Primer and detection method for real-time fluorescent quantitative PCR detection of colletotrichum gloeosporioides of hevea brasiliensis
A technology for real-time fluorescence quantification and glomus anthracnose, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of lack of monitoring means for rubber tree anthracnose pathogens, and achieve fast and quantitative Accurate and repeatable results
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Embodiment 1
[0042] Example 1: Cloning and sequencing of the ITS sequence of Hevea anthracnose
[0043] (1) Preparation of Hevea anthracnose DNA:
[0044] Put Hevea anthracnose bacteria on PDA medium, and culture them at 28° C. in the dark for 5-6 days. The hyphae on the surface of the plate were scraped off with a sterilized glass slide, quickly ground to powder with liquid nitrogen, and the Omega kit was used to extract DNA.
[0045] (2) Amplification of Hevea anthracnose ITS sequence:
[0046] The general primer ITS1 / ITS4 is used to amplify the ITS sequence of Hevea anthracnose by using the ITS sequence amplification of pathogenic fungi. The primer sequences are as shown in SEQ ID No.1 and SEQ ID No.2, specifically:
[0047] ITS1: 5'-TCGTAGGTGAACCTGCGG-3'
[0048] ITS4: 5'-TCCTCCGCTTATTGATATGC-3'
[0049] PCR amplification system: 50ng / μL DNA 1μL, 2× Max Master Mix 25, 2 μL each of 10 μM ITS1 / ITS4, plus sterilized ddH 2 0 to 50 μL. The PCR amplification program was pre-denaturat...
Embodiment 2
[0051] Embodiment 2: Specific detection of Hevea anthracnose primers
[0052] The characteristic of the detection of the specificity of the primers of Hevea anthracnose is that it is pre-detected by conventional PCR and then detected by fluorescent quantitative PCR. This method can reduce the material loss in the screening process of specific primers.
[0053] Materials: DNA from 5 strains of Glucospora anthracnose as positive control, 1 strain of C. acutatum, 1 strain of Oidium heveae, 1 strain of Corynesporacasslicola, The DNA of the rubber tree (Hevea brasiliensis) was subjected to conventional PCR, and the specific information of the strains is shown in Table 1.
[0054] Table 1: Strain specific information
[0055]
[0056] Note: The microorganisms used in the present invention have been recorded in the existing literature, and are also preserved in this laboratory, which can be distributed to the public for verification tests.
[0057] (1) Sample DNA preparation:
...
Embodiment 3
[0076] Example 3: Establishment of Fluorescent Quantitative PCR Standard Curve and Detection of Primer Sensitivity
[0077] (1) Construction of plasmid standards
[0078] The connection system using the ITS fragment of Hevea anthracis amplified in Example 1 and the PMD18-T vector is as follows, and the mixed connection system was incubated at 16°C for 4-6 hours.
[0079] The connection system of ITS fragment of Hevea anthracnose and PMD18-T vector: 1 μL of PMD18-T vector, 3 μL of ITS sequence, 5 μL of Solution Ⅰ, plus sterilized dd H 2 0 to 10 μL.
[0080] Colony PCR detection system: ExTaq 10 μL, 10 μM ITS1 / ITS4 1 μL each, sterilized ddH 2 O 9 μL. Colony PCR detection program: pre-denaturation at 94°C for 4 minutes, denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 42s, a total of 35 cycles, and finally extension at 72°C for 5 minutes, and storage at 12°C.
[0081] The transformation and extraction methods of recombinant plasmids refer to "Mol...
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