A kind of primer, probe and kit for identifying micrococcus based on isothermal amplification technology and its application

A technology of isothermal amplification and technical identification, applied in the field of environmental bacteria detection and identification, can solve the problems of high technical requirements, small identification range, unfavorable promotion, etc., and achieve the effects of high detection sensitivity, wide identification range, and strong promotion and application.

Active Publication Date: 2021-11-30
至微生物智能科技(厦门)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no method to identify environmental Micrococcus at the genus level using isothermal amplification technology. The existing method mainly uses PCR technology to identify Micrococcus luteus. The identification range is small, the technical requirements are high, and the sensitivity is low, which is not conducive to popularization.

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  • A kind of primer, probe and kit for identifying micrococcus based on isothermal amplification technology and its application
  • A kind of primer, probe and kit for identifying micrococcus based on isothermal amplification technology and its application
  • A kind of primer, probe and kit for identifying micrococcus based on isothermal amplification technology and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Design of Micrococcus Specific Primers and Probes

[0075] Specific steps are as follows:

[0076] A. Collect environmental samples and separate environmental bacteria by streaking on a plate.

[0077] B. Sequence the genomic DNA of bacteria isolated from the environment with 16S rDNA universal primers, and determine the species of the isolated bacteria by sequence comparison.

[0078] C. According to the 16S rDNA sequencing results of the isolated Micrococcus and the 16S rDNA gene sequences of Micrococcus yunnanensis, Micrococcus endophytes, Micrococcus luteus and other Micrococci in the NCBI database, design Micrococcus-specific primer pairs in the conserved region, primers The sequence is as follows:

[0079] F: 5'-GATTTATCGGTTTTGGATGGACTCGCGGCCTATC-3' (SEQ ID NO: 1);

[0080] R: 5'-GTGCTTCTTCTGCAGGTACCGTCACTTTCGC-3' (SEQ ID NO: 2);

[0081] D. The sequences of the amplified regions targeted by the primers were compared in NCBI, and the comparison results were al...

Embodiment 2

[0086] Specific detection of micrococci

[0087] A. Standard strains and bacteria of different genera isolated from the environment were shaken overnight.

[0088] Among them, the standard strains include Escherichia coli ATCC25922 (purchased from Microbiologics); Micrococcus luteus CMCC (B) 28001, Pseudomonas aeruginosa ATCC15442, Bacillus cereus CMCC (B) 63303 (purchased from Beijing Biological Collection Center); Staphylococcus aureus NCTC8325, Staphylococcus aureus ATCC6538 (purchased from Shanghai Preservation Biotechnology Center).

[0089] Bacteria of different genera isolated from the environment include 2 strains of Staphylococcus (denoted as P-1, P-2), 1 strain of Pantoea (denoted as F-1), 1 strain of Bacillus (denoted as Y-1), 1 strain of Pseudomonas (denoted as J-1), 2 species of Micrococcus (denoted as W-1, W-2).

[0090] B. Extraction of bacterial genomic DNA from overnight cultures.

[0091] C. Genomic DNA of different bacteria is used as a template, and the ...

Embodiment 3

[0106] Sensitivity testing experiment

[0107] Specific steps are as follows:

[0108] A. Micrococcus luteus CMCC (B) 28001 and a micrococcus isolated from the environment (randomly selected from the micrococcus bacteria isolated from the environment) were shaken overnight.

[0109] B. Extraction of genomic DNA from overnight cultured micrococci.

[0110] C. Carry out 10-fold serial dilution to the extracted Micrococcus luteus and the Micrococcus genomic DNA isolated from the environment.

[0111] D. Perform isothermal amplification using micrococcal genomic DNA with different concentration gradients as a template, and collect fluorescence signals in real time (the negative control is ddH 2 O), wherein Micrococcus luteus 10 0 ~10 -5 The working concentration of each gradient genomic DNA was 13.7×10 0 ng / μL, 13.7×10 -1 ng / μL, 13.7×10 -2 ng / μL, 13.7×10 -3 ng / μL, 13.7×10 -4 ng / μL, 13.7×10 -5 ng / μL.

[0112] Environmental Micrococci (W-1) 100 ~10 -5 The working concent...

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Abstract

The invention provides a primer, a probe, a kit for identifying micrococci based on isothermal amplification technology and applications thereof, belonging to the technical field of environmental bacteria detection and identification. A primer for identifying micrococci based on isothermal amplification technology, comprising a forward primer with a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:2. The primers can only accurately amplify species within the genus Micrococcus, but cannot amplify species outside the genus. Therefore, the primers have strong amplification specificity for species within the genus Micrococcus. The present invention also provides a method for identifying micrococci in the environment. The micrococcus in the environment can be quickly identified through the fluorescence isothermal amplification technology, the operation is simple, the identification range of micrococci is wide, and the minimum detection limit for micrococci can reach Up to 60fg / μL, with high detection sensitivity.

Description

technical field [0001] The invention belongs to the technical field of environmental bacteria detection and identification, and in particular relates to a primer, a probe, a kit for identifying micrococci based on an isothermal amplification technology and an application thereof. Background technique [0002] Micrococcus is a Gram-positive bacterium, an opportunistic pathogenic bacterium that can grow in extreme environments, and under certain conditions can cause skin local tissue infection, or even severe infection, resulting in endocarditis and other diseases. There are many classifications of Micrococcus, and different classifications have different applications in different fields. Under different conditions, different micrococcus species may have good or bad effects on human life, and suitable methods for detection and identification are needed for the prevention and utilization of micrococci. [0003] The 16S rDNA gene is the DNA sequence encoding rRNA in bacteria, w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2521/507C12Q2537/1376C12Q2522/101C12Q2521/319C12Q2563/107
Inventor 不公告发明人
Owner 至微生物智能科技(厦门)有限公司
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