A kind of primer set, method and application for detection of Bacterial angular spot on mangosteen
A technology of bacterial angular leaf spot and primer set, which is applied in the field of molecular biology, can solve problems such as quality reduction, economic loss, and unobvious lesion characteristics, and achieve the effect of simple operation, fast and accurate results
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Embodiment 1
[0032] Example 1 Reaction Condition Construction
[0033] The optimal reaction system for PCR amplification is: 1-3 μL of total DNA of the sample to be tested, 0.2 μL of forward primer, 0.2 μL of reverse primer, 0.2 μL of dNTPs (10 mM), 0.2 μL of EasyTaq DNA Polymerase, 2.0 μL of 10×EasyTaqBuffer, ddH 2 O 14.2 to 16.2 μL. The total reaction volume was 20 μL. The PCR amplification program was as follows: 95°C for 8 min, 95°C for 30s, 60°C for 30s, 72°C for 30s, and 72°C for 10 min, a total of 30 cycles. After the reaction, it was placed in 1.5% agarose gel electrophoresis, 110V, 30min.
[0034]The amplified products are subjected to gel electrophoresis, and judged according to the results of gel electrophoresis: if any one or more of the products with sizes of 297bp, 237bp and 357bp can be amplified, it is considered that the sample contains mangosteen bacteria. Angular spot bacteria. The correct result of primer amplification is as figure 1 shown.
Embodiment 2
[0035] Example 2 Specificity Verification
[0036] Take the total DNA of 6 different bacterial strains of Mango spp. as templates respectively, carry out PCR amplification with the conditions of Example 1, and the products are subjected to gel electrophoresis, and the experimental results are as follows: figure 2 shown.
[0037] Then, the bacterial angular leaf spot of mango fruit, the bacterial leaf spot of rice GX01 strain and the bacterial blight of rice PXO99 were used respectively. A The total DNA of the bacterial strain, the cruciferous black rot fungus 8004 strain, and Escherichia coli was used as a template, and PCR amplification was carried out with the conditions of Example 1, and the product was subjected to gel electrophoresis. The experimental results are as follows image 3 shown.
[0038] The experimental results showed that the target bands could be detected by 6 different strains of the mango bacterium X. figure 2 ), while B. oryzae GX01 strain, B. oryzae...
Embodiment 3
[0039] Example 3 Sensitivity verification
[0040] Dilute the total DNA sample of Mango spp. keratosis spp. serially by 10 times, carry out PCR amplification with the conditions of Example 1, and perform gel electrophoresis on the product. The experimental results are as follows: Figure 4 shown.
[0041] The minimum concentration of the total DNA of Mango japonica bacteria that can be detected by the present invention is: 0.3771 ng / μL. The detection method of the present invention has high sensitivity.
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