Method for detecting clostridium perfringens based on nucleic acid chromatography biosensing technology

A technology of Clostridium perfringens and biotin, applied in measuring devices, analytical materials, instruments, etc., can solve the problems of cumbersome experimental operations, cumbersome screening of microbial antibodies, and low detection specificity

Active Publication Date: 2017-11-10
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The traditional bacterial detection method is mainly based on physiological and biochemical characteristics, but the traditional detection method needs to go through steps such as pre-enrichment, selective plate separation, biochemical identification, etc. It takes 5-7 days from sampling to confirming the result, the detection cycle is long, and the operation is cumbersome , the workload is heavy; using the specificity of antigen-antibody reaction to identify bacteria has a history of more than half a century, but the screening of microbial antibodies is very cumbersome, and the final detection specificity is not high; molecular biology detection technology The continuous improvement and development of the traditional detection method has overcome the problems of cumbersome experimental operation and long time consumption, and has also led to the rapid development of rapid detection methods for microorganisms. However, the disadvantage of molecular biology methods is that it is not easy to analyze the results.

Method used

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  • Method for detecting clostridium perfringens based on nucleic acid chromatography biosensing technology
  • Method for detecting clostridium perfringens based on nucleic acid chromatography biosensing technology
  • Method for detecting clostridium perfringens based on nucleic acid chromatography biosensing technology

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Embodiment 1

[0048] Example 1 Method for detecting Clostridium perfringens based on nucleic acid chromatography biosensing technology

[0049] 1. Experimental materials

[0050] See Table 1 for information on Clostridium perfringens and non-Clostridium perfringens strains used in this example.

[0051] Table 1 Information on Clostridium perfringens and non-Clostridium perfringens used

[0052]

[0053]

[0054] Clostridium perfringens and other bacterial strains were used to determine the specificity of the nanozyme sensor. All strains were stored at -80°C in 20% (v / v) glycerol solution until use. They were then cultured overnight in LB medium for activation. Clostridium perfringens concentrations were determined spectrophotometrically.

[0055] 2. Clostridium perfringens genome extraction

[0056] The bacterial genomic DNA extraction kit from New Industry Company was used, and the specific steps were as follows:

[0057] (1) Take 1.5 mL of bacterial culture solution, centrifug...

Embodiment 2

[0095] The optimization of embodiment 2 nanozyme nucleic acid test strips

[0096] Synthesis of Fe by hydrothermal method 3 o 4 Magnetic particles, and then the magnetic particles were incubated with biotin secondary antibody (goat anti-mouse IgG) to prepare nanozyme probes. Use FITC antibody and biotin antibody to draw lines on the T-line and C-line positions on the NC membrane, and assemble them into nanozyme nucleic acid test strips after drying. In order to improve the sensitivity of the nanozyme sensor, it was systematically analyzed by comparing the performance of membrane materials, the concentration of FITC antibody in the detection area, the amount of nanozyme probe, and the reaction time. The results prove that the performance of the nano-enzyme sensor using Millipore135S nitrocellulose membrane is better ( figure 2 A). Using 1 mg / mL FITC antibody and 1 mg / mL goat anti-mouse IgG, the signal peak area was the highest ( figure 2 B). In addition, the amount of n...

Embodiment 3

[0097] The performance detection of embodiment 3 nano-enzyme sensor

[0098] The principle of the nanozyme sensor is as follows. First, the samples were treated with PMA (step 1). PMA can selectively penetrate the damaged cell membrane of dead cells, bind to the DNA in the cell, and make it unavailable for subsequent LAMP amplification, but if it is the intact cell membrane of living cells, PMA cannot enter the cell. Then, many BIO- and FITC-linked duplex DNAs were generated in a short time using LAMP (step 2). In the presence of target cpa-specific sequence, it is recognized and amplified by four primers. The third is the visual interpretation of the nanozyme nucleic acid test paper (step 3). The FITC antibody and goat anti-mouse IgG were immobilized on the nitrocellulose membrane by physical adsorption to form the detection zone (TL) and quality control zone (CL), respectively. If the sample is positive, after LAMP amplification, the 5' end of the target substance is lab...

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Abstract

The invention relates to a method for detecting clostridium perfringens based on a nucleic acid chromatography biosensing technology. According to a virulence gene cpa of clostridium perfringens, loop-mediated isothermal amplification primers (SEQ ID NO:1-4) are designed, and through the combination with a nano-enzyme nucleic acid test strip, the clostridium perfringens detection method based on an LAMP nano-enzyme sensor is established. The method for detecting clostridium perfringens based on the nucleic acid chromatography biosensing technology can be successfully used for distinguishing live bacterial cells from dead bacterial cells, and the lower limit of detection on clostridium perfringens can reach 10 CFU / mL.

Description

technical field [0001] The invention relates to the technical field of biosensing detection, in particular to a method for detecting Clostridium perfringens based on nucleic acid chromatography biosensing technology. Background technique [0002] Clostridium perfringens (Clostridium perfringens) is the most common Clostridium among the pathogenic bacteria of gas gangrene clinically. Because it can decompose sugar in muscle and connective tissue, it can produce a large amount of gas, causing severe emphysema of the tissue, and then affecting the blood supply, resulting in extensive tissue necrosis. It is a Gram-positive bacterium stubby, with a size of (1-1.5) μm×(3-5) μm. Both ends are blunt and round, arranged singly or in pairs, occasionally chain-like. The spores are elliptical, located in the center or sub-extreme of the thallus, and the diameter of the spores is not larger than the thallus. It is not easy to form spores in general culture, but it is conducive to the f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/569G01N33/543
CPCG01N33/54326G01N33/54346G01N33/558G01N33/5695
Inventor 罗云波许文涛徐瑗聪程楠黄昆仑张莉
Owner CHINA AGRI UNIV
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