Method for simultaneously and rapidly detecting salmonella and shigella dysenteriae

Abstract

The invention belongs to a rapid detection technology of food-borne pathogens, in particular to a loop-mediated isothermal amplification (LAMP) method for simultaneously detecting salmonella and shigella dysenteriae. The method comprises the preparation of reagent comprising sample processing reagent, LAMP reactive reagent, a large fragment of Bst DNA polyase, agarose, bromophenol blue loading buffer solution and ethidium bromide solution and the procedures of the detection of amplification products and sediment generation. The method comprises the following steps: firstly, designing a specific primer; secondly, extracting sample DNA; and thirdly, preparing a reaction system. Compared with the currently adopted traditional bacterial culture method, an immunological detection method and other molecular biology methods, the method has the advantages of simple and rapid operation, no special instrument and reagent, sensitivity and specificity and can be applied to the simultaneous and rapid detection of salmonella and shigella dysenteriae in foods and other samples.

Description

technical field [0001] The invention relates to a loop-mediated isothermal DNA amplification method (loop-mediated isothermal amplification, LAMP) technique for simultaneously detecting Salmonella and Shigella. Background technique [0002] According to the statistics of the National Foodborne Disease Surveillance Network, Salmonella and Shigella are the most common and most important causative substances in foodborne disease incidents caused by microorganisms in my country in the past ten years. These two pathogenic bacteria often appear in meat products, milk and other foods at the same time, so rapid detection of them is very important to effectively control their spread and prevent food poisoning. [0003] Salmonella spp. is a kind of Gram-negative intestinal pathogenic bacteria that widely distributes in nature and can cause human and animals to become ill. It can cause symptoms such as gastroenteritis, typhoid fever, and sepsis in humans. At present, more than 2,000 s...

AI Technical Summary

Problems solved by technology

The former is a national standard detection method in my country. Although the results are reliable, the operation is cumbersome and time-consuming, which can no longer meet the detection needs of a large number of samples. Although the latter can be detected quickly, it is prone to false positives and has poor repeatability.

[0006] Emerging molecular biology methods, including polymerase chain reaction (PCR), nucleic acid sequence-dependent amplification (NASBA), autonomous sequence replication (3SR), and strand displacement techniques (SDA), can amplify Target nucleic acids to similar orders of magnitude have higher detection limits, but they still have some shortcomings that cannot be overcome

Some of them require sophisticated instruments to amplify, and some require sophisticated methods to detect the amplified products because of the low specificity of the selection of the target sequence

Despite its simplicity, the need for high-precision thermal cyclers keeps PCR from being a powerful technique that is widely used

On the other hand, NASBA and 3SR that do not use a thermal cycler have greatly reduced specificity, mainly because a relatively low temperature of 40°C must be used for amplification

SDA largely overcomes these shortcomings by using four primers to amplify under isothermal conditions, but there is still a thin link that must use expensive modified nucleotides as substrates for the amplification reaction