Method for identifying bactrocera minax and primer group used by method
A technology of citrus fruit fly and primer set, which is applied in the biological field, can solve the problems of unsuitable grass-roots departments for popularization and application, difficulty in morphological identification, expensive instruments and equipment, etc., and achieves the effects of avoiding positive contamination of samples, easy popularization and high sensitivity
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Embodiment 1
[0075] Example 1. Acquisition of primer sets and kits for identification of B. tangerines
[0076] 1. Screening of primer sets for the identification of B. citrus
[0077] 1. Using the DNA barcode sequence of COI gene in the mitochondrial genome of B. serrata as the target gene, the primer set BT1, BT2, BT3 and BT4 for identification of B. serrata were designed and synthesized. The primer sequences of each primer set are shown in Table 2.
[0078] Table 2
[0079]
[0080]
[0081] 2. Extract the genomic DNA of the fruit fly sample by using a blood / cell / tissue genomic DNA extraction kit to obtain the genomic DNA of the fruit fly sample.
[0082] The fruit fly samples were B. citrus fruit flies numbered 3 in Table 1, B. citrus fruit flies numbered 8, and fruit flies numbered 18.
[0083] 3. Using the genomic DNA of the fruit fly sample as a template, use primer set BT1, primer set BT2, primer set BT3 or primer set BT4 to perform loop-mediated isothermal amplification t...
Embodiment 2
[0092] Example 2. Application of the kit prepared in Example 1 in identifying whether the fruit fly to be tested is or is a candidate for fruit fly
[0093] 1. Identify whether the fruit fly to be tested is or is a candidate for fruit fly
[0094] 1. Use a blood / cell / tissue genomic DNA extraction kit to extract the genomic DNA of the fruit flies to be tested to obtain the genomic DNA of the fruit flies to be tested.
[0095] 2. Using the genomic DNA of the fruit fly to be tested as a template, using the primer set BT1 to perform loop-mediated isothermal amplification to obtain the amplification product.
[0096] The reaction system was 25 μL, consisting of 12.5 μL Warm Start LAMP 2×Master Mix, 1 μL template, primer mix and sterile ultrapure water. The primer mixture is the mixture composed of each primer in the primer set BT1. In the reaction system, the final concentration of outer primer F3 and outer primer B3 were both 0.2 μM, and the final concentration of inner primer F...
Embodiment 3
[0116] Example 3. Specificity experiment
[0117] The fruit fly 1 to be tested is the fruit fly numbered as 1.
[0118] The fruit fly 2 to be tested is the fruit fly numbered 2.
[0119] The fruit fly 3 to be tested is the fruit fly numbered 3.
[0120] The fruit fly 4 to be tested is the fruit fly numbered 4.
[0121] The fruit fly 5 to be tested is the citrus fruit fly numbered 5.
[0122] The fruit fly 6 to be tested is the citrus fruit fly numbered 6.
[0123] The fruit fly 7 to be tested is the citrus fruit fly numbered 7.
[0124] The fruit fly 8 to be tested is the citrus fruit fly numbered 8.
[0125] The fruit fly 9 to be tested is the citrus fruit fly numbered 9.
[0126] The fruit fly 10 to be tested is the citrus fruit fly numbered 10.
[0127] The fruit fly 11 to be tested is the citrus fruit fly numbered 11.
[0128] The fruit fly 12 to be tested is the citrus fruit fly numbered 12.
[0129] The fruit fly 13 to be tested is the citrus fruit fly numbered ...
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