Multiple real-time fluorescence PCR detection primer composition for identifying streptococcus suis and pig pasteurella multocida and detection method

A technique for Streptococcus suis and Pasteurella spp. is applied in the field of multiplex real-time fluorescent PCR detection primer compositions to achieve the effects of high feasibility and application prospects, good reproducibility, high specificity and specificity

Active Publication Date: 2019-04-23
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there is no dual fluorescent PCR detection method established with Streptococcus suis and Pasteurella multocida as research objects

Method used

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  • Multiple real-time fluorescence PCR detection primer composition for identifying streptococcus suis and pig pasteurella multocida and detection method
  • Multiple real-time fluorescence PCR detection primer composition for identifying streptococcus suis and pig pasteurella multocida and detection method
  • Multiple real-time fluorescence PCR detection primer composition for identifying streptococcus suis and pig pasteurella multocida and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1. DNA extraction of Streptococcus suis samples, Pasteurella multocida samples, and other bacterial samples:

[0053] Bacterial DNA extraction kits were used for extraction, and the specific operation steps were found in the kit instructions. The purity and concentration of the extracted genomic DNA were determined by UV spectrophotometer. The measured OD260 / OD280 values ​​were all about 1.8-1.9, and the concentration was above 10ng / μL, which indicated that the DNA had a high purity and a moderate concentration, which met the requirements of PCR amplification.

[0054] 2. Selection of target genes and design of primers: Streptococcus suis (Suis) and Pasteurella multocida (PM) respectively use Gdh gene, plpEgene and OmlA gene as the nucleotide sequences of specific target gene primers, design The nucleotide sequences of the primers and probes are listed in Table 1.

[0055] Table 1 Nucleotide sequences of primers and probes

[0056]

[0057] 3. Production of standa...

Embodiment 2

[0070] Example 2 specificity verification

[0071] Using the primers and probes designed by the present invention, Streptococcus suis, Pasteurella multocida, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Escherichia coli, Bacillus pyogenes, Staphylococcus aureus, Bacillus subtilis bacillus, Avian bacillus paragallinarum, Bacillus coagulans, Lactobacillus reuteri, Lactobacillus plantarum, Bacillus carinii and Enterococcus faecium were used as templates for real-time fluorescent PCR detection to verify the specificity of their primers and probes . The results are shown in Table 3 and image 3 , the results show that the probes and primers designed in this study have strong specificity.

[0072] Table 3 specificity verification test

[0073]

[0074]

Embodiment 3

[0075] Embodiment 3 Sensitivity experiment

[0076] Quantify the genomic DNA of Streptococcus suis and Pasteurella multocida to 5 ng / μL respectively, and dilute it in a 10× gradient, and take 2.0 μL for each gradient as the template amount, (ie: 10ng, 1ng, 0.1ng, 0.01 ng, 0.001ng, 0.0001ng, 0.00001ng) Real-time fluorescent quantitative PCR detection was performed to evaluate the detection limit of the present invention. See Figure 4-Figure 5 , Streptococcus suis and Pasteurella multocida sensitivity are 0.01ng, the results show that the quantitative detection limit of the method is 0.01ng, illustrating that the method provided by the present invention has very high sensitivity, higher sensitivity than ordinary PCR.

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Abstract

The invention relates to the technical field of molecular biology strain detection, in particular to a multiple real-time fluorescence PCR detection primer composition for identifying streptococcus suis and pig pasteurella multocida and a detection method. Precise quantification can be carried out while qualitative detection of the streptococcus suis and the pig pasteurella multocida is provided,the amplification efficiency, the sensitivity and the accuracy are high, good repeatability is achieved, the detection period is short, detection can be completed within 1.5 hours, real-time monitoring is achieved, high feasibility and broad application prospects are achieved, and the scientific and reliable method is provided for identifying pathogenic microorganisms and reducing breeding economic losses.

Description

technical field [0001] The invention relates to the technical field of molecular biology strain detection, in particular to a multiple real-time fluorescent PCR detection primer composition for identifying Streptococcus suis and Pasteurella multocida, and also relates to a detection method using the detection primer composition. Background technique [0002] Swine Streptococcus suis (Suis-gdh) is an acute zoonotic infectious disease caused by Streptococcus of multiple serogroups. When it broke out, the epidemic was violent and spread rapidly, and the body temperature of sick pigs suddenly rose above 41 °C , almost no typical symptoms are seen, and death occurs within a few hours. Streptococcus suis is a second-class animal disease stipulated in my country, which not only poses a great threat to the world's pig industry, but also endangers the safety of public health. Due to the complex symptoms and pathological changes of the disease, it can only be used as a preliminary di...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/46C12R1/01
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 吴家强于江刘艳艳张玉玉陈智曾昊
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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