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.

Pending Publication Date: 2022-01-07
FOSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to provide a rapid detection system for chicken infectious anemia virus fluorescent quantitative PCR, to solve one or more technical problems in the prior art, to provide at least one beneficial option or to create conditions

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  • Fluorescent quantitative PCR kit and primer for detecting chicken infectious anemia virus
  • Fluorescent quantitative PCR kit and primer for detecting chicken infectious anemia virus
  • Fluorescent quantitative PCR kit and primer for detecting chicken infectious anemia virus

Examples

Experimental program
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Effect test

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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Abstract

The invention discloses a fluorescent quantitative PCR kit and primer for detecting a chicken infectious anemia virus. The primer comprises CIAV-124-F and CIAV-124-R, and is a primer pair capable of specifically amplifying a high-conservative gene segment in a VP1 gene of the CIAV, so that the anti-interference capability of detecting the CIAV by using molecular biology is effectively improved. The fluorescent quantitative PCR kit comprises the primer pair, tests prove that the lowest detection concentration of the fluorescent quantitative PCR kit is 2.0 * 10 <-1> copies/[mu]L, and the detection sensitivity of the fluorescent quantitative PCR kit is improved by 1000 times compared with that of common PCR. Moreover, the specificity is good, and an obvious amplification curve does not appear for common poultry diseases such as AIV-5, AIV-7, ILT, NDV, IBV and Fadv-4.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a fluorescent quantitative PCR kit and primers for detecting chicken infectious anemia virus. Background technique [0002] Chicken infectious anemia (chicken infectious anemia, CIA) is an immunosuppressive disease characterized by aplastic anemia and generalized lymphoid tissue atrophy in chickens caused by chicken infectious anemia virus (CIAV). CIAV is a circular single-stranded DNA virus belonging to the Circoviridae family and the genus Circovirus. It has no envelope and has an icosahedral symmetrical structure. It is one of the viruses with the smallest genome known so far. Since the virus was first reported in Japan in 1979, related reports have also appeared in the United States, Britain, Australia, Germany, Denmark and other countries. In 1992, Chinese scientists isolated the virus for the first time. In recent years, with the in-depth research and data accumul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/6851
CPCC12Q1/701C12Q1/6851C12Q2600/166C12Q2531/113C12Q2545/114C12Q2563/107
Inventor 黄淑坚姜含雨梅堃曾繁聪柯骏鸿罗瑞李文俊姜雪芹黄惠兰
Owner FOSHAN UNIVERSITY
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