Rapid detection and monitoring method for common swine infectious disease pathogens

A technology for infectious diseases and pathogens, applied in the field of molecular biology, to achieve the effects of good detection specificity, good specificity and compatibility, and convenient operation

Pending Publication Date: 2019-09-20
GUANGZHOU PLUSLIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is to overcome the defects and deficiencies of the existing porcine infectious disease pathogen detection technology, research and obtain a porcine infectious disease pathogen nucleic acid detection site based on the CRISPR / Cas12a system, and use the CRISPR / Cas12a system for this site It can realize the nucleic acid detection of porcine infectious disease pathogens, with good specificity, high sensitivity and low false positive

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  • Rapid detection and monitoring method for common swine infectious disease pathogens
  • Rapid detection and monitoring method for common swine infectious disease pathogens
  • Rapid detection and monitoring method for common swine infectious disease pathogens

Examples

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Embodiment 1

[0031] Example 1 Discovery of nucleic acid detection sites for porcine infectious disease pathogens based on the CRISPR / Cas12a system

[0032]We obtained the genome sequence of the porcine infectious disease pathogen, and compared it through bioinformatics analysis to find the specific identification region of each strain of the porcine infectious disease pathogen. The swine infectious disease pathogens we screened in this patent are: African swine fever virus (ASFV), porcine foot and mouth disease virus (Foot and Mouth Disease virus, FMDV), porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), porcine rotavirus group A (Porcine rotavirus group A, PRV), porcine transmissible gastroenteritis virus (Porcine transmissible gastroenteritis virus, TGEV).

[0033] The specific operation method is as follows: search for the whole genome sequence of the pathogen in the NCBI nucleic acid sequence library, obtain all the existing whole genome sequences of the pathogen,...

Embodiment 2

[0038] Example 2 Case of detecting nucleic acid of porcine infectious disease pathogen based on CRISPR / Cas12a system

[0039] 1. CRISPR / Cas12a gene cloning and protein expression

[0040] The Cas12a protein gene derived from Lachnospiraceae bacterium is used, and the codon is optimized to make the gene more suitable for expression in mammalian cells. The optimized Cas12a protein gene was cloned into the pET28a plasmid with a 6-His histidine tag to facilitate protein purification and expression. The Cas12a protein recombinant expression vector was transformed, and the expression strain was BL21 star (DE3).

[0041] The specific protein expression conditions are: in culture solution OD 600 When =0.6, add 0.5mMIPTG and cultivate for 4 hours. Bacteria were collected for protein purification. The purification conditions are: resuspend the bacteria in the lysate (50mM Tris, pH8.0, 300mM NaCl, 5% glycerol, 20mM imidazole), and perform sonication (70% amplitude, 2s On / 4s Off, 3 mi...

Embodiment 3

[0070] Example 3 Detection method of multiple porcine infectious disease pathogen nucleic acid based on CRISPR / Cas12a system

[0071] In order to realize the simultaneous detection of multiple pathogenic bacteria, we have developed a multiplex detection system for the above pathogenic targets. Since African swine fever virus is a DNA virus among the pathogens of porcine infectious diseases, the other four viruses are all RNA viruses, and CRISPR / Cas12a detection can only be performed after RT-RPA. The identified regions of the genomes of these four pathogens and the corresponding gRNA sequences were analyzed. According to parameters such as sequence similarity, GC content, base homogeneity, presence or absence of secondary hairpin structures, and presence or absence of cross-reaction in the same reaction, etc. The reaction system and gRNA combination method were optimized.

[0072] 1. Specific operation: After preparing the gRNA according to the method of step 3 in Example 2, ...

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Abstract

The invention discloses a rapid detection and monitoring method for common swine infectious disease pathogens. A series of specific nucleic acid sequence loci which can be used for detecting the common swine infectious disease pathogens are obtained through researches, a detection technology that can realize qualitative detection of the common swine infectious disease pathogens is developed for the loci, and a convenient, fast and low-cost detection method and detection kit for the swine infectious disease pathogens are constructed, and have good specificity and sensitivity for detection of the swine infectious disease pathogens; multi-locus simultaneous detection can be achieved, and the method and kit have broad application prospects for rapid detection, screening and monitoring of the common swine infectious disease pathogens.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to a rapid detection and monitoring method for pathogens of common infectious diseases in pigs. Background technique [0002] The pathogens of common infectious diseases in pigs are widely distributed and highly infectious. With the adjustment of the rural production structure and the continuous increase of the scale of pig raising, the circulation of live pigs and their products has become increasingly frequent, resulting in frequent outbreaks of pig infectious diseases. In addition, some pig breeding-intensive areas and pig farms have weak detection and epidemic prevention capabilities. Once an infectious disease occurs, if it cannot be diagnosed and controlled in time, it will lead to large-scale disease and death of pigs, which will bring huge economic losses to pig farmers. It will greatly increase the circulation cost of the whole society in epidemi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6806C12N15/113C12R1/93
CPCC12Q1/701C12Q1/6806C12N15/113C12N2310/10C12Q2521/507Y02A50/30
Inventor 刘华勇陈翀谢婵芳
Owner GUANGZHOU PLUSLIFE TECH CO LTD
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