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

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...

Examples

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 ...

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...

Examples

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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