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Method for detecting enterobacter sakazakii based on nucleic acid chromatography biosensing technique

A technology of Enterobacter sakazakii and biotin, which can be used in measurement devices, analytical materials, instruments, etc., can solve the problems of cumbersome operation, long detection cycle, long time consumption, etc.

Active Publication Date: 2017-11-14
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

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  • Method for detecting enterobacter sakazakii based on nucleic acid chromatography biosensing technique
  • Method for detecting enterobacter sakazakii based on nucleic acid chromatography biosensing technique
  • Method for detecting enterobacter sakazakii based on nucleic acid chromatography biosensing technique

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Embodiment 1

[0048] Example 1 Method for detecting Enterobacter sakazakii based on nucleic acid chromatography biosensing technology

[0049] 1. Experimental materials

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

[0051] Information on Enterobacter sakazakii and non-Enterobacter sakazakii used in Table 1

[0052]

[0053] Enterobacter sakazakii 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. The concentration of Enterobacter sakazakii was determined by spectrophotometry.

[0054] 2. Genome extraction of Enterobacter sakazakii

[0055] The bacterial genomic DNA extraction kit from New Industry Company was used, and the specific steps were as follows:

[0056] (1) Take 1.5 mL of bacterial culture solution, centrifuge at 12,000...

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 specific sequence of the target substance ompA, 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 area (TL) and quality control area (CL), respectively. If the sample is positive, after LAMP amplification, the 5' end of the targe...

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Abstract

The invention relates to a method for detecting enterobacter sakazakii based on a nucleic acid chromatography biosensing technique. According to a toxicity gene ompA of the enterobacter sakazakii, a loop-mediated isothermal amplification primer (LAMP) (shown in SEQ ID NO:1-4) is designed, and an enterobacter sakazakii detection method based on an LAMP nano-enzyme sensor is established by combining with a nano-enzyme 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 enterobacter sakazakii 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 Enterobacter sakazakii based on nucleic acid chromatography biosensing technology. Background technique [0002] Enterobacter sakazakii (Cronobacter sp.) is a Gram-negative, periflagellar-dependent, non-spore-forming, facultative anaerobic bacterium. It is an opportunistic pathogen of the genus Enterobacter, which causes foodborne illness leading to meningitis, enterocolitis, and bacteremia in infants, especially neonates, with a mortality rate of 20% in reported cases -50%, survivors often have severe neurologic disease. In recent years, studies on the contamination rate of Enterobacter sakazakii in food (milk powder, chocolate, cereals, potatoes, pasta, etc.) processing plant raw materials, production environment and households have found that Enterobacter sakazakii is widely distributed in nature. In several outbreaks of Enterobacter sakaz...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543
CPCG01N33/54326G01N33/56916
Inventor 罗云波许文涛黄昆仑徐瑗聪张莉程楠
Owner CHINA AGRI UNIV
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