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Standard molecule for wheat tilletia indica mitra detection and construction method thereof

A technology of standard molecules and detection standards, which is applied in the field of standard molecules for the detection of T. indica and its preparation, can solve the problem that the stability of genomic DNA cannot meet long-term and frequent use, the extraction process is complicated, and the promotion and application are restricted. problems, to achieve good uniformity and stability, high purity, and easy to obtain results

Inactive Publication Date: 2011-10-12
SHENZHEN AUDAQUE DATA TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The extraction process is complicated, the preparation is inconvenient, the stability of the extracted genomic DNA cannot meet the needs of long-term and frequent use, and the quality of genomic DNA used by different detection methods or detection laboratories is different, which restricts Popularization and Application of Detection Standards and Other Molecular Detection Methods for Tilletia indica

Method used

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  • Standard molecule for wheat tilletia indica mitra detection and construction method thereof
  • Standard molecule for wheat tilletia indica mitra detection and construction method thereof
  • Standard molecule for wheat tilletia indica mitra detection and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 Construction of Tilletia indica Detection Standard Molecule

[0038] 1. Experimental materials

[0039] 1. Test strains

[0040] Tilletia indica intercepted wheat from India, Brazil and Mexico, and Tilletia rye intercepted wheat from the United States. 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. Experimental 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 Bio...

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 indica DNA and its standard molecules were used as positive controls, Tilletia rye DNA and its cloned plasmids were used as negative controls, and the primers and probes in Table 5 were used to determine the specificity of Tilletia indica standard molecules 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 ...

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Abstract

The invention discloses a standard molecule for wheat tilletia indica mitra detection. The standard molecule is plasmid capable of being replicated, the plasmid contains at least one of sequences A and B, the homology of the A (Seq ID No. 1) and a nucleotide sequence (AF218059) of the last fragment 2.3kb of wheat tilletia indica mitra mitochondria DNA is 99.56 percent, and the homology of the B (Seq ID No. 2) and a DNA sequence (AF399888) of a transcribed spacer in wheat tilletia indica mitra ribosome is 99.85 percent. The invention also discloses a preparation and detection method of the standard molecule. The constructed standard molecule can be used for substituting wheat tilletia indica mitra DNA and used as positive control of wheat tilletia indica mitra molecular detection, is suitable for qualitative polymerase chain reaction (PCR) detection and quantitative PCR detection of wheat tilletia indica mitra, and is suitable for port inspection and quarantine, agricultural production, plant protection and the like.

Description

technical field [0001] The invention relates to a standard molecule for detection in the fields of port inspection and quarantine, agricultural production and plant protection, in particular to a standard molecule for detection of Tilletia indica and a preparation method thereof. Background technique [0002] Tilletia indica Mitra (synonym Neovossia indica (Mitra) Mundkur) is a pathogenic fungus that spreads rapidly and is very harmful. More than 40 countries have listed it as a quarantine pest (OEPP / EPPO, 1980; CABI / EPPO, 1992). So far, in addition to its origin in India, the disease has been reported in Pakistan, Iraq, Nepal, Afghanistan, Mexico, South Africa and the United States (Warham, 1986; Singh et al., 1989; Bonde et al., 1997) , becoming one of the most dangerous fungal diseases threatening world wheat production and trade. [0003] T. indica mainly damages wheat (Triticum aestivum L.) and can also damage durum wheat (T.durum Desf.). It can not only cause the y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12R1/645
Inventor 章桂明李小焦陈枝楠程颖慧王颖向才玉
Owner SHENZHEN AUDAQUE DATA TECH
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