Real-time fluorescence PCR detection method and kit for eucalyptus components
A real-time fluorescence and reagent kit technology, applied in the field of PCR detection, can solve the problems of unsatisfactory demand, high price, and inability to effectively distinguish mixed honey
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0059] Example 1. Determination of target sequence for eucalyptus detection and primer probe design
[0060] Based on the characteristics of the eucalyptus genome and comprehensive analysis of the genomic characteristics of other species closely related to eucalyptus, the inventors determined that the ribosomal protein L16 gene of eucalyptus was used as the specific gene and the specific gene was Design detection primers and probes to analyze the specificity and accuracy of the detection results at multiple specific sites.
[0061] After repeated testing and verification, the inventors determined that the target sequence and specific primer probes for the nectar plant Eucalyptus component detection are as follows:
[0062] F: 5'-ATTGTAGCAACTGAAATCTTTCCTA-3' (SEQ ID NO:1);
[0063] R: 5'-GTATTGATGCTTTATTACACTGCCT-3' (SEQ ID NO: 2);
[0064] P1: 5'-FAM-ATGCAGATAAAGGGAAGGTTGAGAGAA-BHQ1-3' (SEQ ID NO: 3);
[0065] P1: 5'-FAM-TGCAGATGGAGGGAAGGTTGAGAGAA-BHQ1-3' (SEQ ID NO: 4).
[0066] The inv...
Embodiment 2
[0067] Example 2. Universal screening of reference primer probes for real-time fluorescent PCR of nectar plants
[0068] The real-time fluorescence reaction system used is:
[0069]
[0070] Place the mixture reaction tube in the real-time fluorescent PCR machine 7500, the procedure is as follows:
[0071] 50℃ 2min;
[0072] Pre-denaturation at 95°C for 10 minutes;
[0073] 95°C for 15s, 60°C for 1min, a total of 40 cycles;
[0074] The threshold is set to automatic when generating the image.
[0075] When testing for generality, use flowering plants (48 species): acacia, rape, milk vetch, wolfberry, wattle, lychee, longan, eucalyptus grandis, jujube, linden, sunflower, rose, cotton, manuka, apple , Lavender, orange, sweet potato, cabbage, sesame, cucumber, rice, wheat, mulberry, broad bean, wintergreen, magnolia, olive, osmanthus, ginseng, taro, locust tree, sweet potato, camellia, tulip, kiwi, mango, grape, strawberry, Lily, chrysanthemum, rhododendron, chestnut, elm, tomato, poplar, ...
Embodiment 3
[0085] Example 3. Establishment of real-time fluorescent PCR system for eucalyptus honey
[0086] 1. Eucalyptus honey real-time fluorescent PCR reaction system
[0087] The real-time fluorescent PCR reaction system for eucalyptus components is shown in Table 1. Place the mixture reaction tube in the real-time fluorescent PCR machine AB ViiA7.
[0088] The PCR program is as follows:
[0089] 50℃ 2min;
[0090] Pre-denaturation at 95°C for 10 minutes;
[0091] 95℃ for 15s, 60℃ for 1min, 40 cycles.
[0092] The threshold is set to automatic when generating the image.
[0093] The PCR reaction system is shown in Table 1.
[0094] Table 1. Real-time fluorescent PCR reaction system
[0095]
[0096] 2. Eucalyptus primer specificity experiment
[0097] With the above-mentioned real-time fluorescence reaction system and procedures for specific detection, 7 eucalyptus species: Eucalyptus citriodora Hook.f., Eucalyptus globulus Labill., Eucalyptus grandis Hill ex Maiden, and straight DNA of Eucalyptu...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More