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Method for detecting staphylococcus aureus based on nucleic acid chromatography biosensing technique

A staphylococcus, golden yellow technology, used in biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc., can solve the problems of long detection cycle, low detection specificity, and large workload.

Inactive Publication Date: 2017-12-01
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; it has been more than half a century to identify bacteria by using the specificity of antigen-antibody reaction, but the screening of microbial antibodies is very cumbersome, and the final detection specificity is not high; the molecular biological detection technology Continuous improvement and development have overcome the problems of traditional detection methods such as cumbersome experimental operations and long time consumption, and have 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 staphylococcus aureus based on nucleic acid chromatography biosensing technique
  • Method for detecting staphylococcus aureus based on nucleic acid chromatography biosensing technique
  • Method for detecting staphylococcus aureus based on nucleic acid chromatography biosensing technique

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Embodiment 1

[0048] Embodiment 1 The method for detecting Staphylococcus aureus based on nucleic acid chromatography biosensing technology

[0049] 1. Experimental materials

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

[0051] Information on S. aureus and non-S. aureus used in Table 1

[0052]

[0053]

[0054] Staphylococcus aureus 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. Staphylococcus aureus concentration was determined by spectrophotometry.

[0055] 2. Genome extraction of Staphylococcus aureus

[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, centrifuge at 12,000 g for 1 min, and ab...

Embodiment 2

[0094] The optimization of embodiment 2 nanozyme nucleic acid test strips

[0095] 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

[0096] The performance detection of embodiment 3 nano-enzyme sensor

[0097] The principle of the nanozyme sensor is as follows: First, the sample is 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 the target nuc-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 staphylococcus aureus based on a nucleic acid chromatography biosensing technique. The method provided by the invention comprises the following steps: designing a loop-mediated isothermal amplification (LAMP) primer (SEQ ID NO:1-4) according to a virulence gene nuc of the staphylococcus aureus, and establishing a staphylococcus aureus detection method based on an LAMP nano enzyme sensor by combining with a nano nucleic acid test strip. The method provided by the invention can be successfully used for distinguishing viable bacterial cells and dead bacterial cells, and lower detection limit on the staphylococcus aureus can reach 10CFU / mL.

Description

technical field [0001] The invention relates to the technical field of biosensing detection, in particular to a method for detecting Staphylococcus aureus based on nucleic acid chromatography biosensing technology. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is an important human pathogenic bacteria, belonging to the genus Staphylococcus (Staphylococcus). Typical Staphylococcus aureus is spherical, about 0.8 μm in diameter, arranged in grape clusters under the microscope, without spores, flagella, most of them have no capsule, and Gram staining is positive. Staphylococcus aureus is ubiquitous in nature and can be found in air, water, dust, and human and animal waste. The U.S. Centers for Disease Control reports that Staphylococcus aureus is the second-leading infection after E. coli. Staphylococcus aureus enterotoxin has become a worldwide health problem. In the United States, food poisoning caused by Staphylococcus aureus enterotoxin accoun...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/06C12N15/11
CPCC12Q1/6804C12Q1/689C12Q2531/119C12Q2563/173C12Q2565/625
Inventor 罗云波许文涛徐瑗聪张莉黄昆仑程楠
Owner CHINA AGRI UNIV
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