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A kit for identifying bacteria using molecular beacon-melting curve technology and its application

A technology of melting curve and technical identification, which is applied in the field of bacterial detection, can solve the problems that the molecular beacon-melting curve technology has not yet been used, and achieve the effect of large coverage, less misjudgment, and lower detection cost

Active Publication Date: 2020-12-18
HANGZHOU DIAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, molecular beacon-melting curve technology has not been used in the field of clinical bacterial identification

Method used

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  • A kit for identifying bacteria using molecular beacon-melting curve technology and its application
  • A kit for identifying bacteria using molecular beacon-melting curve technology and its application
  • A kit for identifying bacteria using molecular beacon-melting curve technology and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Design of Molecular Beacon Probes.

[0044] (1) download the sequence information of 18 kinds of common bacterial 16s-rDNA of clinical infection that can be identified by the kit of the present invention from the database of the National Center for Biotechnology Information (NCBI) in the United States as the basis for the design of molecular beacon probes;

[0045] The 18 common bacteria in clinical infection include the following bacteria: Acinetobacter baumannii, Aeromonas hydrophila, Burkholderia cepacia, Citrobacter freundii, Enterobacter cloacae, Enterococcus faecium, Enterococcus faecalis, Enterobacter, Escherichia coli, Klebsiella pneumoniae, Listeria monocytogenes, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus aureus, Salmonella, Serratia marcescens, Stenotrophomonas maltophilia bacteria.

[0046] (2) Design molecular beacon probes, the principle is: 1. make it cover part of the conserved region sequence and pa...

Embodiment 2

[0058] Example 2: Establishment and optimization of the high-resolution melting curve identification system of the colony identification kit.

[0059] Conditions were optimized for some important influencing factors of the high-resolution melting curve identification system.

[0060] 1. Method

[0061] (1)Mg 2+ Concentration study: Prepare 2x high-resolution melting curve bacterial identification reaction solution according to the reaction system in Table 1, fix other parameters, and adjust Mg respectively 2+ Concentrations up to 0.1M, 0.05M, 0.03M, 0.01M, 0M, high-resolution melting curve analysis using nucleic acid extracted from E. coli standard strains as a template, and comparing different Mg 2+ The effect of concentration on the height, sharpness and fluency of the peak of the melting curve.

[0062] (2)K + Concentration study: Prepare 2× high-resolution melting curve bacterial identification reaction solution according to the reaction system in Table 1, fix other pa...

Embodiment 3

[0069] Embodiment 3: Composition and detection method of high-resolution melting curve bacterial identification kit

[0070] 1. Composition of the kit (stored at -20°C)

[0071] (1) 2× high resolution melting curve reaction solution: its components are: 0.4M betaine, 1.5% (V / V) DMSO, 80mM

[0072] Tris-HCl, 200 mM potassium chloride, 80 mM magnesium chloride.

[0073] 2. Preparation of positive control substance

[0074] Escherichia coli strain CICC10421=078:K80 was inoculated on a Columbia blood plate, cultured at 37° C. and 70% RH overnight, and nucleic acid was extracted from colonies. Measure the concentration and purity of the extracted nucleic acid to ensure that 1.8 / 1.6 is not lower than 1.8; then adjust the nucleic acid concentration to 50ng / ul with inactivated pure water. Store at -20°C after aliquoting as a positive control for the kit.

[0075] 3. The detection method of the kit is as follows:

[0076] (1) extract the nucleic acid of the sample, which contains ...

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Abstract

The invention discloses a molecular beacon and a kit for quickly identifying various clinically common bacteria and belongs to the technical field of microbiological detection. The sequences SEQ ID of the molecular beacon are as shown in v6p1, v6p2, v1p1 and v1r. Through comparison between various clinically common bacteria 16s ribosome RNA sequences in an NCBI gene sequence database and design of the molecular beacon at a tag sequence, clinically common 18 bacteria, including baumanii, A.hydrophila, burkholderia cepacia, citrobacter freundii, enterobacter cloacae, enterococcus faecium, enterococcus faecalis, enterobacter aerogenes, escherichia coli, klebsiella pneumoniae, listeria monocytogenes, proteus mirabilis, pseudomonas aeruginosa, staphylococcus epidermidis, staphylococcus aureus, salmonella, serratia marcescens and stenotrophomonas maltophilia, can be quickly identified. The invention further discloses the kit containing the molecular beacon and used for detecting various bacteria, and the kit can quickly and accurately identify various clinically common bacteria.

Description

technical field [0001] The invention belongs to the technical field of bacterial detection, and in particular relates to a bacterial rapid identification kit (molecular beacon method); and also relates to a molecular beacon probe with partial specificity for bacterial 16s ribosomal RNA used in the kit. Background technique [0002] The identification of pathogenic bacteria has always been one of the difficulties and priorities in the work of clinical microbiology laboratories. At present, methods such as bacterial isolation and culture, morphological characteristics, biochemical reactions, and immunology are widely used in clinical practice to classify and identify pathogenic bacteria. development requirements. Although the automatic microbial analysis system (AMS) has been popularized in large hospitals, its consumables are expensive and time-consuming is still relatively long. [0003] 16s ribosomal RNA, referred to as 16s rRNA, is a component of the ribosomal 30s subuni...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6816C12Q1/689C12Q1/04C12N15/11
CPCC12Q1/6816C12Q2563/107C12Q2527/107C12Q2545/113Y02A50/30
Inventor 任绪义吕江峰周静
Owner HANGZHOU DIAN BIOTECH CO LTD
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