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Detection primer, probe and detection method for tilletia indica mitra

A technology for detection primers and detection probes, which is applied in the field of detection primers and probes for Tilletia indica, can solve the problem that cannot meet the needs of detection, few, sometimes even only one, several or very small amounts spores and other problems, achieving the effect of shortening the detection time and improving the sensitivity

Inactive Publication Date: 2007-10-17
上海出入境检验检疫局
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Animal and plant quarantine departments, agricultural production departments, plant protection, fungal research departments and other units often obtain very few spores in the samples taken in the country or in the field, sometimes even only one, a few or a very small amount of spores Therefore, the existing method can not meet the needs of animal and plant quarantine inspection department, agricultural production department, plant protection department and fungal scientific research department for the detection of T. indica

Method used

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  • Detection primer, probe and detection method for tilletia indica mitra
  • Detection primer, probe and detection method for tilletia indica mitra
  • Detection primer, probe and detection method for tilletia indica mitra

Examples

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Embodiment 1

[0026] Example 1 Teliospore DNA Acquisition

[0027] Prepare sterilized clean coverslip pieces (2mm×2mm), pick a single teliospore onto one coverslip piece, cover it with another coverslip piece, gently press the coverslip piece to make the spores Crack, and finally transfer the two coverslip pieces together to a small PCR tube containing the PCR reaction solution for amplification.

Embodiment 2

[0028] Example 2 Design of primers and probes

[0029] As shown in Figure 1, the TaqMan-MGB probe TIND for the detection of T. indica was designed for the specific site difference (G / A) of the sequence of the pathogen ITS (internal transcribed spacer, ribosomal gene transcribed spacer) , to establish a single teliospore Real timePCR (real-time fluorescent PCR) detection technology.

[0030] As shown in Figure 2, design the upstream primer D-FP a : 5'-TTCTCTTTTATCCCAACACCCAAACT-3' (SEQ ID NO.2); design downstream primer T-RP b : 5'-CTTATCGCATTTCGCTGCG-3'(SEQ ID NO.3); Design TaqMan-MGB probe TIND: 5'(FAM)-CGGAAGGAACGAGGC-3'(MGB)(SEQ ID NO.4).

Embodiment 3

[0031] Example 3 The first round of amplification (ITS amplification)

[0032] As shown in Figure 1, use primer Til1 (5'-AAGGTTTCTGTAGGTGAACCT-3') (SEQ ID NO.5) / Til4 (5'-GATAT GCTTAAGTTCAGCGGG-3') (SEQ ID NO.6) to carry out PCR amplification of 20 Cycle, the total volume of PCR reaction is 30μl, containing: 10mmol / L Tris-HCl; 50mmol / L KCl (pH8.3); 1.5mmol / L MgCl 2 ; dATP, dGTP, dCTP, dTTP concentration is 100 μmol / L; primer concentration 100nmol / L; 0.5U Taq DNA polymerase (Treasure Bioengineering Co., Ltd.). Add double-distilled water to a final volume of 30 μl, mix and centrifuge, and place in a PCR machine for amplification. Take 1 μl of the product for real-time fluorescent PCR amplification.

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Abstract

The present invention discloses a primer and probe for detecting wheat Tilletia indica, and method. The present primer and prove can specificly distinguish wheat Tilletia indica, shorten detection time and promote the detection sensitivity.

Description

technical field [0001] The invention relates to a detection primer and probe for Tilletia indica; in addition, the invention also relates to a method for detecting Tilletia indica using the primer and probe. Background technique [0002] Tilletia indica Mitra is a dangerous pest of world concern, and it is also a type of dangerous pest announced by my country. It is mainly distributed in India, Pakistan, Iran, Iraq, Nepal, Afghanistan, Brazil, Mexico, and the United States and South Africa. In recent years, with the opening of the grain market, the quantity of imported grain has increased continuously, which has increased the opportunities and risks of the introduction of wheat smut. In particular, the United States, a major grain exporter, also has the occurrence of T. A new problem in the detection of Tilletia indica in wheat. Currently commonly used detection and identification methods are morphological feature identification and conventional PCR technology. Identificat...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 易建平周国梁印丽萍
Owner 上海出入境检验检疫局
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