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Specific primers for detection of riemerella anatipestifer and PCR (Polymerase Chain Reaction) detection kit

A Riemer's duck disease, specific technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, DNA/RNA fragments, etc. Achieving the effect of low cost, short time-consuming and low cost

Inactive Publication Date: 2015-10-07
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The lack of effective cross-immune protection among various serotype strains has brought great difficulty to vaccine prevention

Method used

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  • Specific primers for detection of riemerella anatipestifer and PCR (Polymerase Chain Reaction) detection kit
  • Specific primers for detection of riemerella anatipestifer and PCR (Polymerase Chain Reaction) detection kit
  • Specific primers for detection of riemerella anatipestifer and PCR (Polymerase Chain Reaction) detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A kind of Riemerella anatipestifer PCR detection kit of the present invention comprises:

[0032] 1. 5 tubes of 2×PCR TaqMix (250μl / tube);

[0033] 2. Upstream primer ompA-F 1 branch (50μl / branch);

[0034] 3. Downstream primer ompA-R 1 branch (50μl / branch);

[0035] 4. Positive control DNA 1 branch (250μl / cartridge)

[0036] 5. Negative control DNA 1 branch (250μl / cartridge);

[0037] 6. 1 bottle of sterilized deionized water (1000μl / cartridge).

[0038] The above-mentioned kit can detect 50 samples and store them at -20°C with a shelf life of 1 year.

[0039] Among them, 2×PCR TaqMix was purchased from the market;

[0040] The sequence of the upstream primer ompA-F is:

[0041] 5'-actcaaggaagagcggatca-3',

[0042] The sequence of the downstream primer ompA-R is:

[0043] 5'-gcttcagcagaaccaactcc-3'. The primer sequence can be artificially synthesized.

[0044] Positive control DNA: use the genomic DNA of Riemerella anatipestifer as a template, and use th...

Embodiment 2

[0048] The using method of Riemerella anatipestifer PCR detection kit of the present invention is:

[0049] 1. Total DNA was extracted from pure bacterial culture, brain tissue, heart blood, liver, spleen, lung and other diseased materials of dead ducks. DNA extraction can use commercial kits.

[0050] 2. The 2×PCR TaqMix in the kit, upstream primers ompA-F, downstream primers ompA-R, sterilized deionized water and extracted DNA template were prepared into a PCR reaction solution, the total system was 50 μl. At the same time, use the positive control DNA in the kit as a template to amplify the positive control; use the negative control DNA in the kit as a template to amplify the negative control. The specific preparation method of the PCR reaction solution is shown in Table 1:

[0051] Table 1

[0052]

[0053] 3. Place the above-prepared PCR reaction solution in a PCR instrument to amplify the target fragment. The amplification procedure is as follows: 94°C for...

Embodiment 3

[0058] Specific detection of the kit of the present invention

[0059] Extract the genomic DNA of 20 strains of escherichia coli, 20 strains of Salmonella, 15 strains of Pasteurella, and 35 strains of Riemerella anatipestifer isolated from ducks with a bacterial DNA extraction kit. With these DNA samples as templates, use the Kit for PCR detection. The results showed that Escherichia coli, Salmonella and Pasteurella were all negative, and Riemerella anatipestifer were all positive, indicating that the specificity of the kit was good.

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Abstract

The invention provides a pair of specific primers for detecting riemerella anatipestifer, the nucleotide sequence of the specific primers comprises an upstream primer omp A-F:5minute-actcaaggaagagcggatca-3minute; a downstream primer ompA-R: 5minute-gcttcagcagaaccaactcc-3minute. The invention also provides a PCR detection kit containing the specific primers. The detection kit provided by the invention has the beneficial effects that the operation is simple, the specificity and the sensitivity are high; the result determination is intuitive, and the result can be watched by eyes after cataphoresis.

Description

technical field [0001] The invention relates to specific primers and a PCR detection kit for detecting Riemerella anatipestifer. Background technique [0002] Riemerella anatipestifer ( Riemerella anatipestifer , RA) can cause disease in domestic ducks, geese, turkeys and a variety of poultry and wild birds, and the resulting duck disease is called duck infectious serositis (duck infectious serositis). This bacteria is easy to infect ducks aged 1 to 8 weeks, especially ducklings aged 2 to 3 weeks, and ducks under one week old or over 8 weeks old rarely get sick. The infection rate of this disease can sometimes reach more than 90%, and the mortality rate ranges from 5% to 75%. At present, duck infectious serositis occurs in all duck-raising provinces and regions in my country, and the morbidity and mortality are high. It is one of the important bacterial diseases that endanger the duck industry and causes great economic losses to the duck industry. [0003] There are many ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/04
Inventor 郑福英刘永生陈启伟宫晓炜
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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