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Kit and process for PCR amplification detecting type 2 pig streptococcus virulence gene

A technology for detecting kits and virulence genes, which is applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc. It can solve problems such as difficult emergency detection, PCR reaction and cycle conditions are not the same

Inactive Publication Date: 2007-08-22
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there have been research reports using PCR (polymerase chain reaction) technology to detect SS2 virulence-related genes, but the PCR reactions and cycle conditions used are not the same, and multiple amplifications are required; although there are also reports on multiplex PCR detection, However, it is still not possible to simultaneously detect all 7 important genes of SS2 in one amplification, which is difficult to meet the needs of emergency detection

Method used

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  • Kit and process for PCR amplification detecting type 2 pig streptococcus virulence gene
  • Kit and process for PCR amplification detecting type 2 pig streptococcus virulence gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] SS2 related virulence gene detection kit, including the following components:

[0117] (1) Qiagen DNA extraction reagent

[0118] (2) Multiplex PCR amplification detection reagent: It is made by mixing 10×PCR buffer, deoxynucleoside triphosphate (dNTP) mixture, DNA polymerase and specific amplification primers, etc., with distilled water as a solvent.

[0119] There are 8 pairs of specific amplification primers, and the names of the primers, the size of the sequence amplification products and their management are shown in Table 1.

[0120] Table 1: Primer sequences and groups

[0121]

[0122] The multiplex PCR amplification detection reagent is mixed with distilled water as a solvent by the following components:

[0123] 10×PCR buffer content volume percentage 10%

[0124] Deoxynucleoside triphosphates:

[0125] dATP 200 μM, dCTP 200 μM, dGTP 200 μM, dTTP 200 μM, dUTP 50 μM

[0126] Specific primer tube 1 (100×) Content of each primer 50μM

[0127] Specif...

Embodiment 2

[0152] Relevant virulence gene detection kit, including components such as Example 1, also includes: SS2 DNA control template (DNA ≥ 100ng / μL), source: Streptococcus suis serotype 2 (Streptococcus suis serotype 2) Danish reference strain (Zhejiang University Professor Fang Weihuan) was obtained by extracting DNA by conventional methods.

[0153] PCR specificity test: positive reference strain adopts Streptococcus suisserotype 2 (Streptococcus suisserotype 2) Danish reference strain (i.e. the SS2 DNA control template used in this preferred SS2 virulence gene detection kit); negative reference strain adopts: A) Streptococcus : Streptococcus pneumoniae (Streptococcus pneumoniae, S.pne), beta-hemolytic streptococcus (β-hemolytic streptococcus, S.β-hemo), viridans streptococcus (Streptococcus viridans, S.vi); B) other genera : Neisseria meningitides (Neisseriameningitides, N.m), Staphylococcus epidermidis (S.epi) and Vibrio cholerae (Vibrio cholerae, V.cho), etc., the source of the...

Embodiment 3～7

[0155] Embodiment 3~7 adopts live cell counting method to carry out colony counting to SS2 positive reference strain: get the bacterium suspension of 0.5 McFarland unit, do a series of 10 times of dilutions, then quantitative dilutions are carried out plate culture, according to Count the number of colonies cultured and calculate the number of viable bacteria in the culture. Starting from 10 million, serially dilute 10 times to 1 bacterium, apply the above-mentioned kit, and carry out PCR amplification according to the preferred method of the present invention, to detect the detection ability of the kit used for the sample. The results showed that when specific bands appeared in all 8 target gene fragments, the PCR sensitivity was 78 CFU / reaction. When the DNA template was lower than 7.8 CFU, some bands were not obvious (see Table 4, Figure 1).

[0156] Table 4: PCR Sensitivity Test ×

[0157] Target gene (bp)

[0158] × Note: The strain uses SS2 positive referen...

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Abstract

The (Streptococcus suis Serotype 2 (SS2) virulence gene PCR amplification kit includes a SS2DNA contrast template, a PCR amplification detecting reagent, an agarose gel electrophoresis reagent and a DNA gradient standard. The PCR amplification detecting process includes the measurement of 7 important SS2 genes, diagnosing Streptococcus infection and SS2 infection based on whether to amplify specific Streptococcus gene, specific SS2 gene and 5 relevant important SS2 virulence genes, and predicting the virulence, invasiveness trend and disease prognosis fast and early. The present invention is favorable to the clinical diagnosis of borderline case and early warning, and is hopeful to be applied in emergency detection of human SS2 infection, etc.

Description

(1) Technical field [0001] The invention relates to a type 2 Streptococcus suis virulence gene PCR amplification detection kit and a detection method. (2) Background technology [0002] Streptococcus suis is an important zoonosis, mainly infected by Streptococcus suis Serotype 2 (SS2). Human infection can cause pneumonia, meningitis, bacteremia, septicemia, etc., and severe cases can cause toxic shock syndrome (Toxic Shock Syndrome, TSS), and the latter is the most serious. die soon. Studies have shown that the pathogenicity is closely related to its related virulence factors. Since Danish scholars first reported meningitis caused by human infection with Streptococcus suis in 1968, there have been reports of human infection with Streptococcus suis in many countries and regions around the world. In my country, in 1998 Jiangsu Province reported human infection with streptococcal suis, with 25 cases and 14 deaths; from June to August 2005, an outbreak of human infection with...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12Q1/14
Inventor 杨婷婷程苏云徐宝祥王复甦张政朱水荣
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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