Method for detecting salmonella based on nucleic acid chromatography biosensing technology

A Salmonella and biotin technology, applied in the direction of measuring devices, analytical materials, instruments, etc., can solve the problems of long detection cycle, low detection specificity, heavy workload, etc.

Active Publication Date: 2017-11-10
CHINA AGRI UNIV
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  • 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 ce

Method used

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  • Method for detecting salmonella based on nucleic acid chromatography biosensing technology
  • Method for detecting salmonella based on nucleic acid chromatography biosensing technology
  • Method for detecting salmonella based on nucleic acid chromatography biosensing technology

Examples

Experimental program
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Embodiment 1

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

[0049] 1. Experimental materials

[0050] The information of Salmonella and non-Salmonella strains used in this example is shown in Table 1.

[0051] Table 1 Information on Salmonella and non-Salmonella species used

[0052]

[0053] Salmonella 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. Salmonella concentrations were determined spectrophotometrically.

[0054] 2. Salmonella genome extraction

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

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

[0057] The bacterial genomic ...

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 the specific sequence of the target substance invA, 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 t...

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Abstract

The invention relates to a method for detecting salmonella based on a nucleic acid chromatography biosensing technology. According to a virulence gene invA of salmonella, 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 salmonella detection method based on an LAMP nano-enzyme sensor is established. The method for detecting salmonella 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 salmonella 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 Salmonella based on nucleic acid chromatography biosensing technology. Background technique [0002] Salmonella spp., Gram-negative, short bacilli with blunt ends (thinner than E. coli), scattered (0.7-1.5 μm) × (2-5 μm), without capsule and spores, with flagella all over the body, Motile, most with pili. Salmonellosis is one of the zoonotic diseases of great significance in public health. The pathogenic Salmonella belongs to the Enterobacteriaceae family, including those bacteria that cause food poisoning, gastroenteritis, typhoid fever and paratyphoid fever. In addition to infecting humans, they can also infect many animals including mammals, birds, reptiles, fish, amphibians and insects. Human and animal infection can be asymptomatic or fatal with clinical symptoms, which may aggravate morbidity or mortality, or reduce animal reproductive ...

Claims

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Application Information

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IPC IPC(8): G01N33/558G01N33/569G01N33/543
CPCG01N33/54326G01N33/54346G01N33/558G01N33/56916
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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