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Primer composition for detecting pythium arrhenomane based on loop-mediated isothermal amplification technique and detection method thereof

A technology of primer composition and loop primers, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve the problems of long cycle, low sensitivity, and poor specificity of detection methods, and achieve high accuracy , high sensitivity and easy operation

Active Publication Date: 2018-05-22
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0005] Aiming at the problems of long period, poor specificity and low sensitivity of the Pythium androgen biological detection method in the prior art, the object of the present invention is to provide a LAMP detection primer composition and reagent for Pythium androgen. Box and its visual detection method

Method used

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  • Primer composition for detecting pythium arrhenomane based on loop-mediated isothermal amplification technique and detection method thereof
  • Primer composition for detecting pythium arrhenomane based on loop-mediated isothermal amplification technique and detection method thereof

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Effect test

Embodiment 1

[0024] Embodiment 1: Utilize LAMP method to detect Pythium strongandrum

[0025] LAMP primer composition for detecting Pythium androgenus: the forward inner primer FIP is shown in SEQ ID NO.1, the reverse inner primer BIP is shown in SEQ ID NO.2, and the forward outer primer F3 is shown in SEQ ID NO. .3, reverse outer primer B3 as shown in SEQ ID NO.4, and reverse loop primer LB as shown in SEQ ID NO.5.

[0026] The concentration of each reagent in the LAMP kit for detecting Pythium androgen is: 2.5μL 10×ThermoPol Buffer, 6mmol L -1 MgSO 4 , 1.4mmol·L -1 dNTPs, internal primers FIP and BIP each 1.6 μmol L -1 , 0.2 μmol L for each of the outer primers F3 and B3 -1 , loop primer LB 0.8 μmol L -1 , 0.8mol·L -1 Betaine, 0.1% Triton X-100, 20mmol L-1 Tris-HCl (pH 8.8), 10mmol L -1 KCl, 10mmol·L -1 (NH 4 ) 2 SO 4 , 8U·μL -1 Bst DNApolymerase, 192 μmol L -1 Hydroxynaphthol blue (HNB), 2 μL template DNA, and sterilized water to make up to 25 μL.

[0027] LAMP detection m...

Embodiment 2

[0029] Embodiment 2: the sensitivity test of Pythium strongandros LAMP reaction

[0030] In order to determine the sensitivity of the LAMP detection method, the DNA concentration of the extracted Pythium androgenus was measured by a spectrophotometer and then diluted 10 times, and the DNA concentration range was set to 10ng·μL -1 ~1fg·μL -1 . Take 2 μL of diluted DNA dilutions of each concentration as a template, add 23 μL of the detection solution in the kit to carry out LAMP reaction, and the reaction procedure is: reaction amplification at 64°C for 60 min. The color reaction of HNB shows that when the DNA concentration of Pythium androgen reaches 10pg·μL -1 When, the solution in the reaction tube just can turn sky blue ( figure 2 ).

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Abstract

The invention discloses a primer composition for detecting pythium arrhenomane based on a loop-mediated isothermal amplification technique and a detection method thereof. The detection method comprises the following steps of extracting a genomic DNA (Deoxyribonucleic Acid) of a to-be-detected microbe, taking a genomic DNA solution as a reaction template, and adding a detection solution in an LAMP(Loop-Mediated Isothermal Amplification) kit to carry out an LAMP reaction, wherein the LAMP reaction comprises the procedures of reacting at 64 DEG C for amplification for 60min, afterwards, observing the change of the color of an amplification product, if the color becomes from purple to sky blue, indicating that the pythium arrhenomane exists in a to-be-detected matter, and if the color is notchanged or is still purple, indicating that the pythium arrhenomane does not exist in the to-be-detected matter. Compared with a conventional detection technique of authenticating the pythium arrhenomane according to a morphological character, the detection method has higher accuracy, sensitivity and effectiveness, moreover, is convenient to operate and good in practicability, is used for providing a new technical platform for the detection of the pythium arrhenomane, and can be used for the quick detection of the pythium arrhenomane.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a LAMP detection primer composition, a kit and a visual detection method thereof for Pythium strongandrum. Background technique [0002] Pythium arrhenomane (Pythium arrhenomane) is an important pathogenic fungus of corn stalk rot. The optimum temperature for its growth is 23-25°C. It mainly causes root rot and stem rot of corn, and has great harm to corn production. In view of the harmfulness of Pythium strongandrum, a method that can quickly and accurately identify the pathogen in the early stage of maize disease is needed in production. At present, the identification of corn stalk rot pathogens usually adopts the traditional morphological characteristics identification method, that is, after the diseased samples are collected from the field, the pathogens are isolated and cultured immediately, and then the morphological identification is carried out. This meth...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 窦道龙沈丹宇赵媛媛于佳
Owner NANJING AGRICULTURAL UNIVERSITY
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