LAMP primer composition, kit and detection method for detecting pythium ultimum

The technology of primer composition and detection method is applied in biochemical equipment and methods, recombinant DNA technology, determination/inspection of microorganisms, etc. High accuracy and the effect of improving the reaction rate

Active Publication Date: 2016-12-14
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Aiming at the problems of long period, poor specificity and low sensitivity of the Pythium ultima biological detection method in the prior art, the object of the present invention is to provide a LAMP primer composition, a kit and a method for detecting Pythium ultima. Its detection method

Method used

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  • LAMP primer composition, kit and detection method for detecting pythium ultimum
  • LAMP primer composition, kit and detection method for detecting pythium ultimum
  • LAMP primer composition, kit and detection method for detecting pythium ultimum

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Embodiment 1

[0027] Embodiment 1: Utilize LAMP method to detect Pythium ultima

[0028] LAMP primer composition for detecting Pythium ultima: 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, the reverse outer primer B3 is shown in SEQ ID NO.4, and the forward loop primer LF is shown in SEQ ID NO.5.

[0029] The concentration of each reagent in the LAMP kit for detecting Pythium ultima is: 1.6 μM forward inner primer FIP, 1.6 μM reverse inner primer BIP, 0.2 μM forward outer primer F3, 0.2 μM reverse outer primer B3, 0.8 μM forward Loop primer LF, 0.8M betaine, 1.4mM dNTPs, 20mM Tris-HCl, 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 8mM MgSO 4 , 0.1% Triton X-100, Bst DNA polymerase 320U / ml, 2.4mM Hydroxynapthol Blue (HNB), and use ultrapure water to prepare 1mL detection solution.

[0030] LAMP detection method: extract the DNA of the microorganism to be tested, take 2 μL of the DNA...

Embodiment 2

[0032] Embodiment 2: the sensitivity test of Pythium ultima LAMP reaction

[0033] In order to determine the sensitivity of the LAMP detection method, the DNA concentration of the extracted Pythium ultima was measured with a spectrophotometer and then diluted 10 times. The DNA concentration range was set at 100ng-10fg, and 2 μL of the diluted DNA dilutions of each concentration were taken as Template, add 23 μL kit solution to carry out LAMP reaction, the reaction program is: 64 ° C reaction amplification for 60 min. HNB chromogenic reaction shows: when the DNA concentration of ultimate Pythium reaches 100pg, the solution in the reaction tube just can turn sky blue, further obtains the result consistent with HNB chromogenic reaction by agarose gel electrophoresis ( figure 2 ).

[0034]

[0035]

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Abstract

The invention discloses an LAMP primer composition, kit and detection method for detecting pythium ultimum. The primer composition comprises a positive inner primer FIP, a reverse inner primer BIP, a positive outer primer F3, a reverse outer primer B3 and a positive loop primer LP. The genome DNA of a to-be-detected microorganism serves as a template, and the LAMP primer composition serves as a primer for LAMP reaction; then the color change of an amplification product is observed, if the color changes from purple to sky blue, it represents that pythium ultimum exists in the to-be-detected microorganism, and if the color does not change and is still purple, it represents that pythium ultimum does not exist in the to-be-detected microorganism. Compared with a traditional detection technology that pythium ultimum is verified according to morphological characteristics, the method has higher accuracy, sensitivity and effectiveness, is convenient to operate and good in practicality, provides a new technical platform for pythium ultimum detection, and can be used for high-sensitivity quick detection of pythium ultimum.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a LAMP primer composition, a kit and a detection method for detecting Pythium ultima. Background technique [0002] Pythium ultimum is a common pathogenic Pythium in vegetable Pythium, which belongs to Oomycetes, Perenosporales, Pythiaceae, Pythium Mold (Pythium). Pythium ultima has a wide host range and can infect a variety of Compositae, Fabaceae, Cucurbitaceae, Solanaceae and a few Rosaceae plants, and has become one of the important pathogenic bacteria of many crop seedling diseases. At present, there is no effective prevention and control measures for the disease. Strengthening quarantine and preventing the spread of pathogenic bacteria are the most effective measures to control the disease. To this end, epidemic monitoring should be strengthened, and rapid detection methods should be established to provide evidence for disease risk and research decisions, thereby ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2563/107
Inventor 窦道龙沈丹宇李庆玲
Owner NANJING AGRICULTURAL UNIVERSITY
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