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Double PCR (polymerase chain reaction) method for detecting Streptococcus suis type 2 and Haemophilus parasuis

A technique for Haemophilus suis and Streptococcus suis, which is applied in the field of double PCR for simultaneous detection of Streptococcus suis type 2 and Haemophilus parasuis, and can solve the problems of complicated operation, low specificity and sensitivity, and low separation rate, etc. problem, to achieve the effect of high detection efficiency, high sensitivity and strong specificity

Inactive Publication Date: 2015-04-22
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional detection methods such as bacterial isolation and identification are time-consuming and labor-intensive, especially Haemophilus parasuis has strict nutritional requirements and is difficult to cultivate in experiments. At the same time, the isolation rate in the sampling process is also low. Traditional etiological diagnosis methods are used. It is difficult, and the traditional serotype diagnosis method has many shortcomings such as cumbersome and complicated operation, time-consuming and labor-intensive, low specificity and sensitivity

Method used

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  • Double PCR (polymerase chain reaction) method for detecting Streptococcus suis type 2 and Haemophilus parasuis
  • Double PCR (polymerase chain reaction) method for detecting Streptococcus suis type 2 and Haemophilus parasuis
  • Double PCR (polymerase chain reaction) method for detecting Streptococcus suis type 2 and Haemophilus parasuis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 detects Streptococcus suis type 2 and Haemophilus parasuis duplex PCR method determination of annealing temperature

[0025] 1. Sample pretreatment

[0026] Haemophilus parasuis and Streptococcus suis type 2 preserved in the laboratory were inoculated in TSB medium, and cultured on a shaker at 37°C for 26 hours as a template for sample detection.

[0027] 2. Bacterial DNA Extraction

[0028] The present invention adopts conventional DNA extraction method, and the steps are as follows:

[0029] 2.1 Suspend the above bacterial solution in 1.5ml lysozyme solution (0.15mol / l NaCl, 0.1mol / l NaCl 2 EDTA, 15 mg / ml lysozyme, pH=8);

[0030] 2.2.37℃ warm bath for 2h;

[0031] 2.3. Add 1.5ml 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;

[0032] 2.4 Use equal volumes of phenol: chloroform: isoamyl alcohol to extract each time, add 40 μl of 3mol / l ammo...

Embodiment 2

[0038]Example 2 Detection of Streptococcus suis type 2 and Haemophilus parasuis double PCR method system specificity

[0039] 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, Pasteurella, Staphylococcus aureus, Salmonella, etc. as controls to verify the specificity of the system, and the system can only amplify parasuis. Two target gene fragments of Hemobacterium and Streptococcus suis type 2, while the control group could not amplify the fragments were qualified for specificity.

[0040] 1. Sample pretreatment

[0041] Streak the Escherichia coli, Pasteurella, Staphylococcus aureus, and Salmonella preserved 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 bacterial solution after 12 hours as Control PCR template; Haemophilus parasuis and Streptococcus suis type 2 pr...

Embodiment 3

[0049] Example 3 Detection of Streptococcus suis type 2 and Haemophilus parasuis double PCR method system sensitivity

[0050] Sensitivity is another key factor to measure whether the PCR reaction system is effective. To determine the sensitivity of the established duplex PCR method, the extracted H. parasuis DNA was diluted to: 1.2×10 5 -1.2×10 -4 pg, Streptococcus suis type 2 DNA diluted to: 4.0×10 6 -4.0×10 -5 pg, the established duplex PCR method was used for detection, and the sensitivity of the method was determined.

[0051] 1. Double PCR amplification reaction system amplification

[0052] The PCR method is the same as in Example 2.

[0053] 2. Results

[0054] The content of the extracted Streptococcus suis type 2 DNA was 4.0×10 6 pg, the concentration of the extracted DNA of Haemophilus parasuis was determined to be 1.2×10 5 pg, carry out 10 times of dilution to two kinds of substrate concentrations and detect respectively, as seen from the figure, double PCR...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a double PCR (polymerase chain reaction) method for detecting Streptococcus suis type 2 and Haemophilus parasuis. The method comprises the following steps: respectively screening out Haemophilus parasuis conservative gene segment 16SrRNA and Streptococcus suis type 2 capsular polysaccharide gene cps2J from published documents as two gene target spots for PCR detection; putting 2 pairs of primers for amplifying the 2 gene segments into a PCR reaction system, and directly carrying out one-time detection on Streptococcus suis type 2 and Haemophilus parasuis from the sample by optimizing the annealing temperature, specificity and other parameters. The system of the method can accurately detect the pathogen and analyze the pathogen mixed infection conditions, thereby mastering the development trend and performing the effect of achieving two goals at one stroke.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a multiple PCR detection method, in particular to a double PCR method capable of simultaneously detecting Streptococcus suis type 2 and Haemophilus parasuis. Background technique [0002] Streptococcus suis widely exists in countries with developed animal husbandry. The bacteria can not only cause symptoms such as acute hemorrhagic septicemia, endocarditis, meningitis, arthritis, lymphadenitis, piglet diarrhea, bronchitis, and abortion in pregnant pigs, but also human suppurative arthritis, toxic shock syndrome, etc. Syndrome, meningitis, sequelae of permanent deafness [2], etc., leading to death in severe cases. According to the different capsule antigens, Streptococcus suis can be divided into 35 serotypes, of which Type 1, Type 1 / 2, Type 2, Type 7, Type 9 and Type 14 are the main serotypes of Streptococcus suis, which are prevalent in my country. The main type 2 Streptococcus suis....

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/04C12R1/465C12R1/21
CPCC12Q1/686
Inventor 朱建国韩少鹏李成之
Owner SHANGHAI JIAO TONG UNIV
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