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Standard molecule for detecting tilletia walkeri castebury&carris and construction method thereof

A standard molecule and smut fungus technology, applied in the field of standard molecules, can solve the problems that stability cannot meet long-term and frequent use, lack of comparability and repeatability of test results, restricting the popularization and application of ryegrass smut fungus, etc. Achieving the effects of good uniformity and stability, convenient preparation and easy preparation

Inactive Publication Date: 2013-02-20
深圳出入境检验检疫局动植物检验检疫技术中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the acquisition process of genomic DNA is complicated, the preparation is very inconvenient, and its stability cannot meet the needs of long-term and frequent use. Moreover, due to the different sources of genomic DNA used in different testing laboratories, the extraction quality is different, making different experiments The lack of comparability and repeatability of the test results in the laboratory has restricted the popularization and application of T. ryetilia detection standards and other molecular detection methods.

Method used

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  • Standard molecule for detecting tilletia walkeri castebury&carris and construction method thereof
  • Standard molecule for detecting tilletia walkeri castebury&carris and construction method thereof
  • Standard molecule for detecting tilletia walkeri castebury&carris and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 Construction of Tilletia rye detection standard molecule

[0038] 1. Experimental reagents

[0039] 1. Test strains

[0040]Tilletia indica was intercepted from Brazil, India and Mexico wheat, and Tilletia rye was intercepted from American wheat. The above-mentioned tested strains were all preserved by the Animal and Plant Inspection and Quarantine Technology Research Center of Shenzhen Entry-Exit Inspection and Quarantine Bureau (Table 1) .

[0041] Table 1 Tested strains

[0042]

[0043] Note: Marked "*" is the strain used for cloning in this study.

[0044] 2. Test materials

[0045] Restriction enzyme XbaI, KpnI and restriction enzyme buffer, pMD19-T vector, T4 DNA ligase and its buffer, dNTPs, Taq DNA polymerase and its buffer, DL2000Marker, Agarose Gel DNA Purification Kit and MiniBEST Plasmid Purification Kit was purchased from Dalian Bao Biological Engineering Co., Ltd. DH5α competent cells were purchased from Beijing Tiangen Biological Co...

Embodiment 2

[0066] Embodiment 2 Standard Molecular Performance Evaluation

[0067] 1. Experimental reagents

[0068] Universal Real-time Master Mix was purchased from ABI Company.

[0069] Primers and probes were synthesized by Shanghai Chaoshi Biotechnology Co., Ltd.

[0070] 2. Experimental equipment

[0071] ABI 7700 real-time fluorescent PCR instrument.

[0072] 3. Verification methods and results

[0073] 1. Specificity verification

[0074] Tilletia rye DNA and its standard molecules were used as positive controls, T. indica DNA and its cloned plasmids were used as negative controls, and the primers and probes in Table 5 were used to carry out the standard molecule specificity of T. Real-time fluorescent PCR detection to verify its specificity. The real-time fluorescent PCR detection reaction was carried out with reference to the real-time fluorescent PCR detection method of T. ryetilia established by Zhang Guiming, Zhang Zheng et al. (2005). The reaction system was 10 μL (Ta...

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Abstract

The invention discloses a standard molecule for detecting tilletia walkeri castebury&carris;. The standard molecule is a plasmid which can be duplicated, and the plasmid contains at least one of a sequence A (Seq ID N0.1) and a sequence B (Seq ID N0.2); the sequence A is 99.65% homologous with a 2.3kb nucleotide sequence of the mitochondrial DNA of the tilletia walkeri castebury&carris; and the sequence B is 100% homologous with the DNA sequence of a ribosomal internal transcribed spacer of the tilletia walkeri castebury&carris;. The invention also discloses a preparation method and detection method of the standard molecule. The standard molecule constructed in the invention can serve as a positive control for detecting the molecule of the tilletia walkeri castebury&carris; instead of the DNA of the tilletia walkeri castebury&carris; is suitable for qualitative and quantitative PCR (polymerase chain reaction) detection of the tilletia walkeri castebury&carris; and is suitable for the inspection and quarantine department of the port, the agricultural production department, the plant protection department and the like.

Description

technical field [0001] The invention relates to a standard molecule used for detection in the fields of port inspection and quarantine, agricultural production and plant protection, in particular to a standard molecule used for detection of T. ryesis and a preparation method thereof. Background technique [0002] Tilletia walkeri Castebury&Carris is a fungus that is very similar to Tilletia indica in terms of morphological characteristics, and mainly damages ryegrass. The bacterium is distributed in the United States, Australia, New Zealand, Canada, Denmark, the Netherlands, Japan, Spain and other places, and has not been reported in my country. Tilletia rye was first discovered in the wheat seed washing liquid of Oregon and the southeastern states in the large census of Tilletia indica carried out by the US Department of Agriculture in 1996. In 1999, Castlebury et al. The slight differences with T. indica in morphological characteristics and biology were identified and nam...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/645
Inventor 章桂明李小焦陈枝楠程颖慧王颖向才玉
Owner 深圳出入境检验检疫局动植物检验检疫技术中心
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