Primer pair for detecting Metschnikowia bicuspidate and application of primer pair
A technology of bicuspid plums and primer pairs, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve problems such as low sensitivity, low specificity, and complex processes of bicuspid yeast, Achieve the effect of good sensitivity, good sensitivity, and simple detection method
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Embodiment 1
[0036] Embodiment 1, the design of the specific primers that are used to detect Metschnischiana bitipum of the present invention
[0037] 1. Materials and methods
[0038] 1.1 Sample plasmids and strains
[0039] The PMD19T vector was purchased from Baoriyi Biotechnology Co., Ltd.; Escherichia coli DH5α competent cells were purchased from Quanshijin Biotechnology Co., Ltd.; the positive sample of Metchiella bimini was collected from Panjin, Liaoning, and then obtained by laboratory separation and purification.
[0040] 1.2 Samples and reagents
[0041]DNA extraction kits were purchased from Tiangen (Beijing) Biochemical Technology Co., Ltd., PCR Mix and PCR product recovery kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.; plasmid extraction kits were purchased from Dingguo Biotechnology Co., Ltd.
[0042] 1.3 DNA extraction and purification of target gene fragments
[0043] Extract, recover, purify, and extract plasmids according to the instructions of each...
Embodiment 2
[0048] Example 2, the annealing temperature of the first specific primer PCR amplification of Metchis bicuspii
[0049] Using the nucleic acid of Metchiella bitipina as a template, PCR amplification was carried out, and the reaction system was: 13 μL of 2×PCR Mix, 1 μL of specific primer pair (nmol / L), and 1 μL of DNA template of Metchi bimini.
[0050] Determine the annealing temperature of the primers, and select 45°C, 50°C, and 55°C for PCR amplification according to the reference temperature after primer synthesis. The PCR amplification program is as follows: pre-denaturation at 94°C, 10min; denaturation at 94°C, 1min; annealing at 45°C / 50°C / 55°C, 45s; extension at 72°C, 1min; final extension at 72°C, 10min; and extended for 30 loops. After the PCR reaction was completed, 5 μL of the PCR product was taken for 1.5% agarose gel electrophoresis analysis.
[0051] Test results: Set different annealing temperatures. After the PCR reaction, take 5 μL of the PCR product for 1.5...
Embodiment 3
[0052] Example 3, Sensitivity Identification of Metschia bicuspii-specific Primer Pairs
[0053] Plasmids were extracted from the PCR products recovered by gel cutting in Example 1, and the concentration was measured. The copy number of each sample was calculated according to the DNA copy number calculation formula. The results are shown in Table 2.
[0054] Table 2
[0055]
[0056] Copy number = (6.02×10 14 ×concentration (ng / μL) / (DNA length×660)
[0057] Select Metchimos bicuspii DNA template 3 for dilution, and dilute the copy number to about 8.62×10 9 , 8.62×10 8 , 8.62×10 7 , 8.62×10 6 , 8.62×10 5 , 8.62×10 4 , 8.62×10 3 , 8.62×10 2 , 8.62×10 1 . Take 1 μL of them as a template, and use the first specific primer pair P1 / P2 primers of the present invention to carry out PCR amplification. The reaction conditions are the same as those in Example 2, and the annealing temperature is 50°C. After the end, 5 μL of PCR products were taken for 1.5% agarose gel elect...
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