LAMP primer group for identifying bactrocera minax and application of LAMP primer group
A technology of Bactrocera citrus and primer set, which is applied in the biological field, can solve the problems of unsuitable grass-roots departments for promotion and application, difficulties in morphological identification, expensive instruments and equipment, etc., and achieve the effects of avoiding positive sample contamination, easy promotion, and high sensitivity
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Embodiment 1
[0075] Example 1. Acquisition of primer set and kit for identification of B. citrus fruit fly
[0076] 1. Screening of primer sets for identification of B. citrus fruit fly
[0077] 1. Taking the DNA barcode sequence of COI gene in the mitochondrial genome of B. citrus as the target gene, we designed and synthesized primer sets BM1 and BM2 for identification of B. citrus. 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 are B. citrus fruit flies numbered 3 in Table 1, B. citrus fruit flies numbered 6, and B. citrus fruit flies numbered 18.
[0083] 3. Using the genomic DNA of the fruit fly sample as a template, use primer set BM1 or primer set BM2 to perform loop-mediated isothermal amplification to obtain the amplification pr...
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 of citrus fruit fly
[0093] 1. Identify whether the fruit fly to be tested is or is a candidate for B. citrus fruit fly by agarose gel electrophoresis
[0094] 1. Use a blood / cell / tissue sample 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 BM1 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 DNA template, 9 μL primer mix and 2.5 μL sterile ultrapure water. The primer mixture is the mixture composed of each primer in the primer set BM1. In the reaction system, the final concentration of outer primer F3...
Embodiment 3
[0116] Example 3. Specificity experiment
[0117] The fruit fly 1 to be tested is the citrus fruit fly numbered 5.
[0118] The fruit fly 2 to be tested is the citrus fruit fly numbered 6.
[0119] The fruit fly 3 to be tested is the citrus fruit fly numbered 7.
[0120] The fruit fly 4 to be tested is the citrus fruit fly numbered 8.
[0121] The fruit fly 5 to be tested is the citrus fruit fly numbered 9.
[0122] The fruit fly 6 to be tested is the citrus fruit fly numbered 10.
[0123] The fruit fly 7 to be tested is the citrus fruit fly numbered 11.
[0124] The fruit fly 8 to be tested is the citrus fruit fly numbered 12.
[0125] The fruit fly 9 to be tested is the citrus fruit fly numbered 13.
[0126] The fruit fly 10 to be tested is the citrus fruit fly numbered 14.
[0127] The fruit fly 11 to be tested is the citrus fruit fly numbered 15.
[0128] The fruit fly 12 to be tested is the citrus fruit fly numbered 16.
[0129] The fruit fly 13 to be tested is t...
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