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Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer and application thereof

A technology of Erysipelas suis and porcine erysipelas, which is applied in the field of molecular biology and production, can solve the problems of economic loss of pig farmers and the incomplete purification of the disease, and achieve the effect of good specificity and high sensitivity

Active Publication Date: 2014-12-10
WENS FOOD GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This disease was once known as one of the "three major infectious diseases" in my country. In recent years, with the intensive development of pig raising, porcine erysipelas has gradually faded out of people's field of vision, but the disease has not been completely purified, and it has always occurred in various regions of the country Sporadic occurrences have caused severe economic losses to pig farmers (Gong Pingyang, Wang Lianxiang, Yang Zhixiang. The characteristics and comprehensive prevention and control measures of swine erysipelas in pig farms in Guangdong area [J]. General Animal and Poultry Industry, 2010 ( 11): 11-12.)

Method used

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  • Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer and application thereof
  • Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer and application thereof
  • Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] (1) DNA extraction from samples to be tested: 1.5mL BHI culture solution containing G4T10 strain, 1.5mL BHI culture solution containing GD1201 strain, BHI culture solution was used as negative control, respectively follow the TIANGEN bacterial genomic DNA extraction procedure (extraction reagents were purchased from TIANGEN Company) and store the extracted DNA at -20°C.

[0021] (2) Add 1 μL DNA template to the PCR master mix to prepare a 25 μL reaction system and mix well.

[0022] PCR master mix: 1.5-3 μL of 10-fold diluted PCR buffer, 1.5-3 μL of dNTP mixture, 1 μL of Taq DNA polymerase at a concentration of 2.5M / μL, all shown in SEQ ID NO: 1-2 at a concentration of 10 pmol / μL 2 μL of each primer, ddH 2 O to make up 25 μL.

[0023] (3) Put the PCR reaction system in step (2) on a PCR instrument to perform cycle amplification reaction. The amplification conditions were as follows: pre-denaturation at 95°C for 10 minutes; then 30 cycles of 95°C for 30 s, 58°C for 30...

Embodiment 2

[0027] Example 2 specificity test

[0028] With the BHI culture solution containing G4T10 strain and the BHI culture solution containing GD1201 strain as positive controls, the BHI of Actinobacillus pleuropneumoniae, Haemophilus parasuis, Streptococcus suis, Escherichia coli suis and Pasteurella suis Culture fluid is carried out specific detection, uses the method and primer of embodiment 1, and detection result sees figure 2 . From figure 2 As can be seen from the results, except for the positive control, no bands could be amplified, and the positive disease materials amplified 379bp bands and 498bp bands respectively through PCR, indicating that the primers of SEQ ID NO:1~2 have a high specificity.

Embodiment 3

[0029] Embodiment 3 Sensitivity test

[0030] The DNAs of the two strains (G4T10 strain and GD1201 strain) extracted in Example 1 were serially diluted 10 times with sterile double-distilled water, and the DNA contents of the G4T10 strains were 55.8 μg, 55.8 μg, 0.558 μg, and 0.0558 μg, respectively. , 5.58ng, 0.558ng, 0.0558ng, 5.58pg, and the diluted DNA contents extracted from the GD1201 strain culture samples were 23.8μg, 23.8μg, 0.238μg, 0.0238μg, 2.38ng, 0.238ng, 0.0238ng, 2.38pg, respectively. Take 2 μL of each dilution as a template, perform PCR amplification detection according to the method in Example 1, observe positive bands, and calculate its sensitivity based on the highest dilution of the template amount used for positive expected bands, and the results show that the minimum detection amount 0.56ng and 2.4ng, respectively, see image 3 , Figure 4 .

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Abstract

The invention discloses a Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer and an application thereof, belonging to the fields of molecular biology and production of animal medicines. The nucleotide sequence of the Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer is shown in SEQIDNO:1-2. The Bacillus erysipelatos-suis PCR (Polymerase Chain Reaction) primer in the invention can be used to prepare a detection reagent of Bacillus erysipelatos-suis, can distinguish swine erysipelas attenuated vaccine strain G4T10 strain and other wild prevalent strains, and is contributed to monitoring the epidemic prevention condition of vaccines and diagnosing swine erysipelas condition in production.

Description

technical field [0001] The invention belongs to the field of molecular biology and production of veterinary medicine, and specifically relates to other pigs that are mainly used to distinguish porcine erysipelas attenuated vaccine strain G4T10 and wild virus epidemic strains (including non-porcine erysipelas attenuated vaccine strain G4T10 strains such as virulent epidemic strain GD1201) Erysipelas strain) PCR amplification primers and their applications. Background technique [0002] Swine erysipelas (SE) is caused by Erysipelas suis ( Erysipelothrix rhusiopathiae , ER) is an acute febrile infectious disease characterized by high fever, acute sepsis, skin rash (subacute), chronic verrucous endocarditis and skin necrosis, and multiple non-suppurative arthritis (Wood R.L., Erysipelas, in: Leman, et al. (Eds.), Diseases of swine, Iowa State University Press, Ames, Iowa, 1992, pp. 475–486). Erysipelas suis isolated from pigs with these symptoms were mostly serotypes 1 (subtyp...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/04
Inventor 王雷周庆丰宋延华潘永飞
Owner WENS FOOD GRP CO LTD
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