Detection primer for wolbachia in small brown rice planthopper as well as detection method and detection kit thereof
A detection kit and detection primer technology, which is applied in the field of molecular biology, can solve the problems of non-specific primers and methods for the detection of SBPH, and achieve the effect of simple identification, high sensitivity and high sensitivity
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Embodiment 1
[0038] A detection kit for Wolbachia striatellus described in this embodiment includes detection primers and PCR reagents, and the detection primers are as follows.
[0039] wStriF: 5'-GTAAATACTTCTGAAACAAATG-3'; SEQ ID NO.1
[0040] wStriR: 5'-AAAAATTAAACGCTACTCCA-3'; SEQ ID NO.2.
Embodiment 2
[0041] Example 2: Specificity
[0042] 1. Take six 0.2ml EP tubes filled with 20μl dilution buffer and 0.5μl DNARelease (the formula of dilution buffer is: 30mM NaOH, 0.25mM EDTA, 15mM Tris-HCl, the solvent is water);
[0043] 2. Soak three Guangzhou striatellus (containing Wolbachia striatellus) and three brown planthoppers (containing Wolbachia brown planthopper) in absolute ethanol for 2 minutes, then take out the whole planthopper and put them in In the above 6 EP tubes;
[0044] 3. After transient centrifugation, put it into a PCR instrument for DNA extraction;
[0045] 4. DNA extraction procedure: 55°C, 5min; 98°C, 5min; 30°C, 10s.
[0046]5. After instantaneous centrifugation, the DNA extract was obtained, diluted 10 times, and stored at -20°C to obtain the genomic DNA of Wolbachia from six planthoppers (specifically: three copies of Guangzhou striatellus Wolbachia). The genomic DNA of Baklava, the genomic DNA of three parts of brown planthopper Wolbachia), respectiv...
Embodiment 3
[0058] Example 3: Sensitivity
[0059] Get three parts of Example 2 diluted 10-fold Wolbachia genomic DNA, then carry out 100, 1000, 10000, 100000, 1000000 times of gradient dilution, and then use the diluted dilution as template DNA, according to the implementation The kit described in Example 1 and the PCR method described in Example 2 carry out amplification, and the specific results are as follows figure 2 , image 3 and Figure 4 As shown, the PCR product can be amplified in the diluted solution diluted 10,000 times, and then the concentration is calculated, so that the final detection limit is 55×10 -4 ng / μl (that is, the concentration of the genomic DNA of Wolbachia striatellus in the 10000-fold dilution).
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