Primers and method for detecting alfalfa root rot fungi by virtue of loop-mediated isothermal amplification

A ring-mediated isothermal and ring-mediated constant temperature technology is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., and can solve the problems of long detection time, cumbersome procedures, and low accuracy. Reliable results, high sensitivity and strong specificity are achieved

Active Publication Date: 2017-03-22
INST OF PLANT PROTECTION FAAS
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  • Abstract
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Problems solved by technology

[0007] The purpose of the present invention is to provide primers and methods for the detection of alfalfa root rot by the loop-mediated isothermal amplification method. In the prior art, the detection and identification of alfalfa root rot are mainly based on morphological characteristics, and the method is time-consuming, cumbersome and inexperienced. It is difficult to timely monitor the occurrence of diseases and control the spread and prevalence of pathogenic bacteria, and the existing PCR molecular detection needs to rely on expensive instruments such as amplification instruments, and the detection time is long. Provide A new molecular detection method for alfalfa root rot was developed, and LAMP was used to detect alfalfa root rot. The detection cycle is short, the accuracy is high, and the sensitivity is high.

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  • Primers and method for detecting alfalfa root rot fungi by virtue of loop-mediated isothermal amplification
  • Primers and method for detecting alfalfa root rot fungi by virtue of loop-mediated isothermal amplification
  • Primers and method for detecting alfalfa root rot fungi by virtue of loop-mediated isothermal amplification

Examples

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Embodiment 1

[0029] Example 1: Design of specific primers for the detection of Alfalfa root rot pathogen loop-mediated isothermal amplification (LAMP) and verification of primer specificity

[0030] 1. Extraction of genomic DNA of the tested strains

[0031] The genomic DNA of the tested strain (Table 1) was extracted by the CTAB method. The specific method was as follows: Take a small amount of mycelium powder in a 1.5 mL centrifuge tube (it is better that the mycelium powder just covers the bottom of the semicircle), add 900 µL of 2% CTAB ( cetyltrimethylammonium bromide) extract (2% CTAB; 100 mmol / L Tris-HCl, pH 8.0; 20 mmol / L EDTA, pH 8.0; 1.4 mol / LNaCl) and 90 µL SDS (ten Sodium dialkylbenzene sulfonate) [Note: CTAB, SDS needs to be preheated at 60°C], use a shaker to shake and mix, 60°C water bath for 1h (DNA is released into the buffer), 12000 r min -1 Centrifuge for 15 min; take 700 µL of the supernatant, add an equal volume of phenol, chloroform, isoamyl alcohol (25:24:1), shake ...

Embodiment 2

[0042] Example 2: Detection Sensitivity of Loop-Mediated Isothermal Amplification (LAMP) for Alfalfa Root Rot

[0043] 1. Preparation of genomic DNA at different concentrations

[0044] Genomic DNA of Alfalfa root rot fungus was diluted with sterile ultrapure water, and prepared into series concentrations of 10-fold order of magnitude for future use.

[0045] 2. Sensitivity determination and result observation of LAMP detection method

[0046] Genomic DNA of Alfalfa root rot at different concentrations was used as a template, and the outer primers F3 / B3 and inner primers FIP / BIP were used for LAMP amplification. The LAMP detection reaction system was 25 μL, including 1.0 μL of 5 μM outer primers F3 and B3, 1.0 μL each of 40 μM internal primers FIP and BIP, LAMP reaction mixture [40 mM Tris-HCl, 20 mM (NH 4 ) 2 SO 4 , 20 mM KCl, 16 mM MgSO 4 , 1.6 mol / L Betaine (Betaine), 2.0 mM dNTPs, 0.2% Trion X-100] 12.5 μL, 8 U Bst1.0 μL of polymerase, 1.0 μL of DNA templates of dif...

Embodiment 3

[0049] Example 3: LAMP detection of Alfalfa root rot bacteria in diseased tissues

[0050] Sample collection: Collect roots, stems (4-6cm from the ground surface) and healthy roots and stems with typical symptoms of alfalfa root rot from Shaanxi, Gansu, and Ningxia and bring them back to the laboratory for later use;

[0051] Plant tissue DNA extraction: DNA was extracted by NaOH rapid cleavage method, the specific process is as follows: add 10µL 0.5 mol / L NaOH to each mg of plant tissue, fully grind the tissue into a paste in a mortar and transfer it to a 1.5mL centrifuge tube centrifuge at 12,000 rpm for 6 min, take 5 µl of the supernatant and add 495 µL of 0.1 mol / L Tris-HCl (pH=8.0) to mix well, and take 1.0 µL as a PCR template for amplification.

[0052] Amplification detection and observation: Using the above-mentioned extracted DNA as a template, use outer primers F3 / B3 and inner primers FIP / BIP for LAMP amplification. The LAMP detection reaction system is 25 μL, inclu...

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Abstract

The invention discloses primers and a method for detecting alfalfa root rot fungi by virtue of loop-mediated isothermal amplification. The primers include a group of outer primers F3/B3 and a group of inner primers FIP/BIP, wherein base sequences of the primers are shown as SEQ ID NO.1-4. The method comprises the following steps: extracting DNA of a to-be-detected sample; with the DNA as a template, conducting LAMP isothermal amplification by virtue of the primers; and adding a fluorescent dye to an LAMP amplification product for developing color, observing the color of the LAMP amplification product, and analyzing the product so as to judge whether the alfalfa root rot fungi exist or not. With the application of the primers and the rapid LAMP detection method thereof, the alfalfa root rot fungi can be rapidly, sensitively and accurately detected in production practice; meanwhile, the primers and the rapid LAMP detection method thereof are applicable to the early diagnosis of field diseases as well as the monitoring and identification of fungi; and in addition, the primers and the method are simple and easy to operate, and reliable technology and theoretical basis are provided for the control of alfalfa root rot.

Description

technical field [0001] The invention belongs to the technical field of detection, identification and prevention of crop diseases, in particular to a loop-mediated isothermal amplification (LAMP) detection primer for alfalfa root rot and a rapid detection method thereof, which can be used for rapid, sensitive and specific molecular detection of alfalfa root rot It can also be used for early diagnosis of alfalfa root rot and monitoring and identification of pathogens. Background technique [0002] Alfalfa ( Medicago sativa ) is a perennial herbaceous plant of the genus Alfalfa in the family Leguminosae. Alfalfa has high yield, high protein content, good palatability, and well-developed root system, which has biological nitrogen fixation ability, is beneficial to the growth of other plants and soil and water conservation, and plays an irreplaceable role in the development of animal husbandry production and improvement of ecological environment. At present, the cultivation are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2537/1376
Inventor 兰成忠阮宏椿姚锦爱吴玮
Owner INST OF PLANT PROTECTION FAAS
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