CN105154432A - Method for amplifying four genes of Botrytis cinerea and multiplex PCR (polymerase chain reaction) primer set thereof - Google Patents
Method for amplifying four genes of Botrytis cinerea and multiplex PCR (polymerase chain reaction) primer set thereof Download PDFInfo
- Publication number
- CN105154432A CN105154432A CN201510593450.0A CN201510593450A CN105154432A CN 105154432 A CN105154432 A CN 105154432A CN 201510593450 A CN201510593450 A CN 201510593450A CN 105154432 A CN105154432 A CN 105154432A
- Authority
- CN
- China
- Prior art keywords
- seqidno
- botrytis cinerea
- pcr
- pathogen
- genes
- 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for amplifying four genes of Botrytis cinerea and a multiplex PCR (polymerase chain reaction) primer set thereof, relating to the field of molecular biology. On the basis of drug-resistant mutant sites of known Botrytis cinerea p-benzimidazole, boscalid, iprodione, fenhexamid and many other bactericides, a PCR primer set for simultaneously amplifying four gene segments beta-tub, BsOS1, erg27 and SdhB is established. The extracted Botrytis cinerea genome can be directly used as a template and subjected to amplification by using the designed primer set to obtain the enriched four gene segments. The multiplex PCR process is used for the first time to simultaneously amplify the four drug-resistant mutant genes of Botrytis cinerea, thereby greatly reducing the workload for drug-resistant site detection in the later period. By optimizing the PCR reaction system, the four gene segments are amplified in one PCR process.
Description
Technical field
The present invention relates to biology field, be specifically related to the primer sets of a kind of method of the pathogen of Botrytis cinerea that simultaneously increases and the resistant mutation gene for four kinds of sterilant.
Background technology
Gray mold is common at open country, protecting field and a kind of fungal disease that control is very difficult, and its pathogenic bacterium are the pathogen of Botrytis cinerea (BotrytisCinerea), belongs to Deuteromycotina.The host range of the pathogen of Botrytis cinerea is very extensive, comprises 200 various plants, such as, in the life such as tomato, cucumber, grape common vegetables and fruit, very harmful in agriculture production.Prevention and controls at present for gray mold comprises cultural control, biological control and chemical prevention etc., wherein still based on chemical prevention.Conventional sterilant comprises benzimidazoles, dicarboximide class, N-phenyl urethan class, pyrimidine fungicides etc.
The pathogen of Botrytis cinerea has that breeding is fast, heritable variation large and fitness high, creates resistance to a certain degree to various sterilization agent, even creates multiple resistance.The major cause developed immunity to drugs to sterilant is the sudden change of pharmaceutically-active target point gene origination point, makes sterilant cannot play the effect of its script.The bacterial strain in field is under the selective pressure of various sterilization agent, very likely produce multiple cross resistance, therefore the resistant mutation detection for specific a kind of sterilant of the pathogen of Botrytis cinerea has been not enough to the needs meeting agriculture production, and it is very significant for realizing it to detecting while various sterilization agent resistant mutation.
The reason that the pathogen of Botrytis cinerea produces resistance to benzimidazole germicide and N-pyrimidine fungicides sterilant is that the sudden changes such as E198A, E198V, E198K, F200Y occur 'beta '-tubulin gene (β-tub); Reason dicarboximide fungicide being produced to resistance is that the sudden changes such as I365S, I365R, I365N, Q369P, V368F+Q369H, Q369P+N373S, N373S occur histidine kinase gene (BsOS1); Reason fenhexamid sterilant being produced to resistance is that the sudden changes such as F412S, F412I, F412V occur chlC4 gene (erg27); Reason boscalid amine sterilant being produced to resistance is that the sudden changes such as H272Y, H272R, H272L, P225T, P225F, P225L, N230I occur succinate dehydrogenase iron-sulfur protein gene (SdhB).The present invention is on the basis in the mutational sites such as the resistance of various sterilization agent at known ash botrytis, design primer sets increases β-tub, BsOS1, erg27, SdhB tetra-gene fragments simultaneously, can be used as the template of the high-flux detection methods such as follow-up gene chip, thus realize the pathogen of Botrytis cinerea to detection while four kinds of sterilant resistant mutations.
Summary of the invention
The present invention is at known ash botrytis on the basis of the drug resistance mutant site of the various sterilization agent such as benzimidazoles, boscalid amine class, RP-26019 and fenhexamid, a kind of PCR primer group for the β-tub that increases, BsOS1, erg27, SdhB tetra-kinds of gene fragments simultaneously of foundation.The present invention can directly adopt the pathogen of Botrytis cinerea genome of extraction as template, increases to template by the primer sets of design, obtains four gene fragments of enrichment.The present invention adopts multiple PCR method to increase to four of the pathogen of Botrytis cinerea drug-resistant mutation genes first simultaneously, and for the later stage, the detection in anti-medicine site greatly reduces workload.
The present invention devises the primer sets for the pathogen of Botrytis cinerea β-tub that increases, BsOS1, erg27, SdhB tetra-gene fragments, through the optimization to PCR reaction system, four gene fragments is increased in same PCR reaction.
The advantage that the present invention gives prominence to is: (1) increase the pathogen of Botrytis cinerea β-tub, BsOS1, erg27, SdhB tetra-gene fragments first in same PCR reaction, containing the resistance mutation sites to most sterilant in four gene fragments, for the resistance of late detection the pathogen of Botrytis cinerea to various sterilization agent decreases workload.(2) non-specific amplification in reaction is few, does not affect follow-up detection.
For a method for the pathogen of Botrytis cinerea four genes that increase, it is characterized in that altogether in two steps:
The first step: extract the pathogen of Botrytis cinerea genome
Second step: carry out PCR reaction: adopt four pairs of primers, be respectively used to amplification the pathogen of Botrytis cinerea β-tub, BsOS1, erg27, SdhB gene, four pairs of primers are respectively
SEQIDNO:1CGATACCGTTGTCGAGCCAT
SEQIDNO:2GGTTGCTGAGCTTCAAGGTT
SEQIDNO:3ACACCGACCCAGCACCAGA
SEQIDNO:4TTAGCAATAACCGCCCAAA
SEQIDNO:5CACCCGCGTAGCAAGAGATG
SEQIDNO:6TGAGTCAGGTCTCCCTTTGC
SEQIDNO:7ATCATGCCCTTGAATTTTGTG
SEQIDNO:8CTTTGCCTCCCCTTTCTTCTC
The template that genomic dna containing the testing sample extracted in PCR reaction mixture reacts as PCR, reaction system is as follows,
PCR response procedures: 94 DEG C of 5min; 94 DEG C of 30sec, 59 DEG C of 30sec, 72 DEG C of 20sec; 72 DEG C of 10min.
The object of the present invention is achieved like this more specifically:
A primer sets for amplification four anti-medicine genes involveds of the pathogen of Botrytis cinerea simultaneously, its system comprises four pairs of multiplexed PCR amplification primers, is respectively used to amplification the pathogen of Botrytis cinerea β-tub, BsOS1, erg27, SdhB tetra-gene fragments; PCR reactive component, purchased from invitrogen company, comprises Taq enzyme, and dNTPs, PCR damping fluid is not (containing Mg
2+), MgCl
2.Adopt UNIQ-10 pillar fungal genomic DNA extraction agent box, purchased from Sangon Biotech (Shanghai) Co., Ltd., extract the genomic dna of testing sample.
Primer sequence used is respectively: SEQIDNO:1CGATACCGTTGTCGAGCCAT, SEQIDNO:2GGTTGCTGAGCTTCAAGGTT, for the β-tublin gene that increases; SEQIDNO:3ACACCGACCCAGCACCAGA, SEQIDNO:4TTAGCAATAACCGCCCAAA, for the SdhB gene that increases; SEQIDNO:5CACCCGCGTAGCAAGAGATG, SEQIDNO:6TGAGTCAGGTCTCCCTTTGC, for the BcOS1 gene that increases; SEQIDNO:7ATCATGCCCTTGAATTTTGTG, SEQIDNO:8CTTTGCCTCCCCTTTCTTCTC, for the Erg27 gene that increases.
Carry out experimental group and control group PCR reacts, the template that the genomic dna wherein containing the testing sample extracted in experimental group PCR reaction mixture reacts as PCR, reaction system is as follows,
Distilled water (the ddH of equivalent is added in control group PCR reaction mixture
2o) as the template of PCR reaction, reaction system is as follows:
PCR response procedures: 94 DEG C of 5min; 94 DEG C of 30sec, 59 DEG C of 30sec, 72 DEG C of 20sec, 35cycles; 72 DEG C of 10min.
After PCR terminates, get 5 μ L reaction solutions and carry out sepharose (2%) electrophoresis, detected result.
Accompanying drawing explanation
Fig. 1 embodiment of the present invention electrophoresis detection result figure.
Embodiment
The multiplexed PCR amplification of embodiment 1, bacterial strain sample
Adopt UNIQ-10 pillar fungal genomic DNA extraction agent box, purchased from Sangon Biotech (Shanghai) Co., Ltd., extract the genomic dna of 5 strain bacterial strain to be detected.PCR reactive component, purchased from invitrogen company, comprises Taq enzyme, and dNTPs, PCR damping fluid is not (containing Mg
2+), MgCl
2.
Carry out experimental group and control group PCR reacts, the template that the genomic dna wherein containing the testing sample extracted in experimental group PCR reaction mixture reacts as PCR, reaction system is as follows,
Distilled water (the ddH of equivalent is added in control group PCR reaction mixture
2o) as the template of PCR reaction, reaction system is as follows:
PCR response procedures: 94 DEG C of 5min; 94 DEG C of 30sec, 59 DEG C of 30sec, 72 DEG C of 20sec; 72 DEG C of 10min.
After PCR terminates, get 5 μ L reaction solutions and carry out electrophoresis in sepharose (2%, namely 0.6g solute dissolves is in 30ml solvent), detected result.As shown in Figure 1, loading wells M is DNAmarker, loading wells 1-5 is detection sample, and loading wells 6 is negative control group.According to electrophoresis result, primer of the present invention can go out β-tub, BsOS1, erg27, SdhB tetra-gene fragments by Successful amplification.
Claims (2)
1., for a method for the pathogen of Botrytis cinerea four genes that increase, it is characterized in that altogether in two steps:
The first step: extract the pathogen of Botrytis cinerea genome
Second step: carry out PCR reaction: adopt four pairs of primers, be respectively used to amplification the pathogen of Botrytis cinerea β-tub, BsOS1, erg27, SdhB gene, four pairs of primers are respectively
SEQIDNO:1CGATACCGTTGTCGAGCCAT
SEQIDNO:2GGTTGCTGAGCTTCAAGGTT
SEQIDNO:3ACACCGACCCAGCACCAGA
SEQIDNO:4TTAGCAATAACCGCCCAAA
SEQIDNO:5CACCCGCGTAGCAAGAGATG
SEQIDNO:6TGAGTCAGGTCTCCCTTTGC
SEQIDNO:7ATCATGCCCTTGAATTTTGTG
SEQIDNO:8CTTTGCCTCCCCTTTCTTCTC
The template that genomic dna containing the testing sample extracted in PCR reaction mixture reacts as PCR, reaction system is as follows,
PCR response procedures: 94 DEG C of 5min; 94 DEG C of 30sec, 59 DEG C of 30sec, 72 DEG C of 20sec; 72 DEG C of 10min.
2., for a multiple PCR primer group for the pathogen of Botrytis cinerea four genes that increase, correspond to amplification the pathogen of Botrytis cinerea β-tub, BsOS1, erg27, SdhB gene, four pairs of primers are respectively
SEQIDNO:1CGATACCGTTGTCGAGCCAT
SEQIDNO:2GGTTGCTGAGCTTCAAGGTT
SEQIDNO:3ACACCGACCCAGCACCAGA
SEQIDNO:4TTAGCAATAACCGCCCAAA
SEQIDNO:5CACCCGCGTAGCAAGAGATG
SEQIDNO:6TGAGTCAGGTCTCCCTTTGC
SEQIDNO:7ATCATGCCCTTGAATTTTGTG
SEQIDNO:8CTTTGCCTCCCCTTTCTTCTC。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510593450.0A CN105154432B (en) | 2015-09-17 | 2015-09-17 | A kind of method and its multiple PCR primer group for being used to expand four genes of the pathogen of Botrytis cinerea |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510593450.0A CN105154432B (en) | 2015-09-17 | 2015-09-17 | A kind of method and its multiple PCR primer group for being used to expand four genes of the pathogen of Botrytis cinerea |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105154432A true CN105154432A (en) | 2015-12-16 |
| CN105154432B CN105154432B (en) | 2018-04-06 |
Family
ID=54795477
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510593450.0A Active CN105154432B (en) | 2015-09-17 | 2015-09-17 | A kind of method and its multiple PCR primer group for being used to expand four genes of the pathogen of Botrytis cinerea |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105154432B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119364A (en) * | 2016-07-02 | 2016-11-16 | 北京工业大学 | A kind of based on suspension chip system for the multiple detection method of Botrytis cinerea drug resistance related locus |
| CN106687604A (en) * | 2014-09-11 | 2017-05-17 | 阿格洛法士公司 | Methods for pathogen detection and disease management on meats, plants, or plant parts |
| CN108018374A (en) * | 2017-12-29 | 2018-05-11 | 华中农业大学 | For detecting drug-fast kit of the Botrytis cinerea to benzimidazole germicide |
| CN109338004A (en) * | 2018-11-12 | 2019-02-15 | 上海市农业科学院 | A kind of detection ash arrhizus bacteria is to the combination of the primer of Boscalid resistance, kit and method |
| CN115786560A (en) * | 2022-08-12 | 2023-03-14 | 上海市农业科学院 | Primer group, kit and method for detecting B subunit point mutation type of succinate dehydrogenase of botrytis cinerea |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399776A (en) * | 2011-09-02 | 2012-04-04 | 中国水产科学研究院黄海水产研究所 | Microsatellite marker quadruple PCR primer for identifying Penaeus chinesis family and identification method |
| CN103320534A (en) * | 2013-06-27 | 2013-09-25 | 广西壮族自治区农业科学院植物保护研究所 | Method for synchronously detecting four whitefly transmitted gemini-viruses infecting tomatoes |
| CN103911461A (en) * | 2014-03-13 | 2014-07-09 | 湖南农业大学 | A method of simultaneously detecting a plurality of garlic viruses by quadruple RT-PCR and a primer composition thereof |
| CN103937892A (en) * | 2014-04-21 | 2014-07-23 | 华南农业大学 | Multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit for various duck-derived pathogenic bacteria |
| CN104152541A (en) * | 2014-09-03 | 2014-11-19 | 贵州省烟草科学研究院 | Quadruple PCR primers and method for rapidly detecting transgenic roasted tobacco leaves |
| CN104450966A (en) * | 2014-12-09 | 2015-03-25 | 艾军 | Multiplex PCR kit for simultaneously detecting four viruses carried by ruminants |
-
2015
- 2015-09-17 CN CN201510593450.0A patent/CN105154432B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399776A (en) * | 2011-09-02 | 2012-04-04 | 中国水产科学研究院黄海水产研究所 | Microsatellite marker quadruple PCR primer for identifying Penaeus chinesis family and identification method |
| CN103320534A (en) * | 2013-06-27 | 2013-09-25 | 广西壮族自治区农业科学院植物保护研究所 | Method for synchronously detecting four whitefly transmitted gemini-viruses infecting tomatoes |
| CN103911461A (en) * | 2014-03-13 | 2014-07-09 | 湖南农业大学 | A method of simultaneously detecting a plurality of garlic viruses by quadruple RT-PCR and a primer composition thereof |
| CN103937892A (en) * | 2014-04-21 | 2014-07-23 | 华南农业大学 | Multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit for various duck-derived pathogenic bacteria |
| CN104152541A (en) * | 2014-09-03 | 2014-11-19 | 贵州省烟草科学研究院 | Quadruple PCR primers and method for rapidly detecting transgenic roasted tobacco leaves |
| CN104450966A (en) * | 2014-12-09 | 2015-03-25 | 艾军 | Multiplex PCR kit for simultaneously detecting four viruses carried by ruminants |
Non-Patent Citations (3)
| Title |
|---|
| SHINPEI BANNO ET AL.: "Genotyping of benzimidazole-resistant and dicarboximide-resistant mutations in botrytis cinerea using real-time polymerase chain reaction assays", 《APS JOURNALS》 * |
| Y.N.YIN ET AL.: "Molecular characterizaiton of boscalid resistance in field isolations of botrytis cinerea from apple", 《PHYTOPATHOLOGY》 * |
| 张鑫等: "灰霉病菌抗药位点及其分子检测方法研究进展", 《生物技术进展》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106687604A (en) * | 2014-09-11 | 2017-05-17 | 阿格洛法士公司 | Methods for pathogen detection and disease management on meats, plants, or plant parts |
| CN106119364A (en) * | 2016-07-02 | 2016-11-16 | 北京工业大学 | A kind of based on suspension chip system for the multiple detection method of Botrytis cinerea drug resistance related locus |
| CN108018374A (en) * | 2017-12-29 | 2018-05-11 | 华中农业大学 | For detecting drug-fast kit of the Botrytis cinerea to benzimidazole germicide |
| CN109338004A (en) * | 2018-11-12 | 2019-02-15 | 上海市农业科学院 | A kind of detection ash arrhizus bacteria is to the combination of the primer of Boscalid resistance, kit and method |
| CN115786560A (en) * | 2022-08-12 | 2023-03-14 | 上海市农业科学院 | Primer group, kit and method for detecting B subunit point mutation type of succinate dehydrogenase of botrytis cinerea |
| CN115786560B (en) * | 2022-08-12 | 2024-03-22 | 上海市农业科学院 | Primer group, kit and method for detecting B subunit point mutation type of succinate dehydrogenase of Botrytis cinerea |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105154432B (en) | 2018-04-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105154432A (en) | Method for amplifying four genes of Botrytis cinerea and multiplex PCR (polymerase chain reaction) primer set thereof | |
| Susca et al. | Variation in fumonisin and ochratoxin production associated with differences in biosynthetic gene content in Aspergillus niger and A. welwitschiae isolates from multiple crop and geographic origins | |
| Zhu et al. | Chromosome-level genome map provides insights into diverse defense mechanisms in the medicinal fungus Ganoderma sinense | |
| Johansson et al. | Detection of Chalara fraxinea from tissue of Fraxinus excelsior using species‐specific ITS primers | |
| Murase et al. | Impact of protists on the activity and structure of the bacterial community in a rice field soil | |
| Knauth et al. | Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice | |
| Fraser et al. | Evolution of the mating type locus: insights gained from the dimorphic primary fungal pathogens Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii | |
| Currò et al. | New microsatellite loci for pomegranate, Punica granatum (Lythraceae) | |
| Madania et al. | Morphological and M olecular C haracterization of F usarium I solated F rom M aize in S yria | |
| Roberts et al. | Three novel lineages of ‘Candidatus Liberibacter africanus’ associated with native rutaceous hosts of Trioza erytreae in South Africa | |
| Najafzadeh et al. | Rapid identification of fungal pathogens by rolling circle amplification using Fonsecaea as a model | |
| Machado et al. | The fur gene as a new phylogenetic marker for Vibrionaceae species identification | |
| de Salas et al. | Quorum-sensing mechanisms mediated by farnesol in Ophiostoma piceae: effect on secretion of sterol esterase | |
| Pootakham et al. | Single nucleotide polymorphism marker development in the rubber tree, Hevea brasiliensis (Euphorbiaceae) | |
| Mello et al. | Genetic variability of Tuber uncinatum and its relatedness to other black truffles | |
| Oelschlägel et al. | Styrene oxide isomerase of Sphingopyxis sp. Kp5. 2 | |
| Datta et al. | Amplification of chickpea‐specific SSR primers in Cajanus species and their validity in diversity analysis | |
| Luchi et al. | High-Resolution Melting Analysis: a new molecular approach for the early detection of Diplodia pinea in Austrian pine | |
| Besaury et al. | Expression of copper-resistance genes in microbial communities under copper stress and oxic/anoxic conditions | |
| Brylińska et al. | Evaluation of PCR markers for Phytophthora infestans mating type determination | |
| Singh et al. | Diversification of nitrogen fixing bacterial community using nifH gene as a biomarker in different geographical soils of Western Indian Himalayas | |
| WO2016112179A1 (en) | Methods and kits for detecting fungus and bacteria in cannabis | |
| Louis et al. | Host‐Range Dynamics of Cochliobolus lunatus: From a Biocontrol Agent to a Severe Environmental Threat | |
| KR101212039B1 (en) | Primer set for Gibberella zeae producing deoxynivalenol and kit using the same | |
| Satya et al. | Genetic Analysis of Population Structure Using Peroxidase Gene and Phenylalanine Ammonia‐Lyase Gene‐Based DNA Markers: A Case Study in Jute (Corchorus spp.) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |