Kit for amplifying pseudococcidae mitochondrial genome and application thereof
A technology of mitochondrial genome and mealybug family, which is applied in the genetic field, can solve the problems of lack of specificity, low amplification efficiency, and high template purity requirements, and achieve the effects of high fidelity, strong specificity, and shortened acquisition cycle
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Embodiment 1
[0015] Embodiment 1: the design of primer
[0016] 1. Based on the mitochondrial genome sequences of different Homopteran insects measured in Genbank, the data comes from the National Center for Biotechnology Information (NCBI, http: / www.ncbi.nlm.nih.gov / );
[0017] 2. Use the ClustalX software to compare the genome sequences of different Homoptera insects, and find out the sequence regions that are relatively conservative and have the least base variation; the genome sequence data (partial representative species) of the Homoptera insects used are shown in the table 1.
[0018] Table 1 The present invention adopts mitochondrial genome sequence source (part)
[0019] division
[0020] 3. According to the sequence alignment results, use the primer design software Primer Premier 5 to design 2 pairs of primers in the sequence region with the smallest base variation, F1 (upstream primer): 5'-TAATACCTATGCTTATTAATGAATTTGTACG-3'(SEQID NO:1), R1 (downstream primer): 5'-CTCA...
Embodiment 2
[0021] Example 2 Extraction and Detection of Insect Total Genomic DNA
[0022] 1. Material treatment: Wash the adults of fine mealybugs, long mealybugs, paired mealybugs, Beardsley mealybugs and sea mealybugs with distilled water, divide them into 3 parts on average, and place them in centrifuge tubes respectively.
[0023] 2. Preparation of lysate: Prepare a sufficient amount of lysate according to 400 μl of each centrifuge tube (add 20 μl of proteinase K).
[0024] 3. Sample grinding: add 200 μl of lysate to the above three centrifuge tubes respectively, grind the sample with scissors and grinding rods until clarified, and then add the remaining lysate into the centrifuge tubes in equal amounts.
[0025] 4. Sample digestion: put the above-mentioned centrifuge tube into a water bath that has been preheated to 55°C, and take a water bath for 3 hours. Shake gently once every 15 minutes.
[0026] 5. Extraction: phenol extraction with an equal volume of about 400 μl; take the s...
Embodiment 3
[0031] Example 3 Amplification of mitochondrial genome long fragments for different mealybugs
[0032] 1. the genomic DNA of the mealybugs, mealybugs, mealybugs, Beardsley mealybugs, and sea mealybugs extracted in Example 2 are amplified with long fragments using the universal primers of the present invention;
[0033] PCR amplification system is 20μl, including 5μl Mg 2+ 10×buffer, 2.5μl of 2.5mM dNTP, 2μl of 10μmol upstream primer, 2μl of 10μmol downstream primer, 0.3μl of 5U / μl rTaq enzyme, 1μl of DNA template, and the rest is sterile distilled water ;
[0034] Among them, the amplification program was: pre-denaturation at 94°C for 1 min, denaturation at 92°C for 8 s, annealing at 45°C-55°C for 90 s, extension at 72°C for 1 min, a total of 35 cycles, extension at 72°C for 10 min, and storage at 4°C.
[0035] 2. Use 1% agarose gel electrophoresis to detect the PCR products, observe the results with the UVP ultraviolet gel imaging system, and take pictures and save them (se...
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