Primers and method for detection of Alfalfa root rot by loop-mediated isothermal amplification
A ring-mediated isotherm and ring-mediated constant temperature technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of long detection time, low accuracy, and cumbersome procedures, etc. Achieve reliable results, high sensitivity, and strong specificity
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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 strains (Table 1) was extracted by the CTAB method. The specific method is as follows: Take a small amount of mycelium powder in a 1.5mL 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 g...
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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