Double detection kit for citrus anthracnose and citrus foot rot and its application
A citrus anthracnose, double detection technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial determination/inspection, etc. To achieve the effect of simple absolute quantitative analysis, strong resistance to impurity interference, good detection accuracy and reproducibility
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Embodiment 1
[0040] Example 1: Primer screening and testing.
[0041] Use the primer primer5 software to detect the complementarity between the primers and the annealing temperature of the primers, and select a primer pair with an appropriate annealing temperature and easy identification of the amplified target fragment. The present invention designs two pairs of primers and probes respectively for the citrus anthracnose fungus and the citrus foot rot fungus.
[0042] This example gives the process of screening the best primers, and the alternative primers used for screening are as follows, see Table 1.
[0043] Table 1 Alternative primer sequences for P. citrus anthracnose and P. citrus foot rot
[0044]
[0045] Alternative primer and probe combinations include: TJB 1-F+ TJB 1-R+ TJB 1-P, TJB 2-F+ TJB2-R+ TJB 2-P, JFB1-F+ JFB 1-R+ JFB 1-P, JFB 2-F+ JFB 2-R+ JFB2-P, using probe method fluorescent quantitative PCR for specificity analysis, 20 μL reaction system, Premix Ex Taq buffer 1...
Embodiment 2
[0048] Example 2: Primer specificity analysis
[0049] In order to verify the specificity of the quantitative detection method of the present invention to the detection of citrus anthracnose bacteria and citrus foot rot bacteria, use the primers of TJB 1-F+ TJB 1-R+ TJB 1-P and JFB1-F+ JFB 1-R+ JFB 1-P respectively Combined with the probe to detect citrus anthracnose and citrus foot rot disease samples. Use digital quantitative PCR system formula: 2×3D Digital PCRMaster Mix 8.0μL, 10μM probes and primers TJB 1-F, TJB 1-R, TJB 1-P, JFB1-F, JFB 1-R, JFB1-P each 0.2 μL, 3 μL of citrus disease-like DNA template, and ddH for the rest 2 O to make up to 16 μL. The digital PCR amplification reaction program was as follows: pre-denaturation at 96°C for 10 min; denaturation at 98°C for 30 s; extension at 60°C for 2 min, a total of 40 cycles were performed, and the reaction was stopped at 10°C. The samples of citrus canker disease, citrus anthracnose, citrus foot rot, citrus decay, ci...
Embodiment 3
[0052] Example 3: Effects of different annealing temperatures on double digital PCR reactions of citrus anthracnose and citrus foot rot.
[0053] Primer and probe combinations of TJB 1-F+ TJB 1-R+ TJB 1-P, and JFB1-F+ JFB 1-R+ JFB 1-P were used. Use digital quantitative PCR system formula: 2×3D Digital PCR Master Mix 8.0μL, 10μM probes and primers TJB 1-F, TJB 1-R, TJB 1-P, JFB1-F, JFB 1-R, JFB 1- 0.2 μL for each of P, 3 μL of DNA templates for composite infection of citrus anthracnose and citrus foot rot, and ddH 2 O to make up to 16 μL. The PCR amplification reaction program was as follows: pre-denaturation at 96°C for 10 min, denaturation at 98°C for 30 s, and then annealing and extension for 2 min under a temperature gradient (56, 58, 60, 62) respectively, for a total of 40 cycles; finally, the reaction was stopped at 10°C.
[0054] Results: At different annealing temperatures, the detection values in the FAM signal channel or VIC detection channel had little differenc...
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