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Method for detecting brucella abortus attenuated vaccine strain S19

A technology of attenuated vaccine and Brucella, which is applied in the field of microbial detection, can solve the problems of no detection method for identifying S19 strain, and achieve the effect of simple operation and strong specificity

Inactive Publication Date: 2018-05-18
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, this technology has been applied in the rapid detection of some pathogens, but there is no detection method based on RPA technology to identify the S19 strain

Method used

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  • Method for detecting brucella abortus attenuated vaccine strain S19
  • Method for detecting brucella abortus attenuated vaccine strain S19
  • Method for detecting brucella abortus attenuated vaccine strain S19

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, be used to detect the design of the RPA primer set of S19 strain

[0027] The S19 strain of Brucella bovis was isolated from milk in the United States in 1923. After natural attenuation in the laboratory, it became an attenuated strain with stable virulence. S19 strain is sensitive to erythritol. Compared with other Brucella genomes, S19 strain has significant deletion of EryC gene. The Omp2 gene is a highly conserved gene in Brucella, which exists in all types of Brucella genomes. The present invention uses Oligo The software first designed the general detection primer Omp2 for Brucella strains based on the Omp2 gene, and then designed the S19 strain identification primer S19E based on the EryC gene. Using the above two sets of primers can effectively identify the S19 strain. Designing and screening suitable primers is the key to the RPA reaction. Factors such as base composition, GC content, secondary structure formation, and Tm value need to be consi...

Embodiment 2

[0031] Embodiment 2, the effect detection of Brucella bovis attenuated vaccine strain S19RPA

[0032] 1.1 Test material

[0033] Strains: Common Brucella species, biotype reference strains and Brucella vaccine strains used, see Table 2; four non-Brucella reference strains: Escherichia coli K99, Pasteurella C48-1, Streptococcus suis ST171, Pseudomonas aeruginosa DI-1.

[0034] Table 2: Brucella strains used and accession numbers

[0035]

[0036] 1.2 Bacterial DNA Extraction

[0037] Inoculate the above-mentioned strains into tryptic agar medium, culture at 37°C for 24-72 hours, without adding or adding 5-10% CO 2 . After the cultured colonies were washed with physiological saline containing 0.5% formaldehyde, they were inactivated at 37°C for 24 h, and the bacterial DNA was extracted with a bacterial genomic DNA extraction kit (QIAGEN Company), and the concentration of bacterial genomic DNA was measured by a micronucleic acid protein analyzer, and frozen at -20 ℃, set ...

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Abstract

The invention provides a method for detecting a brucella abortus attenuated vaccine strain S19 and a primer group used for detection, wherein the sequence of the adopted primer group is SEQ ID NO: 1-4. The primer group and method have advantages that 1) the primer group and method are efficient and sensitive, amplification can be completed in 30 min, and the lowest detection limit of an amplification template is 1.0*10<-3> ng / [mu]l; 2) the primer group and method are high in specificity, and two pairs of PRA primer groups specifically identify and detect S19 and don't cause any cross reactionwith brucellin of other species and biological types, brucellin vaccine strains and common non-brucellin strains; 3) the primer group and method are simple to operate, the prepared RPA reaction systemundergoes a reaction in a 39-degree constant-temperature condition to undergo a reaction, and a PCR instrument is not required; and 4) the primer group and method are high in flux, a large amount ofsamples can be detected in one time in a water bath or a constant-temperature incubator, and are not limited by the quantity of test holes of the PCR instrument.

Description

technical field [0001] The invention belongs to the technical field of microbial detection, and in particular relates to a rapid identification method of Brucella bovis attenuated vaccine strain S19 using a recombinase polymerase amplification technology (recombinase polymerase amplification, RPA). Background technique [0002] Brucellosis (referred to as brucellosis) is a zoonotic infectious disease that is widely prevalent in the world caused by Brucella, and poses a serious threat to animal husbandry and public health safety. In recent years, the situation of brucellosis prevention and control in my country has been severe, showing a trend of shifting from pastoral areas to non-pastoral areas, and from occupational groups to non-occupational groups. Immunization of livestock with attenuated vaccine can curb the spread of brucellosis to humans, but the immunization of attenuated vaccine will interfere with the diagnosis and monitoring of brucellosis. It is of great signif...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2522/101C12Q2531/119
Inventor 陈义平秦立得南文龙谭鹏飞巩明霞张凤霞
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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