Salmonella nucleic acid rapid detection kit, test strip and detection method

A Salmonella nucleic acid and detection kit technology, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/inspection, etc., can solve the problem of inability to use test strips for detection and reflection without obvious simplification of inspection operation steps Advantages and other issues, to achieve the effect of simple operation, high sensitivity, and avoid pollution

Active Publication Date: 2017-05-10
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
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Problems solved by technology

[0007] Invention patents "Salmonella constant temperature fluorescent detection primer set, kit and detection method that can avoid false negatives" (publication number CN105219870A) and "Strand Displacement Constant Temperature Amplification (SDA) rapid detection kit and detection method of Salmonella enteritidis" (publication No.: CN102382884A) describes the amplification of Salmonella DNA nucleic acid sequence under constant temperature conditions based on different amplification principles, but this type of method still needs to be combined with gel electrophoresis and imaging systems to process and analyze the experimental data, and there is no obvious simplification of the inspection operation steps and inability to take advantage of its strengths in public health emergencies
However, since this amplification system requires the participation of multiple internal and external primers, it is difficult to avoid the formation of non-specific amplification products such as primer dimers, so this type of amplification result is not suitable for the detection of test strips

Method used

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  • Salmonella nucleic acid rapid detection kit, test strip and detection method
  • Salmonella nucleic acid rapid detection kit, test strip and detection method
  • Salmonella nucleic acid rapid detection kit, test strip and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Optimization of constant temperature amplification system for Salmonella helicase

[0057] With primer concentration, MgSO 4 , dNTP and reaction temperature were used as variables to carry out orthogonal experiments on Salmonella, and the primer concentrations were 75nm, 80nm and 85nm; MgSO 4 The concentrations were 3mM, 3.5mM and 4mM; the dNTP addition amounts were 2.5μL, 3.5μL and 4.5μL; the reaction temperatures were 63°C, 64°C and 65°C. The amplified product was amplified by agarose gel electrophoresis, and the amplification effect was observed with a gel imager. After verification, a 50 μL total reaction system for the amplification of Salmonella was obtained, including 1 μL of DNA template, 80 nM primer concentration, MgSO 4 The concentration was 3.5 mM, the amount of dNTP added was 4.5 μL, and finally 3.5 μL Enzyme Mix was added, and the reaction was placed at 64°C for 60 min.

Embodiment 2

[0059] Preparation of Salmonella Nucleic Acid Thin Film Chromatography Test Strips

[0060] 1. Preparation of colloidal gold-labeled antibody

[0061] (1) Take 1mL of 10nm colloidal gold solution and place it in a 1.5mL clean centrifuge tube, add a certain amount of K dropwise 2 CO 3 The solution adjusts the colloidal gold solution to the optimum pH;

[0062] (2) Take 15 μL of the antibody with a concentration of 1 μg / μL and add it dropwise to the colloidal gold solution, shake it rapidly for 5 minutes, and let the mixed solution stand at 4°C for 1 hour;

[0063] (3) Add 20 μL of 20% BSA solution and 10 μL of 20% PEG 20000 solution dropwise, and let the mixed solution stand at 4°C for 30 minutes;

[0064] (4) Centrifuge at 2000rpm and 4°C for 15 minutes, carefully transfer the supernatant to a clean centrifuge tube, and discard the unlabeled colloidal gold precipitate;

[0065] (5) Centrifuge the supernatant at 10,000 rpm and 4°C for 30 minutes, discard the supernatant, sa...

Embodiment 3

[0076] Establishment of detection method for Salmonella nucleic acid thin film chromatography test strips

[0077] (1) Add 1 μL of Salmonella DNA as a template into the PCR tube containing the constant temperature amplification reaction system of Salmonella helicase, and at the same time use the same amount of sterile double-distilled water as a negative control, and place the above reaction system at 68°C for 60 minutes to carry out the detection of Salmonella Isothermal amplification of the helicase.

[0078] (2) Select PBS buffer solution (pH 7.4) with 0.1% BSA and 0.1% Tween 20 as the loading developing solution, take 10 μL of the amplification product diluted 10 times with the loading developing solution, and drop it on the colloidal gold test strip for detection. The test needs to be repeated three times, and the test results are observed after 5 minutes. If the amplified product of Salmonella is positive, both the test line and the quality control line are red; when th...

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Abstract

The invention relates to a salmonella nucleic acid rapid detection kit, a salmonella nucleic acid rapid detection test strip and a salmonella nucleic acid rapid detection method, and relates to design of salmonella specific primers, optimization of helicase isothermal amplification conditions and establishment of a nucleic acid film chromatography detection test strip, and visual observation of a nucleic acid detection result is achieved. Just one thermostat is adopted in a whole reaction process, and from pretreatment to detection completion on a single sample, the detection result can be obtained just by 1h; the method, on detecting salmonella, is 4.5*10<1>CFU/mL in sensitivity, and the method can avoid a cross reaction with other common food-borne pathogenic bacteria; and the detection test strip is simple and convenient to operate, high in sensitivity, strong in specificity and high in efficiency, and the detection strip is especially suitable for on-site detection and clinical diagnosis in grassroots units.

Description

technical field [0001] The invention relates to a rapid detection method for constant temperature amplification products of nucleic acid, which uses nucleic acid thin film chromatography detection test strips to detect target sequences, and is applied in the detection process of food-borne pathogenic bacteria, and specifically relates to a constant temperature amplification of Salmonella helicase And nucleic acid thin film chromatography test strips. [0002] technical background [0003] Analyzing the food safety incidents that occurred in the past, food poisoning caused by bacterial contamination has a high frequency of occurrence, a large degree of impact, and a wide range of coverage. The incidence of human and animal infections caused by food-borne pathogens remains high. The common pathogens are mainly Salmonella, Escherichia coli O157, Staphylococcus aureus, Listeria and so on. As a pathogenic bacterium that can cause zoonotic diseases, Salmonella exists widely in nat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12R1/42
CPCC12Q1/6804C12Q1/689C12Q2521/513C12Q2563/131C12Q2565/625
Inventor 杜欣军周天骄李萍王硕
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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