Visual cascade amplification functional nucleic acid sensor for quantitatively detecting salmonellae

A technology of functional nucleic acid and Salmonella, which is applied in the field of preparation of functional nucleic acid sensors, can solve the problems of long detection time and achieve the effect of short detection time, simple operation, and qualitative judgment of detection time by naked eyes

Inactive Publication Date: 2019-03-15
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In recent years, with the rapid development of biotechnology, new technologies and methods have been widely used in the field of food microbiological testing, an...

Method used

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  • Visual cascade amplification functional nucleic acid sensor for quantitatively detecting salmonellae
  • Visual cascade amplification functional nucleic acid sensor for quantitatively detecting salmonellae
  • Visual cascade amplification functional nucleic acid sensor for quantitatively detecting salmonellae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1, Development and Screening of Primers for Recombinase Polymerase Constant Temperature Amplification Technology (RPA)

[0063] RPA amplification was carried out using the TwistAmp Basic kit. The system for each reaction was 50 μL, and the specific addition amount was shown in Table 1. The mixture except 280 mM magnesium acetate was added to the TwistAmp Basic reaction tube with lyophilized enzyme powder, so that The powder was completely dissolved in the tube.

[0064] Table 1, RPA gel detection reaction system

[0065] RPA reaction system

Volume (μL)

reaction buffer

29.5

RPA-upstream primer (10 μM)

2.4

RPA-downstream primer (10 μM)

2.4

DNA template

1

Magnesium acetate (280mM)

2.5

wxya 2 o

12.2

total

50

[0066] Design 9 pairs of primers to screen for better primers, and select a relatively better pair of primers for the next experiment. RPA primers are usually 30 to 35 ...

Embodiment 2

[0071] Embodiment two, the optimization of RPA reaction time and primer concentration

[0072] Set the reaction time gradient as: 10min, 20min, 30min. After the RPA reaction, perform agarose gel electrophoresis analysis. By comparing the brightness of the bands with different reaction times, select the appropriate RPA reaction time for subsequent experiments.

[0073] figure 2 , 3 , 4 respectively show the gel electrophoresis images of different concentrations of primers reacted for 10min, 20min and 30min, wherein the primer concentrations of lanes 1 to 8 are: 0.1μM, 0.25μM, 0.5μM, 1μM, 2μM, 3μM, 4μM, 5 μM.

[0074] It can be seen that under the same reaction time, as the primer concentration increases, the brightness of the product bands gradually increases, indicating that the amount of RPA products gradually increases, but there are certain differences in the products of RPA at different reaction times; in addition, it can be seen from the figure The change of the prime...

Embodiment 3

[0075] Embodiment three, the optimization of RPA reaction temperature

[0076] RPA performs nucleic acid amplification under a single temperature condition, unlike PCR, which can avoid the amplification of non-target bands caused by the combination of primers through heating cycles, especially when there is no template or low concentration of template, the reaction ends It will generate non-specific signals, reduce reaction efficiency, and affect experimental results.

[0077] In this application, there will be non-specific bands at the position of about 500bp. Try to reduce the generation of non-specific bands by changing the primer concentration and reaction temperature. It is found that different reaction temperatures have certain effects on RPA amplification. In order to reduce non-specific bands For the generation of heterosexual bands, it is necessary to select an appropriate reaction temperature for the next experiment.

[0078] Figure 5 The gel electrophoresis of RP...

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Abstract

The invention discloses a visual cascade amplification functional nucleic acid sensor for quantitatively detecting salmonellae. A preparation method of the sensor comprises the following steps: extracting strain DNA; developing and screening a primer for an RPA reaction; optimizing reaction time and a reaction temperature of the RPA reaction and determining primer concentration; and optimizing a TdT reaction condition. By the label-free visual cascade amplification functional nucleic acid sensor for sensitively and selectively detecting the salmonellae, which is designed by the method disclosed by the invention, specificity detection on the salmonellae can be completed in short time; and the visual cascade amplification functional nucleic acid sensor has the advantages of visualization, high sensitivity, high universality and the like.

Description

technical field [0001] The invention relates to the technical field of food detection, in particular to a preparation method and application of a functional nucleic acid sensor for detecting Salmonella. Background technique [0002] Salmonella, a rod-shaped Gram-negative bacterium, is one of the main zoonotic pathogens in the environment. Most Salmonella serotypes can cause gastroenteritis and other diseases. According to statistics, about 1 million people in the United States have enteritis caused by Salmonella, and about 450 of them died. A small number of serotypes can cause enteric fever, such as Salmonella typhi, Salmonella paratyphi type A, type B and type C, etc. Salmonella is a common food-borne pathogen that can be transmitted by many nutritious foods such as meat, eggs, milk, cakes, and meat products. [0003] In recent years, with the rapid development of biotechnology, new technologies and methods have been widely used in the field of food microbiological testi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6825C12Q1/6851C12Q1/689C12Q1/04C12R1/42
CPCC12Q1/6825C12Q1/6851C12Q1/689C12Q2521/507C12Q2522/101
Inventor 罗云波许文涛田洪涛张媛田晶晶黄昆仑商颖
Owner CHINA AGRI UNIV
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