Fluorescent quantitative PCR kit and primer for detecting chicken infectious anemia virus
A chicken infectious anemia and kit technology is applied in the field of detection of chicken infectious anemia virus fluorescence quantitative PCR kits and primers, and achieves the effects of good specificity and improved anti-interference ability.
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Embodiment 1
[0036] Embodiment 1, the preparation of positive standard plasmid
[0037] (1) PCR amplification and purification of the target fragment
[0038] Use specific primers CIAV-124-F and CIAV-124-R to carry out PCR amplification on the viral genome, and the conventional PCR reaction system is shown in Table 1:
[0039] Table 1 Conventional PCR reaction system
[0040]
[0041] The reaction program was: maintain 95°C for 5 min; maintain 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, repeating 35 cycles; and maintain 72°C for 10 min.
[0042] After the PCR program is over, the products in the tube are subjected to gel electrophoresis. After the electrophoresis is completed, the target band is recovered by cutting the gel, and the gel recovery is performed using the Omega gel recovery kit. Specific steps are as follows:
[0043] ① Cut the DNA band of the single target from the agarose gel, put it into a clean centrifuge tube, weigh and calculate the weight of the gel.
[0044...
Embodiment 2
[0081] Embodiment 2, construction of standard curve
[0082] take 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 , the positive standard plasmid obtained in Example 1 of 7 gradients was used as a template to check and detect the amplification effect of the fluorescent quantitative PCR kit for chicken infectious anemia virus. The reaction system of the described detection chicken infectious anemia virus fluorescent quantitative PCR kit is as shown in table 4, and the results are as follows figure 1 As shown, the standard curve equation is Y=-3.2514X+37.091; the correlation coefficient R 2 =0.9995; Amplification efficiency E=103.03%, and the results show that there is a good linear relationship between the standard products of different concentrations, wherein the X-axis is the copy number of the positive standard plasmid, and the Y-axis is the cycle threshold, which is in line with the expected results.
[0083] Table 4 Fluorescent quantitative PCR kit reaction system
[008...
Embodiment 3
[0086] Embodiment 3, sensitivity test
[0087] Take the copy number as 10 9 、10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 、10 0 、10 -1 , the positive standard plasmid is used as a template for real-time fluorescence quantitative PCR amplification, and the same plasmid is carried out for ordinary PCR amplification to measure the detection of chicken infectious anemia virus fluorescent quantitative PCR kit described in Example 2 compared to ordinary PCR The lowest copy number of positive plasmids that can be detected. The amplification results of the kit are as follows: figure 2 As shown, the curves marked 1~11 in the figure are 2.0×10 9 ~2.0×10 -1 Copies / μL positive standard plasmid sample, the curve labeled 12 is the negative control. The electrophoresis results of ordinary PCR amplification are as follows: image 3 As shown, the label M is 500DNAmarker, and the lanes labeled 1-11 are 2.0×10 9 ~2.0×10 -1 Copies / μL of the positive standard plasmid sample, th...
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