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LAMP (loop-mediated isothermal amplification) primer group for quickly detecting sporisorium scitamineum, kit and detection method thereof

A technology of sugarcane smut bacteria and a kit, which is applied in the biological field, can solve the problems of low specificity, cumbersome steps, and high cost, and achieve the effects of high sensitivity, strong specificity, and low cost

Active Publication Date: 2014-09-10
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims at the disadvantages of high cost, low specificity, cumbersome steps and easy pollution in the detection method of sugarcane smut in the prior art, and provides a LAMP primer set for rapid detection of sugarcane smut

Method used

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  • LAMP (loop-mediated isothermal amplification) primer group for quickly detecting sporisorium scitamineum, kit and detection method thereof
  • LAMP (loop-mediated isothermal amplification) primer group for quickly detecting sporisorium scitamineum, kit and detection method thereof
  • LAMP (loop-mediated isothermal amplification) primer group for quickly detecting sporisorium scitamineum, kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Primer Design

[0057] The present invention redesigns primers according to the ITS sequence of ribosomal gene transcription spacer of sugarcane smut, and has a pair of outer primers F3 / B3 and a pair of inner primers FIP / BIP. The primer sequences are shown in SEQ ID NO: 1-4.

[0058] The sequences of the above 2 pairs of primers are as follows:

[0059] F3: 5`-GGTGTTCAGAAGCACTCCAA-3`;

[0060] B3: 5`-ATTACGAAAGAGCTGGCGG-3`;

[0061] FIP: 5`-CTTTTGGCCCATCTTCCCTGCCGTCGCGTCCAGCTTCTTG-3`;

[0062] BIP: 5`-CTTCGTCCGTCTTTGCCTGTCATCGGTAGTGAGGGTTTTGC-3`.

Embodiment 2

[0063] Example 2 Loop-mediated isothermal amplification (LAMP) rapid detection method for sugarcane smut

[0064] Using the primer pair described in Example 1, a rapid detection method for sugarcane smut loop-mediated isothermal amplification (LAMP) was established. The specific steps are as follows:

[0065] S1. Genomic DNA extraction: In April 2014, 9 heart leaves of sugarcane variety Xintaitang No. 22 sugarcane seedlings were collected in the field (with early symptoms of sugarcane smut, but the smut was not extracted). The total DNA of the sugarcane smut genome was extracted from the heart leaves of small seedlings, and the samples were obtained and numbered as samples 2-10 in sequence;

[0066] S2. Prepare the reaction system: 25 μL of the total system, including 2.5 μL of genomic DNA, 0.5 μL of Bst DNA polymerase (8U / μL), 19 μL of detection base solution, 0.5 μL of 0.2 μmol / L F3 and B3 primers, 1.6 μmol / L 1 μL each of FIP and BIP primers. In addition, add 50 μL of st...

Embodiment 3

[0091] Example 3 Establishment of a Loop-Mediated Isothermal Amplification (LAMP) Rapid Detection Kit for Sugarcane Smut

[0092] Prepare the sugarcane smut loop-mediated isothermal amplification (LAMP) rapid detection kit according to the following composition:

[0093] Contains Genomic DNA Extraction Solution, LAMP Reaction Solution, Positive Control Substance, Negative Control Substance, Stabilizer Solution, Chromogenic Solution.

[0094] Among them, the LAMP reaction solution contains two pairs of specific primers F3 / B3 (outer primer pair) and FIP / BIP (inner primer pair), Bst DNA polymerase, and detection base solution.

[0095] Among them, the detection base liquid is dNTPs, MgSO 4 and betaine, the formula is a final concentration of 1.3~1.5 mmol / L dNTPs, 0.6~0.9 mmol / L betaine and 7~9 mmol / L MgSO 4 .

[0096] Wherein, the genomic DNA extraction solution described in the kit is a CTAB extraction solution, and its formula is: 2% CTAB, 20mmol / L EDTA, 100 mmol / L Tris-HC...

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Abstract

The invention relates to the field of biotechnology and particularly discloses an LAMP (loop-mediated isothermal amplification) primer group for quickly detecting sporisorium scitamineum, a kit and a detection method thereof. In the invention, a primer group F3 / B3 and FIP / BIP is designed according to the ribosome gene internal transcribed spacer (ITS) sequence of sporisorium scitamineum, the sporisorium scitamineum is detected by the primer group or a kit containing the primer group through LAMP and the detection has the advantages of low cost, easy operation, simplicity, high speed, visual detection result, judgment with naked eyes, high sensitivity, high specificity and the like; and the LAMP primer group, the kit and the detection method disclosed by the invention are applicable to the sugarcane introduction quarantine in the basic-level departments, quality detection on detoxified seedlings, field early detection of culmicolous smut and the like, and has extensive and practical application value.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, to a LAMP primer set, a kit and a detection method for rapidly detecting sugarcane smut. Background technique [0002] by Ustilago cane whip ( Sporisorium scitamineum ) caused by sugarcane smut is a worldwide important sugarcane disease, and it is also the most economically harmful sugarcane disease in my country. The pathogen spreads and infects during the storage and planting of sugarcane. The most typical symptom of sugarcane smut is the black whip produced by the variation of the growth point of the sugarcane stem. It is the easiest disease to diagnose among sugarcane diseases, but its onset incubation period is long, and the early asymptomatic sugarcane seedlings or sugarcane seed stems are difficult to treat by general methods. Accurately diagnose whether it is infected or carries sugarcane smut. Therefore, exploring its rapid detection technology is extremely important for...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 沈万宽徐刚红罗明珠
Owner SOUTH CHINA AGRI UNIV
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