Double PCR (polymerase chain reaction) detection method for haemophilus parasuis

A technology for the detection of Haemophilus suis and a detection method, which is applied in the field of double PCR capable of detecting Haemophilus parasuis, can solve the problems of cumbersome and complicated operation, low separation rate, low specificity and sensitivity, and achieve improved detection efficiency, Good reproducibility and strong specific effect

Inactive Publication Date: 2015-01-21
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Haemophilus parasuis has strict nutritional requirements, and it is difficult to cultivate in experiments. At the same time, the isolation rate in the sampling process is also low. It is difficult to use traditional etiological diagnosis methods, and the traditional serotype diagnosis methods are cumbersome to operate. Complicated, time-consuming and labor-intensive, low specificity and sensitivity, etc.

Method used

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  • Double PCR (polymerase chain reaction) detection method for haemophilus parasuis
  • Double PCR (polymerase chain reaction) detection method for haemophilus parasuis
  • Double PCR (polymerase chain reaction) detection method for haemophilus parasuis

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Effect test

Embodiment 1

[0018] Embodiment 1 detects the determination of the annealing temperature of Haemophilus parasuis double PCR method

[0019] 1 Bacterial DNA Extraction

[0020] The present invention adopts conventional DNA extraction method, and the steps are as follows:

[0021] 1.1 Suspend the above bacterial solution in 1.5ml lysozyme solution (0.15mol / lNaCl, 0.1mol / lNaCl 2 EDTA, 15 mg / ml lysozyme, pH=8);

[0022] 1.2.37℃ warm bath for 2h;

[0023] 1.3. Add 1.5ml of 10% SDS (0.1mol / l NaCl, 0.5mol / l Tris, 10% SDS, pH=8) and mix by inverting repeatedly for 10min, centrifuge at 10000r / min for 10min, and take the supernatant;

[0024] 1.4 Use equal volumes of phenol: chloroform: isoamyl alcohol to extract once each, add 40 μl of 3mol / l ammonium acetate and 3ml of absolute ethanol to the water phase, precipitate DNA at -20°C for 1h, and centrifuge at 12000r / min for 10min;

[0025] 1.5 Collect the DNA precipitate and dissolve it with 100 μl TE.

[0026] 2 Establishment of double PCR amplif...

Embodiment 2

[0030] Example 2 Detection of Haemophilus parasuis double PCR method system specificity

[0031] Specificity is another key factor in the PCR reaction system. In order to ensure the specificity of the established double PCR system, the present invention uses Escherichia coli, Salmonella, Haemophilus, Streptococcus type 2, etc. as controls to verify the specificity of the system, and the system can only amplify parasuis Two target gene fragments of Haemobacter, while the control group could not amplify the fragments were qualified for specificity. Streak the Salmonella and Escherichia coli stored in the laboratory on the LB medium, culture overnight at 37°C and inoculate a single colony in 2ml LB liquid medium, and take the Salmonella and Escherichia coli liquid as the control PCR template after 12 hours; Mycoplasma hyopneumoniae, Pasteurella multocida, and Streptococcus type 2 stored in the room were streaked on TSA medium, and after being cultured at 37°C for 24 hours, a sin...

Embodiment 3 2

[0038] Embodiment 3 Double PCR method detects Haemophilus parasuis in the actual sample

[0039] In order to further verify the practicability of the double PCR method for detecting Haemophilus parasuis provided by the present invention, the present invention carried out bacterial detection on the lung tissue and joint fluid of sick pigs with respiratory symptoms and (or) joint swelling collected from Pudong pig farms. For isolation, the disease material was first streaked on TSA medium, cultured at 37°C for 24 hours, and then monoclonal colonies were picked and inoculated in TSB medium. After 12 hours, the bacterial liquid was taken as the DNA sample of Pasteurella multocida.

[0040] 1 Bacterial DNA Extraction

[0041] With embodiment 1.

[0042] 2 Double PCR amplification reaction system amplification

[0043] The double PCR reaction system is as follows: 1 μl of cDNA template, 0.5 μl of 2 pairs of upstream and downstream primers, 12.5 μl of PCR Mix, and 25 μl of ultrapur...

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Abstract

The invention belongs to the biotechnical field and particularly relates to a double PCR (polymerase chain reaction) detection method for haemophilus parasuis. The double PCR method for directly detecting haemophilus parasuis from a sample at one time is established by using a conservative 16SrRNA gene with a pathogen genotype characteristic as well as a virulence factor coding gene vtaA gene as two gene targets for PCR detection by way of putting two pairs of primers for amplifying two gene segments in a PCR reaction system through adjustment and optimization of parameters such as annealing temperature and specificity. The method system provided by the invention can be used for detecting diseased pig lung tissues with respiratory symptom and (or) arthrocele and haemophilus parasuis of a synovial fluid. The detection method not only can be used for detecting a pathogen, but also can be used for analyzing structural variation of the virulence factor coding gene, so that the variation trend is mastered, thereby playing a role in killing two birds with one stone.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a double PCR method capable of detecting Haemophilus parasuis. Background technique [0002] Haemophilus parasuis (HPS) is a polymorphic, non-hemolytic, non-motile, Gram-negative bacterium belonging to the genus Haemophilus in the family Pasteurellaceae, and is responsible for the disease of pigs. disease) pathogenic bacteria, widely present in the upper respiratory tract of ordinary pigs, can cause polyserositis, arthritis and meningitis in pigs. With the development of the pig industry, the disease caused by the infection of this bacterium has become an important bacterial disease affecting the global pig industry, which has brought huge economic losses to the breeding industry, and has caused a high degree of research on Haemophilus parasuis in various countries. Pay attention, my country is a big pig raising country, and it is of great significance to study the rapid and accurate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q2537/143
Inventor 朱建国韩少鹏
Owner SHANGHAI JIAO TONG UNIV
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