Duplex PCR primers for actinobacillus pleuropneumoniae and pasteurella multocida

A porcine pleuropneumonia and actinobacillus technology, applied in microorganisms, recombinant DNA technology, microorganism-based methods, etc., can solve the problems of high morbidity and mortality, difficulty in differential diagnosis, economic losses in the pig industry, etc., and achieve strong specificity. Sex and sensitivity, simple effect

Inactive Publication Date: 2017-07-28
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both App and Pm are important pathogenic bacteria commonly found in the respiratory system of pigs. During the acute outbreak period, they have high morbidity and mortality, causing large economic losses to the pig industry.
The clinical symptoms and lung lesions of pigs infected with these two bacteria are very similar, and often accompanied by co-infection with multiple pathogens, which brings certain difficulties to clinical differential diagnosis

Method used

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  • Duplex PCR primers for actinobacillus pleuropneumoniae and pasteurella multocida
  • Duplex PCR primers for actinobacillus pleuropneumoniae and pasteurella multocida
  • Duplex PCR primers for actinobacillus pleuropneumoniae and pasteurella multocida

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Detection of specificity of primers A1 and A2 of Actinobacillus pleuropneumoniae

[0018] The DNA of App bacteria, DNA of Pm bacteria, DNA of Haemophilus parasuis and DNA of Escherichia coli were extracted as templates for PCR detection.

[0019] Using the extracted DNA of the sample to be tested as a template, the total volume of the PCR reaction of a single pair of primers is 20 μL: add 10 μL of 2×EasyTaq SuperMix, and the concentrations of primers A1 and A2 are both 10 uM Add 1.0 μL each, extract 3 μL of the DNA template of the sample to be tested, and supplement with sterile deionized water to a final volume of 20 μL.

[0020] Specific results such as figure 1 , in which lane 1: DNA Marker DL2000; lane 2: detection of DNA of App bacteria, there is a band at 924bp, indicating that App bacteria is positive; lane 3: detection of DNA of Pm bacteria, no band display, indicating that App bacteria is negative Swimming lane 4: detection of DNA of Haemophilus pa...

Embodiment 2

[0021] Example 2 Detection of the specificity of Pasteurella multocida primers A1 and A2

[0022] The DNA of Pm bacteria, App bacteria DNA, Haemophilus parasuis DNA and Escherichia coli DNA were extracted as templates for PCR detection.

[0023] Using the extracted DNA of the sample to be tested as a template, the total volume of the PCR reaction of a single pair of primers is 20 μL: add 10 μL of 2×EasyTaq SuperMix, and the concentrations of primers P1 and P2 are both 10 uM Add 1.0 μL each, extract 3 μL of the DNA template of the sample to be tested, and supplement with sterile deionized water to a final volume of 20 μL.

[0024] Specific results such as figure 2 , in which lane 1: DNA Marker DL2000; lane 2: detection of DNA of Pm bacteria, a band at 119 bp, indicating that Pm bacteria are positive; lane 3: detection of DNA of App bacteria, no band display, indicating that Pm bacteria are negative Swimming lane 4: detection of DNA of Haemophilus parasuis, no band display, i...

Embodiment 3

[0025] Example 3 Double PCR detects the specificity of App and Pm

[0026] The DNA of App bacteria, DNA of Pm bacteria, DNA of Haemophilus parasuis, DNA of Escherichia coli and sterile deionized water were used as templates for PCR detection.

[0027] The total volume of the double PCR reaction is 20 μL: 10 μL of 2×Easy Taq SuperMix is ​​added, and the concentrations of primers A1 and A2 are both 10 uM Add 1.0 μL each, the concentrations of primers P1 and P2 are both 10 uM Add 0.5 μL each, extract 3 μL of the DNA template of the sample to be tested, and add sterile deionized water to a final volume of 20 μL.

[0028] The reaction conditions were as follows: pre-denaturation at 94 °C for 10 min, followed by cycling, the cycle parameters were 30 sec at 94 °C, annealing at 57 °C for 30 sec, 45 sec at 72 °C, and extension at 72 °C for 10 min after 35 cycles. At the end of the reaction, 5 μL of the PCR reaction product was electrophoresed on a 1% agarose gel, using DNA Marker DL2...

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Abstract

The invention provides duplex PCR primers for actinobacillus pleuropneumoniae and pasteurella multocida. The primers are shown as the following: A1:5'-CACGCAGGCGGTTGATTAAG-3', A2:5'-TTGCTACGACTTCACCCCAG-3'; P1:5'-TAAAGCGTGGGGAGCAAACA-3', P2:5'-CAGGCGGTCGATTTATCACG-3'. The invention also establishes a duplex PCR detection method for simultaneous differential diagnosis of actinobacillus pleuropneumoniae and pasteurella multocida, and the method has high accuracy and sensitivity, can achieve precise differential diagnosis of the causes of porcine pulmonary infection, and accordingly implement a right therapeutic schedule to save economic losses.

Description

technical field [0001] The invention relates to double PCR primers for detecting Actinobacillus pleuropneumoniae and Pasteurella multocida and its application, belonging to the technical field of animal husbandry and veterinary medicine. Background technique [0002] Actinobacillus pleuropneumoniae (App) is a highly contagious pathogen that can cause hemorrhagic, suppurative and fibrinous pneumonia in pigs. Pasteurella multocida (Pm) can cause porcine pneumonia and atrophic rhinitis in pigs, mainly causing acute sepsis in adult pigs. Both App and Pm are common important pathogenic bacteria in the respiratory system of pigs. During the acute outbreak period, they have high morbidity and mortality, and cause large economic losses to the pig industry. The clinical symptoms and lung lesions of pigs infected with these two bacteria are very similar, and often accompanied by co-infection with multiple pathogens, which brings certain difficulties to clinical differential diagnosis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686C12Q2600/16
Inventor 吴学敏陈秋勇陈如敬王隆柏车勇良周伦江王晨燕刘玉涛严山
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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