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Identification of animal-derived food pathogens and its drug resistance and virulence gene detection composite chip

A drug resistance gene and virulence gene technology, applied in the biological field, can solve the problems of heavy laboratory workload, low test detection sensitivity, long test cycle, etc.

Active Publication Date: 2019-12-13
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the traditional biochemical methods for pathogenic bacteria identification, toxin identification, and drug resistance identification in animal-derived foods have long test cycles, heavy laboratory workload, and low detection sensitivity of individual tests.
Based on the above characteristics, it is impossible to efficiently and accurately respond to public health emergencies

Method used

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  • Identification of animal-derived food pathogens and its drug resistance and virulence gene detection composite chip
  • Identification of animal-derived food pathogens and its drug resistance and virulence gene detection composite chip
  • Identification of animal-derived food pathogens and its drug resistance and virulence gene detection composite chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Embodiment 1, the preparation of composite detection gene chip

[0090] In order to simultaneously achieve the triple purpose of pathogenic bacteria identification, virulence gene identification and drug resistance gene identification, a microarray detection platform for simultaneous detection of pathogenic bacteria and their related virulence and drug resistance genes in 14 animal-derived foods was established. The 171 oligonucleotide probes placed on the composite detection gene chip involved and included specific genes for identification of pathogenic bacteria in 14 common animal-sourced foods, 16S rRNA genes of 11 pathogenic bacteria, and the 16S rRNA genes of some bacteria. Virulence genes, as well as common drug resistance genes involved in the field of drug resistance surveillance in my country. Some of the detection probes can be flexibly applied to high-sensitivity multiplex PCR amplification and 16SrRNA-PCR single-amplification chip detection. The characterist...

Embodiment 2

[0123] Embodiment 2, the application of composite detection gene chip

[0124] 1. Obtaining samples to be hybridized

[0125] 1. Extraction of sample nucleic acid

[0126] According to the determination of the pre-detection object of the gene chip, the The Genomic DNA Purification Kit was used to extract the DNA of the bacteria to be tested shown in Table 2 according to the research instructions to obtain the genomic DNA of each bacteria.

[0127] The genomic DNA of each bacterium was subjected to the following experiments:

[0128] 2. Random primer PCR amplification of samples to be hybridized

[0129] 1) PrimerA reaction:

[0130] DNA reaction solution 1: 400ng of genomic DNA, 2.5 μL of PrimerA (PrimerA: GTTTCCCAGTCACGATCNNNNNNNNNN (sequence 172)), 6 μL of MQ H 2 After O mixing, pre-denature at 95°C for 5 minutes, and then place it on ice immediately to obtain DNA reaction solution 1;

[0131] Mixture 2: Mix 2.5 μL dNTP (Takara Company Code: R001A)), 23 μL MQ H 2 O m...

Embodiment 3

[0192] Embodiment 3, compound detection gene chip carries out blind test test

[0193] 1. Obtaining samples to be hybridized

[0194] 1. Extraction of sample nucleic acid

[0195] The detection strain used for the blind test was a strain of MRSA (methicillin-resistant Staphylococcus aureus) isolated from commercial pigs, and another strain was a strain of Salmonella isolated from the chicken processing chain.

[0196] Genomic DNA of MRSA and Salmonella was extracted.

[0197] 2. Random primer PCR amplification of the sample to be hybridized: the same as in Example 2.

[0198] 3. Fluorescent labeling: the same as in Example 2.

[0199] 2. Chip hybridization: the same as in Example 2.

[0200] 3. Chip detection

[0201] Method is identical with embodiment 2, and result is as image 3 As shown in A.

[0202] According to the chip detection results, the identification of the two bacteria was accurately identified, and at the same time, the detection results of the drug-resi...

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Abstract

The invention provides an animal-derived food pathogen identification and drug-resistant and toxic gene detection composite chip. The invention provides a probe for identifying the animal-derived food pathogen, the pathogen drug-resistant gene and / or drug toxicity gene. The probe consists of single-chain DNA (deoxyribonucleic acid) molecules shown by the sequence 1 to sequence 171. Experiment results show that the animal-derived food pathogen identification and drug-resistant and toxic gene detection composite chip provided by the invention can effectively achieve the integral goal of simultaneously identifying the bacteria, the toxicity of the bacteria and the drug-resistant gene.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the identification of pathogenic bacteria in animal source food and a composite chip for detecting drug resistance and virulence genes. Background technique [0002] Diseases caused by pathogenic bacteria in foods of animal origin have been one of the public health concerns of developing and developed countries. The control of food contamination and foodborne diseases is listed as a priority strategic area by the World Health Organization (WHO). As the largest developing country in my country, the establishment of foodborne disease surveillance network is still not perfect. In recent years, microbial pathogens have been one of the main causes of foodborne disease outbreaks. The biotoxins of various food-borne pathogens are closely related to the pathogenicity of bacteria. At the same time, many of the virulence genes can also be used as identification genes of food-borne pathogens....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12N15/11
CPCC12Q1/6837C12Q1/689C12Q2563/107C12Q2531/113
Inventor 吴聪明崔明全汪洋
Owner CHINA AGRI UNIV
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