CN102533942A - Specific primers for amplifying Verticillium lecanii - Google Patents
Specific primers for amplifying Verticillium lecanii Download PDFInfo
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- CN102533942A CN102533942A CN2010105814690A CN201010581469A CN102533942A CN 102533942 A CN102533942 A CN 102533942A CN 2010105814690 A CN2010105814690 A CN 2010105814690A CN 201010581469 A CN201010581469 A CN 201010581469A CN 102533942 A CN102533942 A CN 102533942A
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- verticillium lecanii
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Abstract
The invention relates to a pair of specific primers for amplifying Verticillium lecanii. The invention solves the problem of lack of primers for amplifying the Verticillium lecanii target sequence from a sample DNA. The specific primers for amplifying Verticillium lecanii are (5'-CGGCGTCCGGACGCGGACCCAG-3' and VR(5'-CCCCAACGCCGACTTCCCCGAG-3'). The primers provided by the invention can accurately amplify the target segment of Verticillium lecanii.
Description
Technical field
The present invention relates to the Auele Specific Primer of a kind of Verticillium lecanii that is used to increase, can be used for Molecular Detection Verticillium lecanii in the sample.
Background technology
Verticillium lecanii (Verticillium lecanii) is all insect pathogenic fungus very widely of a kind of regional distribution and host range; Find first in the Ceylon in 1861 by Nivter that the earliest China is that the damp field of nineteen fifty-nine Japan obtains Verticillium lecanii double lucky from the sample in Taiwan, the separation.Verticillium lecanii belongs to Verticillium always and belongs to; But according to strain morphology difference and molecular genetic analysis; Now it is changed to Lecanicillium and belong to, it is very abundant to comprise kind, can infect pathogenic fungi, insect and pathogenic nematode on the various crop.
Verticillium lecanii has been proved to be a kind of microbial pesticide that has potentiality, insects such as states such as America and Europe, the FSU are applied to successfully that the greenhouse anti-eliminates aphis, trialeurodes vaporariorum and thrips.But; Verticillium lecanii is different with the characteristic of common entomiasis fungi muscardine and green muscardine fungus; Can't use " greater wax moth lures the collection method " (greater wax moth is insensitive to Verticillium lecanii) and " colony counting method " (lack the selectivity of Verticillium lecanii is cultivated) that the Verticillium lecanii in the sample is effectively studied; Can only do simple assessment to the popular situation of Verticillium lecanii through the catch an illness quantity of worm corpse of nature, this has seriously restricted such application of useful fungi in the integrated pest control system.
Summary of the invention
The present invention amplifies the problem of Verticillium lecanii target sequence in order to solve present shortage primer from sample DNA, and the Auele Specific Primer of a kind of Verticillium lecanii that is used to increase is provided.
The present invention's be used to increase Auele Specific Primer of Verticillium lecanii is: forward primer 5 '-CGGCGTCCGGACGCGGACCCAG-3 ' and reverse primer 5 '-CCCCAACGCCGACTTCCCCGAG-3 '.With the primer of the present invention to called after VF/VR.
Primer of the present invention is that the characteristics that the sequence base according to fungi rrna rDNA there are differences design.Use primer VF/VR of the present invention and can from all kinds of sample DNAs, directly amplify the Verticillium lecanii purpose fragment that length is 299bp.
Description of drawings
Fig. 1 be among the embodiment 1 the VF/VR primer to the gel electrophoresis figure of 7 kind fungal DNA amplified productions.
Fig. 2 be among the embodiment 2 the VF/VR primer to the gel electrophoresis figure of pedotheque DNA cloning product.
Embodiment
Respectively with Verticillium lecanii (Verticillium lecanii); Big beautiful Verticillium (Verticillium dahliae); Beauveria bassiana (Beauveria bassiana); Metarhizium anisopliae (Metarhizium anisopliae); Rose cigarette Paecilomyces varioti (Paecilomyces fumosoroseus); Point sickle spore bacterium (Fusarium oxysporum); It is the specificity proof test that template is carried out primer VF/VR that 7 of botrytis cinerea (Botrytis cinrea) and alternaric bacterias (Alternaria alternata) belong to fungal DNA.
The pcr amplification system is: 25 μ L are by 0.5 μ LDNA template (about 10ng), 0.5 μ L forward primer VF (10 μ M), 0.5 μ L reverse primer VR (10 μ M), 0.5 μ L dNTPs (10mmol/L), 2.5 μ L, 10 * PCR buffer (with MgCl2), 0.2 μ l TaqDNA polysaccharase (5U/ μ L), and the sterilization distilled water is supplied 25 μ L.The pcr amplification reaction condition is: preparatory 94 ℃ of 3min of sex change, and 94 ℃ of 30s of sex change, the 55 ℃ of 30s that anneal extend 72 ℃ of 45s, totally 35 circulations, 72 ℃ are extended 5min, 4 ℃ of preservations.
The sequence that this embodiment amplifies is carried out the agarose gel electrophoresis detection; Detected result is as shown in Figure 1; The M swimming lane is standard BM2000 among Fig. 1; The 1-4 swimming lane is respectively the amplification that contains Verticillium lecanii (Verticillium lecanii) 4 strain different strains DNA; The 5-11 swimming lane is respectively the amplification that contains big beautiful Verticillium (Verticillium dahliae), beauveria bassiana (Beauveria bassiana), Metarhizium anisopliae (Metarhizium anisopliae), rose cigarette Paecilomyces varioti (Paecilomyces fumosoroseus), sharp sickle spore bacterium (Fusarium oxysporum), botrytis cinerea (Botrytis cinrea) and alternaric bacteria (Alternaria alternata) DNA, and No. 12 swimming lanes are not for containing the amplification of DNA blank.As can be seen from Figure 1 this embodiment be used to increase Auele Specific Primer VF/VR of Verticillium lecanii can amplify the target sequence fragment of Verticillium lecanii accurately, and fails from nearly edge fungal DNA and blank, to amplify nucleic acid fragment.1,2,3, No. 4 corresponding sequence fragment of swimming lane is connected back transformed into escherichia coli DH5 α competent cell order-checking respectively at the T carrier; Sequencing result is: sequence length is 299bp; And analyze through blast, be the distinguished sequence of Verticillium lecanii (Verticillium lecanii).
Use primer VF/VR 24 parts of soil samples (picking up from Hebei Langfang City and Wuhan City, Hubei) DNA is carried out the pcr amplification test.The pcr amplification system is: 25 μ L are by 0.5 μ L dna profiling (about 10ng), 0.5 μ L forward primer VF (10 μ M), 0.5 μ L reverse primer VR (10 μ M), 0.5 μ L dNTPs (10mmol/L), 2.5 μ L, 10 * PCR buffer (with MgCl
2), 0.2 μ l TaqDNA polysaccharase (5U/ μ L), the sterilization distilled water supply 25 μ L.The pcr amplification reaction condition is: preparatory 94 ℃ of 3min of sex change, and 94 ℃ of 30s of sex change, the 55 ℃ of 30s that anneal extend 72 ℃ of 45s, totally 35 circulations, 72 ℃ are extended 5min, 4 ℃ of preservations.
The sequence that this embodiment amplifies is carried out the agarose gel electrophoresis detection; Detected result is as shown in Figure 2; The M swimming lane is standard BM2000 among Fig. 2; The 1-12 swimming lane is respectively the amplification of Hebei Langfang City pedotheque DNA, and the 13-24 swimming lane is respectively the amplification of the pedotheque DNA of Wuhan City, Hubei.As can be seen from Figure 2 this embodiment be used for increasing Auele Specific Primer VF/VR of Verticillium lecanii can amplify the target sequence fragment of the Verticillium lecanii of all pedotheque DNA accurately.The sequence fragment that 2,4,8,16, No. 24 swimming lanes of random choose are corresponding connects back transformed into escherichia coli DH5 α competent cell order-checking respectively at the T carrier; Sequencing result is: sequence length is 299bp; And analyze through blast, be the distinguished sequence of Verticillium lecanii (Verticillium lecanii).
Claims (1)
1. the Auele Specific Primer of the Verticillium lecanii that is used to increase, the Auele Specific Primer of the Verticillium lecanii that it is characterized in that being used to increasing is:
Forward primer VF:5 '-CGGCGTCCGGACGCGGACCCAG-3 '
Reverse primer VR:5 '-CCCCAACGCCGACTTCCCCGAG-3 '.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010105814690A CN102533942A (en) | 2010-12-10 | 2010-12-10 | Specific primers for amplifying Verticillium lecanii |
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| CN2010105814690A CN102533942A (en) | 2010-12-10 | 2010-12-10 | Specific primers for amplifying Verticillium lecanii |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898236A (en) * | 2014-04-24 | 2014-07-02 | 青海春天药用资源科技利用有限公司 | Primer pair, kit and method for detecting verticillium lecanii ferment powder added in cordyceps sinensis ultra-fine powder |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW201006388A (en) * | 2008-08-11 | 2010-02-16 | Ishihara Sangyo Kaisha | Lecanicillium muscarium strain v-5, pest extermination method using the same, and microorganism pesticide comprising the same |
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2010
- 2010-12-10 CN CN2010105814690A patent/CN102533942A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW201006388A (en) * | 2008-08-11 | 2010-02-16 | Ishihara Sangyo Kaisha | Lecanicillium muscarium strain v-5, pest extermination method using the same, and microorganism pesticide comprising the same |
Non-Patent Citations (2)
| Title |
|---|
| GENBANK: "AJ292382", 《GENBANK》, 24 February 2001 (2001-02-24) * |
| 阎峻: "生理生化方法在蜡蚧轮枝菌及一些相关菌聚类分析上的应用", 《中国虫生真菌研究与应用》, 31 December 1991 (1991-12-31), pages 179 - 185 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898236A (en) * | 2014-04-24 | 2014-07-02 | 青海春天药用资源科技利用有限公司 | Primer pair, kit and method for detecting verticillium lecanii ferment powder added in cordyceps sinensis ultra-fine powder |
| CN103898236B (en) * | 2014-04-24 | 2015-05-27 | 青海春天药用资源科技利用有限公司 | Primer pair, kit and method for detecting verticillium lecanii ferment powder added in cordyceps sinensis ultra-fine powder |
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Application publication date: 20120704 |