LAMP detection kit and detection method for dendrobium officinale-phytophthora nicotianae

A technology of Phytophthora Tobacco and a detection kit, which is applied in the field of microbial detection, can solve the problems of easily missing the best control period, being easily infected by other bacteria, and poor accuracy, achieving high specificity, simple identification, and short time-consuming Effect

Active Publication Date: 2015-04-29
SHANXI REALLY TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods take a long time, easily miss the best control period, and are susceptible to infection by other bacteria during the isolation and culture process, so the accuracy is poor

Method used

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  • LAMP detection kit and detection method for dendrobium officinale-phytophthora nicotianae
  • LAMP detection kit and detection method for dendrobium officinale-phytophthora nicotianae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 Preparation of Dendrobium candidum LAMP detection kit of the present invention 1.1 Reagent

[0038] Primers were synthesized by Tiangen Biochemical Technology (Beijing) Co., Ltd.; Bst DNA polymerase and 10×ThermoPol reaction buffer were purchased from NEB; SYBR Green I was purchased from Invitrogen; other PCR reagents and reagents required for preparing CTAB extraction buffer were purchased from From Sigma.

[0039] 1.2 Preparation of the kit:

[0040] Obtain following reagent, to prepare Dendrobium officinale LAMP detection kit of the present invention:

[0041] CTAB extraction buffer: prepared according to the following formula: 100mM Tris-HCl pH 8.0, 50mM EDTA, 1M NaCl, 1% (v / v) β-mercaptoethanol, 2% CTAB, and finally adjust the pH to 7.0-7.5;

[0042] Reaction buffer: prepared according to the following formula: 10mM dNTP, 10×ThermoPol reaction buffer, 50mM MgSO 4 , 5mM betaine;

[0043]Primer: outer primer F3, its nucleotide sequence is as shown in...

Embodiment 2

[0048] Embodiment 2 Phytophthora Tobacco Specific Detection

[0049] 2.1 LAMP-specific detection

[0050] The strains to be tested included 6 strains of Phytophthora nicotianae obtained from the cultivation base of Dendrobium officinale in Yiwu, Zhejiang Province, and 1 strain each of Phytophthora cucumber, Phytophthora capsici and Phytophthora perisha.

[0051] Use the kit prepared in Example 1 to detect Phytophthora tabacum according to the following steps:

[0052] (1) Extract the DNA of the sample to be tested: Inoculate the strain to be tested on a PDA plate, culture it by streaking, collect and centrifuge, add 50 μl of CTAB extraction buffer, and extract DNA according to the conventional CTAB method;

[0053] (2) Establish a LAMP reaction system: Prepare a 25 μl reaction system in a PCR tube, including 1 μl of each of the four primers, 2.5 μl of reaction buffer, 1 μl of 8 U / μl Bst DNA polymerase, 2 μl of template DNA obtained in step (1), and add water Supplement to 25...

Embodiment 3

[0057] The PCR tubes of the 6 strains of Phytophthora nicotiana all showed green color, while the color results of Phytophthora cucumber, Phytophthora capsici and Phytophthora perforatum were all orange, indicating that the primers had strong specificity. Embodiment 3 Phytophthora Tobacco Sensitivity Detection

[0058] 3.1 LAMP sensitivity test

[0059] Phytophthora nicotiana DNA was extracted according to the step (1) of Example 2, and diluted into 7 gradients of 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, and 100fg with a 10-fold concentration serial dilution method.

[0060] LAMP reaction system and reaction conditions and result analysis are the same as the steps (2)-(4) of embodiment 2

[0061] 3.2 Test results

[0062] Except that the PCR tube with a DNA concentration of 100fg shows orange, the PCR tubes with other concentrations all show green, indicating that the minimum detection limit of the detection method of the present invention reaches 1pgDNA, and the sensitivity is v...

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Abstract

The invention relates to an LAMP detection kit for dendrobium officinale-phytophthora nicotianae, which comprises a primer, a reaction buffer solution, a Bst DNA polymerase and a nucleic acid dye. The invention also relates to an LAMP detection method for dendrobium officinale-phytophthora nicotianae. After the kit disclosed by the invention is adopted for carrying out LAMP detection, the high-specificity, rapid, efficient, high-sensitivity and simple molecular detection on dendrobium officinale-phytophthora nicotianae can be realized.

Description

technical field [0001] The invention relates to the field of microbial detection, in particular to a detection kit and a detection method for Phytophthora nicotiana LAMP of Dendrobium officinale. Background technique [0002] Dendrobium candidum, also known as black knot grass, is the most rare and valuable species of medicinal plants in the genus Dendrobium. Its stems are used as medicine, which has the effects of benefiting the stomach and promoting body fluid, nourishing yin and clearing heat, and regulating immunity. In recent years, it has been widely used in the fields of disease treatment and prevention, nutrition and health care, and beauty. It has huge market demand and important economic value. Wild Dendrobium candidum is mainly distributed in southern my country, and is on the verge of extinction due to slow growth, low self-reproductive capacity, small number, and over-harvesting. In order to meet the ever-increasing social needs, artificial cultivation of Dendr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 邓继红
Owner SHANXI REALLY TECH
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