Probe combination, chip, kit and method for detecting pathogenic bacteria

A kit and positive control probe technology, applied in the field of microbial detection, can solve the problems of lack of standardized processing of fluorescent signals and inability to meet the design requirements of bacterial detection gene chips, etc.

Active Publication Date: 2021-09-10
壹健生物科技苏州有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Can not meet the design requirements of large-scale bacterial detection gene chip
In addition, the existing chip design method does not consider the regional inhomogeneity of the fluorescent signal on the microarray to design the arrangement of various probes, and the existing chip design method lacks the standardized processing of the fluorescent signal

Method used

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  • Probe combination, chip, kit and method for detecting pathogenic bacteria
  • Probe combination, chip, kit and method for detecting pathogenic bacteria
  • Probe combination, chip, kit and method for detecting pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Design of specific probes for 4 kinds of food-borne pathogenic bacteria

[0068] In this embodiment, probes are designed for 4 kinds of food-borne pathogenic bacteria: Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Shigella sonnei. The specific steps are as follows:

[0069] (1) Genome database construction of four food-borne pathogenic bacteria: the reference genome sequence contained in the database is used for the construction of specific probes for four food-borne pathogenic bacteria. The inventors obtained from NCBI NT repository (ftp: / / ftp.ncbi.nlm.nih.gov / blast / db / FASTA / nt.gz), RefSeq and GenBank repository (ftp: / / ftp.ncbi.nlm.nih.gov / genomes) and other authoritative databases in the field obtained the genome sequences of 4 kinds of food-borne pathogenic bacteria. The inventors further removed redundant repetitions and genome sequences with low reliability. The removal principle is: each bacterium The upper limit of the number of ...

Embodiment 2

[0085] Embodiment 2 gene chip and its preparation

[0086] Utilize the probe gene chip of the design of embodiment 1, this gene chip also comprises control probe and global quality control probe except comprising above-mentioned probe for 4 kinds of food-borne pathogenic bacteria, and its design method is as follows:

[0087] 1. Design of control probes and global quality control probes

[0088] Negative control probe design: The inventor used computer simulation to generate 10,000 50nt artificial sequences, and used blastn to compare the sequence set to the NCBI NT library (ftp: / / ftp.ncbi.nlm.nih.gov / blast / db / FASTA / nt.gz), retaining the sequence sets with consecutively aligned bases less than 18nt and identity less than 85, and a total of 424 sequences were obtained. Randomly select a sequence as a negative control probe, in this embodiment, the selected negative control probe sequence is as follows:

[0089] Probe No.25 (SEQ ID No.25):

[0090] CAATGGCCAATTAAGATCATATCGAG...

Embodiment 3

[0109] Embodiment 3 Utilizes the gene chip to detect the sample to be tested

[0110] In this example, the inventors used the gene chip prepared in Example 2 containing 24 probes for 4 kinds of food-borne pathogenic bacteria to detect the samples to be tested. In this example, fresh fruits, vegetables, meat and poultry (often susceptible to bacterial contamination) each collected a sample (sample 1-4), the sample information is shown in Table 3:

[0111] The sample information of table 3 embodiment 3

[0112] sample name Sample category sample 1 peaches sample 2 Chinese cabbage sample 3 raw pork Sample 4 chicken

[0113] The detection specifically includes the following steps:

[0114] 1. Preparation of genomic DNA (gDNA) from the sample to be tested

[0115] The inventor used the DNA extraction kit of Agilent CGH MicroArray to extract gDNA from the sample to be tested:

[0116] (1) Nucleic acid extraction and quality inspection:...

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Abstract

The invention discloses a probe combination, and belongs to the technical field of microbiological detection. The probe combination comprises probes for detecting at least two pathogenic bacteria of escherichia coli, staphylococcus aureus, listeria monocytogenes and shigella sonnei. The invention also discloses a gene chip prepared by using the probe combination, a kit and a detection method. The method is used for detecting four food-borne pathogenic bacteria, is strong in specificity, high in sensitivity, good in accuracy, convenient and rapid, is particularly suitable for detecting samples with low bacterial concentration, and provides better technical support for infection diagnosis and treatment and food safety detection.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, and in particular relates to a probe combination, a chip, a kit and a method for detecting pathogenic bacteria. Background technique [0002] Foodborne bacterial infection is one of the most concerned public health problems in the world today. After bacteria pollute food and drinking water through various channels, they enter the human body by eating food and water contaminated by foodborne pathogenic bacteria. cause foodborne infections in humans. In recent years, due to the rapid growth of global food trade, bacterial diseases caused by food-borne bacterial infections have continued to occur, and the situation of bacterial drug resistance has become increasingly severe. In the face of many food-borne bacterial infectious diseases (especially super Drug-resistant bacteria) are often helpless and have jeopardized the control of foodborne pathogens. [0003] Foodborne pathogenic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6837C12Q1/04C40B40/06C12N15/11C12R1/19C12R1/445C12R1/01
CPCC12Q1/689C12Q1/6837C40B40/06
Inventor 林德春金桃蒋华
Owner 壹健生物科技苏州有限公司
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