Method for detecting bactrocera dorsalis by utilizing TaqMan probe as well as primers and probe for detection
A method for detection of Bacteroides tangerines and probes, which is applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to achieve the effect of simple and effective detection method, strong repeatability and good specificity.
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Embodiment 1
[0026] 1. Sample template: 6 copies of fruit fly DNA of known species, numbers are: 1, 2, 3, 4, 5, 6, and the sample names are Bactrocera dorsalis, Bactrocera guava, Bactrocera spp., B. squash, B. sichuanensis and B. melon, among which the first five species were provided by the Port Laboratory of Sichuan International Travel Health Care Center, and B. melon was provided by the Department of Entomology, School of Resources and Environment, South China Agricultural University. See Table 1 for details.
[0027] Table 1 Sample information
[0028] Sample serial number
sample name
1
Bactrocera dorsalis
2
Guava Bactrocera correcta
3
Bactrocera scutellata
4
Bactrocera tau
5
Bactrocera ater
6
Bactrocera cucurbitae
[0029] 2, a kind of method utilizing TaqMan probe to detect Bactrocera dorsalis, comprises the following steps:
[0030] (1) Design primers and probes:
[0031] Primers were designed accordi...
Embodiment 2
[0038] The optimization of annealing temperature in the fluorescent quantitative PCR experiment of embodiment 2
[0039] The selected annealing temperatures were: 55.8°C, 56.8°C, 57.8°C, and 58.8°C, and the differences were observed. The cycle threshold (Ct) results of different annealing temperatures are shown in Table 2 and figure 1 .
[0040] Table 2 Ct values at different annealing temperatures
[0041] Annealing temperature (℃)
[0042] The results show that the same concentration of guava real rope DNA template has different cycle thresholds (Ct) at different annealing temperatures. When the annealing temperature is 55.8°C, the cycle threshold is the smallest. Therefore, the optimal annealing temperature is 55.8°C.
Embodiment 3
[0043] Embodiment 3 specificity and sensitivity experiment
[0044] Specificity experiment: with the Bactrocera dorsalis DNA template as a positive control, with the detection method in Example 1 combined with the annealing temperature in Example 2, the DNA templates of 6 samples were detected by fluorescent quantitative PCR, and the test results are shown in figure 2 ;
[0045] Depend on figure 2 It can be seen that only Bacteralis dorsalis has specific amplification, and the other 5 species of fruit flies have no amplification, indicating that the detection method has good specificity.
[0046] Sensitivity test: Bacteralis dorsalis DNA (original concentration was 100ng / μL) was serially diluted 10 times, diluted 6 times, and 1 μL of each diluted sample was used as a template for fluorescent quantitative PCR detection. The detection results are shown in Table 3 and image 3 .
[0047] Table 3 10-fold serial dilution sample detection results
[0048] Concentratio...
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