caused. In severe cases, the incidence of ear diseases can reach 50% -80%, even resulting in the loss of individual orchards (Qian constant et al 2015).
At present, the most effective prevention and treatment method for the disease still depends on chemical prevention and treatment, and benzimidazoles (MBC) occupy an important position in bactericides, and comprise the varieties of carbendazim, probenazole, benomyl, thiabendazole, fuberidazole and the like. However, Botrytis cinerea can produce a large amount of spores due to its extremely high genetic diversity (Pierre et al 2002), has a short generation time (Petsikos-Panayotarou et al 2003) and has a wide range of hosts (Yourman et al 2008), so that it is easy to develop resistance to fungicides, and the action Committee for resistance to Fungicides (FRAC) assesses Botrytis cinerea as a high-risk pathogen that is easy to develop resistance to pesticides. The detection of the occurrence frequency of drug-resistant strains in the botrytis cinerea population not only provides technical support for reasonably selecting pesticides to control the occurrence of diseases, but also provides powerful guarantee for delaying the development of the drug-resistant strains and preventing the epidemic harm of the drug-resistant diseases. A large number of studies have shown that the pathogenic fungi develop resistance to benzimidazole agents mainly due to point mutation of the beta tubulin (beta-tubulin) gene (TUB2) in the VII linkage group, and mutation of the 198 th glutamic acid point of the protein transcribed and translated into alanine (E198A) in the gene results in the development of high resistance of Botrytis cinerea to benzimidazole fungicides. Therefore, establishing a rapid detection technology of the botrytis cinerea for the drug resistance of the benzimidazole bactericide is particularly important in the treatment of the drug resistance of the botrytis cinerea.
Currently, the commonly used drug resistance detection methods are mainly classified into PCR based on gene mutation and the traditional colony diameter method based on hyphal growth inhibition. The traditional colony diameter method needs to perform pure culture on pathogenic bacteria collected in the field in a sterile environment, perform culture on a series of culture media containing different medicament concentrations, detect and calculate the inhibitory median concentration (EC) of pesticide on the pathogenic bacteria50) Or the Minimum Inhibitory Concentration (MIC). However, the method requires a long time period, is large in workload, and has a limited number of measurement samples, so that the existence of drug-resistant strains is difficult to find in the early stage of disease occurrence. The PCR technology based on gene mutation designs corresponding primers according to known point mutation causing drug resistance, but the required instruments are expensive, the technical requirements for operators are high, and the PCR technology is not suitable for popularization in agricultural departments such as prefectures, cities and counties.
Compared with the traditional PCR method, the Recombinase-mediated isothermal Amplification (RPA) technology has the characteristics of higher Amplification efficiency, no need of expensive instruments, good specificity and the like, and the method is suitable for conventional reaction temperature.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a composition for detecting the drug resistance of botrytis cinerea benzimidazole bactericide, which comprises a primer pair for detecting botrytis cinerea, wherein the primer pair is directed against the TUB2 gene of botrytis cinerea. A series of RPA primers capable of distinguishing sensitive strains from strains resistant to E198A are designed, and multiple designed primers are optimized and screened through experiments, so that a pair of special primers for detecting strains resistant to MBC bactericides by the Botrytis cinerea through RPA is obtained.
The primer pair has nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and specifically comprises the following components:
an upstream primer: 5'-GTCCATCAATTGGTTGAGAACTCTGAAAC-3', respectively;
a downstream primer: 5'-GCAAATCCAACCATGAAGAAATGGAGACG-3' are provided.
The invention also aims to provide a kit for detecting the drug resistance of botrytis cinerea benzimidazole bactericide, wherein the kit comprises the composition.
In the kit, FAM is added to the 5 'end of the upstream primer, and Biotin is added to the 5' end of the downstream primer.
Wherein FAM represents carboxyfluorescein, and Biotin represents Biotin.
In the kit, the final concentration of the primer pair during amplification is 0.2. mu.M.
The kit also comprises standard positive control DNA, buffer solution, an RPA amplification enzyme system and a lateral flow chromatography test strip.
In the kit, the RPA amplification enzyme system comprises recombinase, single-stranded DNA binding protein, DNA polymerase, recombinase-assisted aggregation protein and endonuclease.
In the kit, the lateral flow chromatography test strip comprises a sample pad, a combination pad, an interpretation zone and an absorption pad which are sequentially arranged;
the binding pad comprises a gold-labeled antibody, e.g., the binding pad is gold-labeled streptavidin;
the interpretation zone comprises a quality control line and a detection line;
the detection line includes an anti-fluorescein antibody, e.g., the anti-fluorescein antibody is a mouse anti-fluorescein monoclonal antibody, and the quality control line includes BSA biotin.
The kit also comprises lysis solution.
The invention also provides application of the composition or the kit in detection of drug resistance of botrytis cinerea benzimidazole bactericide.
The invention also provides a method for detecting the drug resistance of botrytis cinerea benzimidazole, which comprises the following steps:
(1) extracting DNA of a sample to be detected;
(2) taking DNA as a template to carry out RPA amplification, wherein an upstream primer is as follows: 5'-GTCCATCAATTGGTTGAGAACTCTGAAAC-3', respectively; the downstream primer is: 5'-GCAAATCCAACCATGAAGAAATGGAGACG-3', respectively; a modification group FAM is added at the 5 'end of the upstream primer, and a modification group Biotin is added at the 5' end of the downstream primer;
(3) detecting the amplification product by using a lateral flow chromatography paper strip; when two brown strips appear on the test strip, one is positioned in the quality control area, and the other is positioned in the detection area, the result is positive, and the result shows that the sample contains benzimidazole drug-resistant botrytis strain; when only the quality control area of the test strip has a brown strip and the detection area has no strip, the result is negative, which indicates that the sample does not contain benzimidazole drug-resistant botrytis strain.
In the above method, in step 2), the method for RPA amplification comprises: mixing the RPA amplification enzyme system with Buffer A, adding an upstream primer and a downstream primer, adding a sample DNA to be detected, adding Buffer B, uniformly mixing, centrifuging, and reacting.
In the above method, in step 2), the reaction system for RPA amplification is: buffer A29.4. mu.l, 10. mu.M upstream primer 1.0. mu.l, 10. mu.M downstream primer 1.0. mu.l, sample DNA 1.0. mu.l; buffer B2.5 μ l, sterile water to 50 μ l; the reaction strip body is as follows: centrifuging at 5000 × g for 10s, and reacting at 25-40 deg.C for 10-30 min.
In the method, the pathogenic bacteria DNA can be obtained from the plant to be detected or the hypha of the pathogenic bacteria to be detected by culture-free extraction.
Compared with the prior art, the primer and the method can be used for quickly detecting MBC resistant botrytis cinerea in the bacterium-carrying tissues, the detection process can be completed in less than 20min, the pathogenic bacteria do not need to be separated, purified and cultured, the time is saved, the time required by the method is far shorter than that of the conventional PCR (more than one day from the preparation of DNA to the sequencing analysis) and the LAMP detection technology (60-80min), and the method has great significance for reasonably selecting the medicament in the field.
The method is simpler and convenient to carry, the enzyme and other reagents required by amplification can be stored in a freeze-dried manner and can be placed for a long time at normal temperature, and only a hydrolysis buffer solution, a primer, a template and a reaction starting solution need to be added during amplification, so that professional testers are not required. The amplification of the template nucleic acid can be realized under the constant temperature condition of about 37 ℃ by only one water bath, the nucleic acid melting and annealing can be realized without expensive PCR instruments, and the kit is very suitable for the rapid detection requirement of the base layer on pathogenic bacteria. And the result is real and reliable, and the conformity with the common PCR sequencing analysis result is 100%. Strong specificity and high sensitivity.
The detection sensitivity of the primer and the kit is 102Copy/. mu.l, and has a wide detection range of at least 107–102Samples in the copy/. mu.l range can be detected. The kit is used for respectively detecting the common pathogenic bacteria and the closely-sourced species of botrytis cinerea and the botrytis cinerea MBC sensitive and E198A resistant strains on grapes and strawberries, and as a result, only the botrytis cinerea MBC resistant strains can be well amplified, and other pathogenic bacteria cannot be detected, so that the kit is proved to have good specificity.
Drawings
FIG. 1 is a schematic diagram of the alignment and primer design of E198A and different genotype sequences, in which the single underline represents the site of nucleotide diversity resulting in different genotypes and the double underline represents the position of mismatched bases designed to increase primer specificity.
FIG. 2 is an electrophoretogram of specific RPA primers for nucleic acid amplification of different genotype Botrytis cinerea strains; m is DL2000 Marker; sequentially selecting nucleic acids with genotypes of E198A, E198V, E198K and MBC sensitive botrytis strain for amplification, and setting clear water control; wherein, the strain with the genotype of E198A is Tg16, Tg67, WH7, YN4 and YN 5; the MBC sensitive strains are YN80, LB12 and SD 124; the strains with the genotype of E198V are WH89, YN2 and WH 85; the strains with genotype E198K were WH105, YN155, YN 401.
FIG. 3 is a diagram showing the structure of the flow-through chromatography test strip.
FIG. 4 is a schematic diagram of the detection of a lateral flow chromatography test strip.
FIG. 5 is a graph of the effect of different temperatures on the results of the RPA-LFD reaction; the test temperature is 20, 25, 30, 35, 37, 40, 45, 50 and 55 degrees in sequence, one line on the test strip is a quality control line which represents that the test strip is normal, and two lines represent that the reaction normally occurs, namely an E198A resistant strain appears.
FIG. 6 shows the sensitivity test of the RPD-LFS and PCR detection methods; 1-8 the DNA concentrations added were in the order: 107、106、105、104、103、10210, 1 copies/. mu.l; the upper diagram shows the detection of the RPA-LFD methodThe result is; the lower panel shows the results of the general PCR assay; one line on the test strip is a quality control line, which represents that the test strip is normal, and two lines represent that the reaction normally occurs, namely, the E198A resistant strain appears.
FIG. 7 shows the specific detection of the RPD-LFS primer; downy mildew (Plasmopara spp.), powdery mildew (Uncinula spp.), Alternaria alternata (Alternaria spp.), Botrytis cinerea (B.porri), Sclerotium sclerotiorum (Sclerotium sclerotiorum), Colletotrichum gloeosporioides (Colletotrichum acum), Colletotrichum gloeosporioides (Colletotrichum gloeosporiodes), Monilinia persicae (Monilinia fructicola), MBC resistant botrytis cinerea strain E198A (B.cinerea E198A) and MBC sensitive botrytis cinerea strain (B.cinerea ldtype) are detected in sequence; the E198A type griseus strain is Tg16, and the MBC sensitive type griseus strain is YN 80.
FIG. 8 shows the specific detection of the RPD-LFS primer on Botrytis cinerea (B.cinerea).
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The materials and devices used in the present invention are commercially available unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1 design and preparation of primers
The inventor compares the nucleotide sequence information of the TUB2 genes of wild type, E198A type, E198V type and E198K type strains by sequencing and analyzing the TUB2 sequence information of botrytis cinerea MBC sensitive and resistant strains collected in grape and tomato gardens and comparing the sequence information with the sequences XM _024690731 (sensitive type), MH680905(E198A type), MG949126(E198V type) and MG949128(E198A type) in Genbank, and designs a series of primers aiming at the nucleotide difference sites, as shown in FIG. 1.
The length of the special primer for RPA detection designed by the invention is 27-35bp, which is longer than that of the general PCR detection primer. Too short primers can reduce the recombination rate and affect the sensitivity of the RPA reaction.
Because the primer design principles of RPA amplification and PCR amplification are different and no clear primer design principle exists for a while, a plurality of specific upstream primers and a plurality of specific downstream primers which can distinguish the E198A strain and other genotypes are designed. In the specific upstream primer, we try to increase several base pairs, adjust the positions of the mismatched bases and adjust different mismatched bases, and simultaneously adjust the length of the upstream primer to increase the specificity and sensitivity. Finally, we selected the primer pair with the best amplification specificity, the least primer dimer and the highest sensitivity among hundreds of different combinations of primers.
Using this primer pair, amplification was performed by RPA, and the amplification product was purified and subjected to electrophoresis, wherein the size of the band of interest for amplification was 244bp, as shown in FIG. 2, and the specific sequence is shown in Table 1 and synthesized by Ongjingkai (Beijing).
Wherein, the reaction system for RPA amplification is as follows: buffer A29.4. mu.l, 10. mu.M upstream primer 1.0. mu.l, 10. mu.M downstream primer 1.0. mu.l, sample DNA 1.0. mu.l; buffer B2.5 μ l, sterile water to 50 μ l; the reaction strip body is as follows: centrifuging at 5000 × g for 10s, and reacting at 25-40 deg.C for 10-30 min.
A rapid DNA extraction method comprises, for example, adopting a Optimalaceae plant amplification kit (Optimalaceae, Beijing), and rapidly extracting pathogenic bacteria DNA from plant or pathogenic bacteria hypha to be detected without culture. The specific method comprises the following steps: a small amount of hyphae was lysed in 10uL of Lyse and Go lysate (Thermo Fisher Scientific Co., USA) at 100 ℃ for 5min, and 1. mu.l of the supernatant was taken as a solution of Botrytis cinerea genomic DNA.
The electrophoresis method comprises the following steps: after the RPA reaction, 50. mu.l of phenol/chloroform (1:1) solution was added to the RPA amplification product, mixed well, centrifuged at 10000 × g for 1min, 5. mu.l of the supernatant was electrophoresed on 1.0% agar gel, and the results were observed on a gel imaging system. The detection voltage of the agar gel electrophoresis detection is 100V, and the detection time is 30 min.
TABLE 1 primers for the specific amplification of the strain type E198A, a Botrytis cinerea MBC resistant strain
| Primer types | Primer name | Primer sequence (5 '-3') |
| Upstream primer | E198A-F | [5’FAM]-GTCCATCAATTGGTTGAGAACTCTGAAAC |
| Downstream primer | E198A-R | [5’Biotin]-GCAAATCCAACCATGAAGAAATGGAGACG |
Example 2 detection kit for rapidly detecting Botrytis cinerea MBC resistant strains and establishment of detection method
A kit for detecting the drug resistance of botrytis cinerea benzimidazole fungicides comprises the following components: the primer pair of example 1 further comprises a standard positive control DNA, a buffer, an RPA amplification enzyme system, and a lateral flow chromatography test strip.
The RPA amplification enzyme system comprises recombinase, single-stranded DNA binding protein, DNA polymerase, recombinase auxiliary aggregation protein and endonuclease;
specifically, the method comprises the following steps:
(1) the standard positive control DNA is a positive plasmid, such as Pmd 18-TubA. When used, 1-10 pg/. mu.L is used as a positive template.
The preparation method of the Pmd18-TubA comprises the following steps: genomic DNA of Botrytis cinerea strain TG67, which is known to contain an E198A mutation in the TUB2 gene, was amplified by the primer pair TubA1378F 19/TubA 2314R20 (5'-GATCTCCAACTTGAGCGTA-3'/5'-TGG AAACCTTACCACGGCTA-3'). The amplification product was purified by easy pure PCR purification kit (all-grass, beijing). The purified amplification product was subsequently subjected to TA cloning into the vector pMD18-T (TA cloning kit from TaKaRa, Dalian) according to the procedures of the kit pMD18-T instructions, and the resistance was Amp resistance. The plasmid was extracted by a plasmid extraction kit (purchased from Onghamia corporation, Beijing).
(2) The RPA amplification enzyme system can use a reaction tube containing a dry powder reagent of the RPA amplification enzyme system, and the reaction system comprises the following components in final concentration: 200 ng/. mu.L of recombinase, 200 ng/. mu.L single-stranded DNA binding protein, 100 ng/. mu.L DNA polymerase, 80 ng/. mu.L recombinase accessory protein and 100ng/mL endonuclease IV, which are all purchased from Weifang Anpu future Biotech Co., Ltd;
(3) 48 parts of upstream primer E198A-F10. mu.M and downstream primer E198A-R10. mu.M (Table 1);
(4) 1.2mL of buffer (3% polyethylene glycol by mass concentration, Tris 30mM) 2 tubes;
(5) b buffer (280mM magnesium acetate) 150. mu.L, 1 tube;
(6) the flow-through test strip is, for example, 48 copies (purchased from Weifang Anpu future Biotechnology Ltd.).
When in use, the reaction system for RPA amplification is as follows: buffer A29.4. mu.l, 10. mu.M upstream primer 1.0. mu.l, 10. mu.M downstream primer 1.0. mu.l, sample DNA 1.0. mu.l; buffer B2.5 μ l, sterile water to 50 μ l; the reaction strip body is as follows: centrifuging at 5000 × g for 10s, and reacting at 25-40 deg.C for 10-30 min.
The flow-measuring chromatography test strip comprises a sample pad, a combination pad, an interpretation zone and an absorption pad which are arranged in sequence; wherein the binding pad comprises gold-labeled streptavidin; the interpretation zone comprises a quality control line and a detection line; the detection line includes an anti-fluorescein antibody, e.g., a mouse anti-fluorescein monoclonal antibody, and the quality control line includes BSA biotin, as shown in fig. 3.
During detection, the sample end of the flow-measuring chromatography test strip is inserted into a reaction product, when an amplification product exists, the biotin modified on the product is combined with the colloidal gold of the streptavidin modified on the gold label, and when the amplification product reaches a detection line, the anti-fluorescein antibody on the detection line captures the product labeled with the hydroxyl Fluorescein (FAM), so that color development is realized; the excessive colloidal gold is combined with biotin to develop color when going to the quality control line.
The results were interpreted as shown in FIG. 4:
positive: the Test strip has two red strips, one is located in a Control Line (C Line) and the other is located in a Test Line (T Line). The positive result shows that the sample is the MBC drug-resistant strain containing E198A, and the content of the MBC drug-resistant strain is more than or equal to the lowest detection amount of the test strip. Negative: a red strip appears in the quality control area (line C) of the test strip, and no strip exists in the detection area (line T). And a negative result indicates that the sample does not contain the target nucleic acid to be detected or the content of the target nucleic acid is lower than the minimum detection amount of the test strip. And (4) invalidation: no strip appears in the quality control area (line C) and the detection area (line T) of the test strip. This result suggests that the test strip used is ineffective, damaged or mishandled.
Further, the kit may further comprise: nucleic acid lysates, for example, 250 μ L1 tubes of ultrafast nucleic acid releasing agent (LyseGo, available from Thermo Fisher Scientific, USA).
Example 3 Rapid detection of Botrytis cinerea MBC resistant strains
Strain information
Grape botrytis strains in Hubei, Shandong, Shanxi, Yunnan and other provinces are respectively collected, and the sensitivity types of the grape botrytis strains to MBC bactericides and the TUB2 gene sequences of the grape botrytis strains are determined.
Pathogen DNA extraction
The pathogenic bacteria DNA can be obtained by rapid extraction method, such as by using a Potentilla plant amplification kit (Potentilla corporation, Beijing), from the plant or the pathogenic bacteria hypha to be detected.
The specific method comprises the following steps: a small amount of hyphae was lysed in 10uL of Lyse and Go lysate (Thermo Fisher Scientific Co., USA) at 100 ℃ for 5min, and 1. mu.l of the supernatant was taken as a solution of Botrytis cinerea genomic DNA.
Optimization of amplification conditions in RPA assay
The extracted and purified pathogenic bacteria DNA is used as a template for amplification, and the experimental system is as follows: mu.l Buffer A (Anpu future), 1.0. mu.l 10. mu.M forward primer, 1.0. mu.l 10. mu.M reverse primer, 1.0. mu.l DNA; 2.5. mu.l of Buffer B (Anpu future) for a total of 50. mu.l of amplification system.
Using the above DNA as a template and the RPA primer designed in example 1, an RPA reaction was carried out in the reaction system of example 1, and different temperatures of 20, 25, 30, 35, 37, 40, 45, 50, and 55 ℃ were set, and the influence of the different temperatures on the reaction results was examined. The results of the experiment are shown in FIG. 5, which shows that the bands are clear at reaction temperatures of 25-40 deg.C, and 37 deg.C was selected as the reaction temperature.
When the reaction time of the RPA primer was determined, the reaction was added according to the above system, and the amplification difference was examined at different times when the temperature was set at 37 ℃. We set the time to 5min, 10min, 15min, 20min, 30min, respectively. The results show that at 10min of amplification, there is a band on the test strip, and when the reaction time is between 10-30min, there is no observable difference in the test strip, so we chose 10min as the incubation time for this assay.
EXAMPLE 4 sensitive and specific detection of the kit
In the case of the sensitivity test of the RPA-LFD reaction, the reaction mixture was added in the same manner as in example 2, and the plasmid Pmd18-TubA was used as shown in example 2. Calculating the copy number by measuring the concentration with a nucleic acid analyzer, and using PCR-grade H2Sequentially diluting O to a concentration gradient of 107Copy/. mu.l, 106Copy/. mu.l, 105Copy/. mu.l, 104Copy/. mu.l, 103Copy/. mu.l, 102Copy/. mu.l, 10 copies/. mu.l, 1 copy/. mu.l, totaling 8 concentration gradients of DNA template.
The RPA detection was performed on DNA templates of different concentrations according to the primers, reaction system and reaction conditions used in example 3 above. The results are shown in FIG. 6, from which it can be seen that the sensitivity of the method is 102Copy/. mu.l, and has a wide detection range of at least 107-102Copy/. mu.l rangeThe samples in the enclosure can be detected. The same test is repeated for 4 times, and the result shows that the detection results of the 4 tests are consistent and can be well detected by 10 times at least2DNA template of one copy/. mu.l, with good reproducibility, and no longer amplified a positive result with each test when the concentration was reduced to 10 copies/. mu.l.
In the specificity test of the RPA reaction, 5 strains of E198A point mutation-containing grifola frondosa, 3 strains of E198V point mutation-containing grifola frondosa, 3 strains of E198K point mutation-containing grifola frondosa, 3 strains of wild type strain without point mutation, and a clear water control (FIG. 2) were used. In addition, grape pathogens which are common include downy mildew (Plasmopara spp.), powdery mildew (Uncinum spp.), Alternaria spp, Gracilaria verrucosa (B.porri), Sclerotinia sclerotiorum (Colletotrichum acutum), Colletotrichum gloeosporioides (Colletotrichum acremonium), Monilinia persicinum (Monilinia fructicola). The amplification reaction was carried out at 37 ℃ for 10 min. The result shows that only the MBC resistant strain containing E198A can be amplified to generate a strip on the test strip, and the DNA of other pathogenic bacteria cannot be amplified to generate a strip. Therefore, the method has good specificity, and the result is shown in FIG. 7. Meanwhile, the present inventors also examined other point mutations including E198V and E198K at position 198 of β -tubulin (protein encoded by TUB2 gene), and found that only E198A mutation could be detected (FIG. 8).
Example 5 amplification of Botrytis cinerea (B.cinerea) hyphae directly with RPA-LFD specific primers
By using the RPA detection method of example 2, mycelia or spores were directly collected from diseased fruits, and grifola frondosa strains (n ═ 136) collected from Shandong province, Hubei province and Yunnan province were detected, and the detection results were compared with the results after PCR sequencing, and among the 136 strains, the RPA detection results were 95 positive samples, which were completely identical to the results after PCR sequencing. Thus, the accuracy of the RPA detection method was 100% based on 136 samples tested.
Table 2 compares the field sample detection results of the RPA-LFS method and the conventional PCR method
In conclusion, the method can quickly diagnose the botrytis cinerea MBC resistant strain containing the E198A point mutation, and the detection method is simple and quick, and the detection result is real and reliable.
SEQUENCE LISTING
<110> institute of plant protection of Chinese academy of agricultural sciences
<120> composition and method for detecting drug resistance of botrytis cinerea benzimidazole bactericide
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 29
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 1
gtccatcaat tggttgagaa ctctgaaac 29
<210> 2
<211> 29
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 2
gcaaatccaa ccatgaagaa atggagacg 29
Claims (10)
1. The composition for detecting the drug resistance of the botrytis cinerea benzimidazole bactericide is characterized by comprising a primer pair for detecting the botrytis cinerea, wherein the sequence of the primer pair is as follows:
an upstream primer: 5'-GTCCATCAATTGGTTGAGAACTCTGAAAC-3', respectively;
a downstream primer: 5'-GCAAATCCAACCATGAAGAAATGGAGACG-3' are provided.
2. A kit for detecting resistance to botrytis cinerea benzimidazole fungicides comprising the composition of claim 1.
3. The kit according to claim 2, wherein FAM is added to the 5 'end of the upstream primer, and Biotin is added to the 5' end of the downstream primer.
4. The kit of claim 2, wherein the primer pair has a final concentration of 0.2 μ M during amplification.
5. The kit of claim 2, further comprising a standard positive control DNA, a buffer, an RPA amplification enzyme system, and a lateral flow chromatography strip.
6. The kit of claim 5, wherein the lateral flow chromatography test strip comprises a sample pad, a binding pad, an interpretation zone and an absorption pad arranged in sequence, wherein the binding pad comprises a gold-labeled antibody, the interpretation zone comprises a quality control line and a detection line, the detection line comprises an anti-fluorescein antibody, and the quality control line comprises BSA biotin.
7. Use of a composition according to any one of claims 1 to 2 or a kit according to any one of claims 3 to 6 for detecting resistance to botrytis cinerea benzimidazole fungicide.
8. A method for detecting the drug resistance of botrytis cinerea benzimidazole is characterized by comprising the following steps:
(1) extracting DNA of a sample to be detected;
(2) taking DNA as a template to carry out RPA amplification, wherein an upstream primer is as follows: 5'-GTCCATCAATTGGTTGAGAACTCTGAAAC-3', respectively; the downstream primer is: 5'-GCAAATCCAACCATGAAGAAATGGAGACG-3', respectively; a modification group FAM is added at the 5 'end of the upstream primer, and a modification group Biotin is added at the 5' end of the downstream primer;
(3) detecting the amplification product by using a lateral flow chromatography paper strip; when two brown strips appear on the test strip, one is positioned in the quality control area, and the other is positioned in the detection area, the result is positive, and the result shows that the sample contains benzimidazole drug-resistant botrytis strain; when only the quality control area of the test strip has a brown strip and the detection area has no strip, the result is negative, which indicates that the sample does not contain benzimidazole drug-resistant botrytis strain.
9. The method according to claim 8, wherein in step 2), the method for RPA amplification comprises: mixing the RPA amplification enzyme system with Buffer A, adding an upstream primer and a downstream primer, adding a sample DNA to be detected, adding Buffer B, uniformly mixing, centrifuging, and reacting.
10. The method according to claim 9, wherein in step 2), the reaction system for RPA amplification is: buffer A29.4. mu.l, 10. mu.M upstream primer 1.0. mu.l, 10. mu.M downstream primer 1.0. mu.l, sample DNA 1.0. mu.l; buffer B2.5 μ l, sterile water to 50 μ l; the reaction strip body is as follows: centrifuging at 5000 × g for 10s, and reacting at 25-40 deg.C for 10-30 min.
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