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Rapid detection for duplex PCR primer pair of bacillus erysipelatos-suis and streptococcus suis, and kit, and method

A technology of Erysipelas swine and detection kits, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., can solve problems such as waste, and achieve the effects of saving time, simple operation, and saving detection costs.

Pending Publication Date: 2018-04-20
HUAZHONG AGRI UNIV
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  • Claims
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AI Technical Summary

Problems solved by technology

[0004] Clinical PCR method is commonly used to detect the presence of Erysipelobacter suis and Streptococcus suis in samples, but multiple single PCR reactions will cause unnecessary waste, and there are a large number of mixed infection cases clinically. Infected samples, a single PCR reaction is difficult to perform comprehensive and rapid detection

Method used

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  • Rapid detection for duplex PCR primer pair of bacillus erysipelatos-suis and streptococcus suis, and kit, and method
  • Rapid detection for duplex PCR primer pair of bacillus erysipelatos-suis and streptococcus suis, and kit, and method
  • Rapid detection for duplex PCR primer pair of bacillus erysipelatos-suis and streptococcus suis, and kit, and method

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Experimental program
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Effect test

Embodiment 1 2

[0040] The preparation of embodiment 1 duplex PCR primer pair and test kit

[0041] Primer Premier 6 software was used to design PCR amplification primers for the SpaA gene and the gdh gene (the sequences of which are SEQ ID No.1 and SEQ ID No.2, respectively), and Wuhan Qingke Bioengineering Co., Ltd. was commissioned to synthesize the primers. ddH after primer synthesis 2 O was diluted to 50 μM, and the primer sequences were as follows:

[0042]

[0043] Each kit detects 100 samples, and the kit is assembled according to the following contents (the entire operation requires a sterile environment):

[0044] 1. 2×PCR reaction buffer (containing DNA polymerase EasyTaq, 2.5mM Mg2+ concentration magnesium chloride, dNTPs): 1.25mL

[0045] 2. Primer mixture (containing spaA-1F, spaA-1R, gdh-1F and gdh-1R at a concentration of 50 μM): 400 μL

[0046] 3. Positive control solution: 100μL

[0047] 4. ddH 2 O: 1mL

[0048] a. The PCR reaction system is:

[0049]

[0050] b...

Embodiment 2

[0057] Example 2: Preparation of Erysipelas suis and Streptococcus suis double PCR detection kit and detection of its positive control

[0058] The isolated and identified Erysipelas suis SE10 and Streptococcus suis SC19 were extracted respectively, and the genomes of the two bacteria were extracted by boiling method; 2 pairs of primers were synthesized according to the following sequence, diluted to 50 μM for use.

[0059] spaA-1F: 5'-CAGCAATGCCACTACAAACAG-3',

[0060] spaA-1R: 5'-TACAACTTGAATTTGGCGATT-3',

[0061] gdh-1F: 5'-AAGCAGTCAAAGCCCCGC-3',

[0062] gdh-1R: 5'-GGAGGCGTTTGTATTGACC-3';

[0063]Use DNA polymerase EasyTaq from Dalian Bao Biology Co., Ltd. and its supporting 10×Buffer, and the reaction system is: 4 μl of primer mixture (containing 1 μl each of spaA-1F, spaA-1R, gdh-1F, and gdh-1R), PCR buffer 19.5μl (including 2μl of dNTPs, 2.5μl of 10×Buffer, ddH 2 O 15 μl), EasyTaq (Dalian Bao Biological Company) 0.5 μl, sample DNA 1 μl. Use a micropipette to mix we...

Embodiment 3

[0066] Embodiment 3: Detection of clinical samples by Erysipelas suis and Streptococcus suis double PCR detection kit

[0067] 511 samples of heart, lung, kidney, liver, spleen, brain and joints collected from slaughterhouses with this detection kit, the detection steps are as follows:

[0068] Extraction of DNA from specimen: Homogenate 10 g of diseased tissue, incubate at 37°C for 5 hours, and treat with proteinase K (25g / L) in a water bath at 64-65°C for 2 hours. Take 1ml of the digested product, heat it in boiling water for 5 minutes, quickly place it in an ice bath for 5 minutes, centrifuge at 12000rpm for 2 minutes, and take the supernatant as the sample DNA.

[0069] PCR amplification: PCR reaction is performed using the extracted genome of the disease material as a template, and a positive control and a negative control are set at the same time. See Example 1 for the PCR reaction system and reaction conditions.

[0070] PCR amplification product analysis: After the P...

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Abstract

The invention discloses rapid detection for a duplex PCR primer pair of bacillus erysipelatos-suis and streptococcus suis, and a kit and a method. Through PCR amplification to DNA of a to-be-detectedsample, the kit can rapidly detect bacillus erysipelatos-suis and streptococcus suis in the sample. The duplex PCR is based on common PCR, allows a pair of primers to be added in the reaction system,and is used to detect single infection and mixed infection of the two kinds of bacteria in a clinic sample. Therefore, a duplex PCR detection kit and a detection method of the two kinds of pathogenicbacteria are established and have a great significance on fast accurate identification of pathogenic bacteria in a clinic sample and molecular epidemiological investigation on pathogenic bacteria.

Description

technical field [0001] The invention relates to a PCR detection kit, in particular to a double PCR primer pair, kit and method for rapid detection of Erysipelas suis and Streptococcus suis. Background technique [0002] Erysipelas suis can cause septicemia, polyarthritis and endocarditis in pigs. Humans are susceptible to Erysipelas suis, manifested as skin type or sepsis, which is called "like erysipelas". According to the National Pig Epidemic Report released by the Ministry of Agriculture in 2013, the number of swine erysipelas cases is much higher than that of swine fever, highly pathogenic PRRS, porcine cysticercosis, anthrax, porcine pneumonia and brucellosis. Streptococcus suis is the most important pathogen of porcine streptococcal disease in China. [0003] There are 25 serotypes of E. suis, and the main serotypes prevalent in my country are 1a and 2. Wang Lei and others used the SpaA gene to design PCR primers, which can quickly identify Erysipelas suis and disti...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/14C12Q1/04C12N15/11C12R1/46C12R1/01
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
Inventor 金梅林吴超康超朱伟峰李敬涛孙小美
Owner HUAZHONG AGRI UNIV
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