Clostridium perfringens dry-powdered lamp rapid detection kit and method of use thereof

Abstract

The invention discloses a clostridium perfringens dry powder LAMP rapid detection kit and a using method thereof. The kit comprises lyophilized isothermal amplification agent, reconstitution solution, positive control dry powder, negative control, developing solution, lysate and confining liquid. A primer has good specificity and high sensitivity as well as extremely high reaction efficiency, the detection time can be greatly saved, the operation is simple, expensive instruments are not needed, and the lowest detection limit is up to 11 CFU / Test. The kit has the potential of conducting real-time monitoring, and is more convenient to transport, thus providing convenience for the base detection work of remote regions. The kit can be widely applied to laboratories and on-site rapid detection in the fields of food, beverage, drinking water and the like, and has wide application prospects and good economic and social benefits.

Description

technical field [0001] The invention and microbial molecular biology detection reagents specifically relate to a dry powdered Clostridium perfringens nucleic acid rapid diagnostic kit based on LAMP technology and a use method thereof. Background technique [0002] Clostridium perfringens, once known as Clostridium welchii or Clostridium perfringens, is the most common type of Clostridium among clinical gas gangrene pathogens, because it can decompose sugar in muscle and connective tissue, producing a large amount of gas, It leads to severe emphysema of the tissue, which in turn affects the blood supply and causes extensive necrosis of the tissue. In addition, this bacteria can form a capsule in the body, so it is called Clostridium aerogenes. Clostridium perfringens is widely distributed in human and animal feces, soil, sewage and other external environments. The enterotoxin produced by it is the main factor causing food poisoning. After heating the water contaminated by Clo...

AI Technical Summary

Problems solved by technology

In practical applications, the corresponding solution reaction system must be prepared before detection, and there are many components in it, which can easily lead to reagent contamination, detection interval contamination, and result errors caused by frequent operations. In addition, some reagents in the reaction system need to be frozen. , For example, the required Bst DNA polymerase needs to be stored strictly at -20°C. If the temperature is too high, it will affect its enzyme activity and cause the detection reagent to fail.

Therefore, the high-temperature long-distance transportation and on-site application of the reagent are inconvenient, and the corresponding cost is also increased.

At the same time, the current detection method has not been popularized in the fields of food safety and medicine, especially in the rapid detection of water bodies. As a non-standard method, the existing LAMP products are only for the detection process, and the methods of sample pretreatment are all no mention

Moreover, there are few rapid diagnostic kits for Clostridium perfringens based on ring-mediated constant temperature amplification technology on the market, and similar dry powder products that can be used for normal temperature transportation have not yet appeared.

[0004] The main difficulties in the development of the dry powder type LAMP rapid detection kit are as follows: 1) Keep the activity of BstDNA polymerase during the freeze-drying process without loss; 2) Design suitable primers; 3) The color development is not obvious enough, making it difficult to distinguish the results

[0005] In order to maintain the activity of Bst DNA polymerase, adding a lyoprotectant is a more conventional choice. The commonly used lyoprotectant is trehalose, but its protective effect is relatively limited, and it is difficult to guarantee product quality

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