Kit for rapidly detecting staphylococcus aureus and application thereof

A staphylococcus, golden yellow technology, applied in the application field of microbial molecular detection technology, can solve the problems of economic loss in the breeding industry, achieve low detection cost, good specificity, and ensure safety

Inactive Publication Date: 2015-03-04
WUHAN MINGMAN GENE ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, Staphylococcus aureus is also an important pathogen that causes mastitis in dairy cows, causing huge economic losses to the breeding industry

Method used

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  • Kit for rapidly detecting staphylococcus aureus and application thereof
  • Kit for rapidly detecting staphylococcus aureus and application thereof
  • Kit for rapidly detecting staphylococcus aureus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A visual Staphylococcus aureus loop-mediated isothermal amplification (LAMP) detection kit

[0031] The kit consists of a reaction premix, a fluorescent chromogen, and a test tube with a sterilized filter paper on the inner cover. The reaction premix includes Bst DNA polymerase, reaction buffer, dNTP, magnesium sulfate, and a specific primer set. , betaine and sterile double distilled water.

[0032] The specific primer set includes outer primers F3 and B3, inner primers FIP and BIP, the nucleotide sequences of which are shown in SEQ ID NO 1-4.

Embodiment 2

[0034] Optimization of a visual Staphylococcus aureus loop-mediated isothermal amplification (LAMP) detection kit

[0035] The optimization program of the kit of the present invention is: screening the most suitable specific primer group, the optimum concentration ratio of internal and external primers, the optimum reaction temperature, the concentration of magnesium sulfate, the concentration of betaine, the concentration of dNTP, and the reaction time. The amplified product was electrophoresed on a 2% (w / v, the same below) agarose gel, and placed in a gel imaging system for imaging. The best solution was determined according to the intensity of the characteristic ladder-like band of LAMP displayed on the electrophoresis picture. condition. Finally, optimize the amount of fluorescent chromogen. The optimization of the optimum reaction temperature is taken as an example to illustrate in detail below.

[0036] 1. Preparation of bacterial DNA template:

[0037] For aseptic op...

Embodiment 3

[0045] Application of a visual Staphylococcus aureus loop-mediated isothermal amplification (LAMP) detection kit

[0046] Combined with the detection sensitivity of the kit of the present invention and the requirements of relevant detection standards, the processing of the samples to be tested is optimized.

[0047] 1. Extraction of bacterial DNA in the sample to be tested:

[0048] For aseptic operation, weigh 25g or 25ml of the sample to be tested into 225ml sterilized LB liquid medium, and incubate at 36±1°C for 16±1 hours; for aseptic operation, take 0.1ml to 0.9ml from the above culture medium for sterilization In physiological saline; keep warm in boiling water at 100°C for 5 minutes, cool at room temperature and set aside at 4°C for use on the same day.

[0049] 2. LAMP amplification:

[0050] For aseptic operation, take 5 μl of the above-prepared boiled dilution supernatant into detection tubes filled with reaction premix and fluorescent chromogenic reagent. React i...

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PUM

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Abstract

The invention discloses a visual kit for detecting staphylococcus aureus through loop-mediated isothermal amplification (LAMP) and application thereof. The kit comprises a premixed reaction liquid, a fluorescent color developing agent and a detection tube, wherein sterilized filter paper is attached to an inner cap of the detection tube; the premixed reaction liquid comprises BstDNA (bacillus stearothermophilus deoxyribonucleic acid) polymerase, a reaction buffer, dNTP (deoxy-ribonucleoside triphosphate), magnesium sulfate, a specific primer group, betaine and sterile double distilled water. Staphylococcus aureus in samples can be rapidly detected by using the kit through simple treatment of the samples. The kit has the advantages of simplicity and convenience in operation, rapidness, convenience in result determination, strong specificity, high sensitivity, low cost, and the like.

Description

technical field [0001] The invention belongs to the application field of microbial molecular detection technology, and in particular relates to a high-sensitivity and high-specificity visualized staphylococcus aureus loop-mediated isothermal amplification (LAMP) detection kit and its application. Background technique [0002] Staphylococcus aureus is an important zoonotic pathogen causing a variety of serious infections. Staphylococcus aureus is widely distributed in nature, and there are many chances of food contamination. It is one of the main pathogens causing food poisoning, and it is a common food poisoning pathogen that cannot be detected in food hygiene standards. With the widespread use of antibiotics, a large number of drug-resistant Staphylococcus aureus have emerged. The diseases caused by Staphylococcus aureus have become a global public health problem, seriously endangering human safety and health. At the same time, Staphylococcus aureus is also an important p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12R1/445
CPCC12Q1/6851C12Q1/689C12Q2531/119
Inventor 张辉陈曦
Owner WUHAN MINGMAN GENE ENG CO LTD
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