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Primer composition for assistant identification of porcine infectious pleuropneumonia ApxI toxin and application thereof

A primer composition and a technology of pleuropneumonia, applied in the biological field, can solve the problems of inability to provide accurate diagnosis and identification of antiviral factors, and achieve the effects of easy operation and application at the grassroots level, low cost and strong specificity

Pending Publication Date: 2018-04-20
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the patent only detects the ApxIVA toxin, it is still impossible to identify which type of the main antiviral factor belongs to ApxI, ApxII, and ApxIII clinically, and there is a defect that it cannot provide accurate diagnosis

Method used

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  • Primer composition for assistant identification of porcine infectious pleuropneumonia ApxI toxin and application thereof
  • Primer composition for assistant identification of porcine infectious pleuropneumonia ApxI toxin and application thereof
  • Primer composition for assistant identification of porcine infectious pleuropneumonia ApxI toxin and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The preparation of embodiment 1 primer composition

[0057] The primers shown in Table 1 were synthesized, and the nucleotide sequences of 1-F, 1-R, 2-F, and 2-R were shown in Table 1.

[0058] Table 1

[0059] Primer name

[0060] The partial gene sequence of ApxIA (Gene bank: AF 240779.1) is shown in sequence 5 of the sequence list (sequence length 513bp), and the primers are located in figure 1 , the partial gene sequence of ApxIVA (GenBank: FJ 848574.1) is shown in sequence 6 of the sequence table (sequence length 1092bp), and the primers are located in figure 2 .

Embodiment 2

[0061] Example 2 Primer Sensitivity Detection

[0062] Bacterial cultivation and DNA extraction

[0063] (1) Culture and DNA extraction of 1.5mL ApxⅠA standard bacteria and ApxIVA standard bacteria

[0064] Take 4 test tubes, add 7mL TSB, 5μl NAD, 300μl bovine serum, two of them add 50μl ApxIA standard bacteria solution, two of them add 50μl ApxIVA standard and mix well. Four test tubes were placed on a shaker at the same time, and incubated at 37°C for 12 hours at 200r / min.

[0065] Take 1.5mL ApxⅠA standard bacterial solution and ApxIVA standard bacterial solution respectively in two EP tubes, centrifuge at 12000r / min for 2min, and discard the supernatant. Repeat the operation twice. The DNA of the two bacteria was extracted according to the instructions of the DNA extraction kit. The DNA concentration of ApxⅣVA bacteria was measured to be 150 μg / mL; the DNA concentration of ApxⅠA bacteria was 240 μg / mL.

[0066] (2) Culture of other bacteria and extraction of DNA

[0...

Embodiment 3

[0075] The influence of embodiment 3 annealing temperature on PCR result

[0076] In the PCR reaction conditions, 6 different annealing temperatures were designed, namely 50°C, 52°C, 54°C, 56°C, 58°C, and 60°C. Using the reaction system in Table 2, the effects of different annealing temperatures on the PCR results of ApxIA and ApxIVA genes were determined. According to the results at different annealing temperatures, the effects of annealing temperature on the PCR reactions of toxins ApxIA and ApxIVA are different. Depend on Figure 5 It can be seen that the annealing temperature has a great influence on ApxIA. Although the target gene fragment can be obtained at all temperatures between 50°C and 60°C, the brightness of the bands at 50°C and 60°C is obviously not as bright as that at the intermediate temperature. bright. Depend on Figure 6 It can be seen that the change of annealing temperature between 50°C and 60°C has little effect on the ApxIVA fragment.

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Abstract

The invention provides a primer composition for assistant identification of porcine infectious pleuropneumonia ApxI toxin and application thereof, and belongs to the technical field of biology. A specific primer pair provided by the invention consists of single stranded DNA (deoxyribonucleic acid) molecules shown as a sequence table 1 to 4. The specific primer pair provided by the invention can beused for identifying porcine infectious pleuropneumonia actinobacillus toxin. The primer composition provided by the invention is combined with double PCR (polymerase chain reaction), so that the ApxI A and Apx IV A toxin can be simultaneously identified; the characteristics of high sensitivity, high specificity, short time, low cost and the like are realized; the primer composition can be usedfor clinical sample detection; the operation is simple; the popularization is easy; the basic level operation and the application are convenient.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer composition for assisting identification of porcine infectious pleuropneumonia ApxI toxin and application thereof. Background technique [0002] Porcine infectious pleuropneumonia is a highly contagious infectious disease of pigs caused by Actinobacillus pleuropneumoniae (APP). One of the three major respiratory infectious diseases. In clinical autopsy, pulmonary hemorrhage, necrosis and fibrinous exudation are the main lesions, and the fatality rate of acute cases is high. The season of onset of this disease is obvious, and it occurs frequently in harsh weather such as winter with low temperature, early spring and late autumn. Pigs of any age are susceptible to the disease, with the most susceptible being 3 months old. Pigs infected with Actinobacillus pleuropneumoniae are often prone to other bacterial diseases clinically, such as Haemophilus parasuis, Pasteurella multo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686C12Q2537/143
Inventor 刘平胡国良郭小权曹华斌刘佩李麟吴聪张彩英幸程鸿邹振兴韩银华王玲
Owner JIANGXI AGRICULTURAL UNIVERSITY
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