Pseudomonas aeruginosa dry powder lamp rapid detection kit and its application method

Abstract

The invention discloses a patina pseudomonas dry powder LAMP rapid detection reagent box and a rapid detection method based on the patina pseudomonas dry powder LAMP rapid detection reagent box. The reagent box comprises a freeze-drying isothermy expansion agent, reconstitution fluid, positive control dry powder, passive control, a colorimetric solution, a lysis solution and confining liquid. The freeze-drying isothermy expansion agent contains dNTPs, Bst DNA polymerases, expansion primers and a freeze-drying protective agent. The primers in the reagent box correspond to eight sectors of the patina pseudomonas specificity gene sequence, the colorimetric solution is added after reaction, and the passive and positive color developing difference is obvious. The reagent box has the advantages of being high in specificity, high in sensitivity, easy to operate, simple in result judgment and the like, and is suitable for safe and fast quality control detection of food safety, beverages, water, cosmetics and other raw and auxiliary materials. The reagent box is convenient to transport at normal temperature, and simple and fast in use; special apparatus is not needed; the cost is lowered; the product application range is widened; and the reagent box is more suitable for on-site quick detection and correlative detection of a basic-level remote region, and has wide application prospects.

Description

technical field [0001] The invention relates to a dry powdered LAMP rapid detection reagent and a rapid detection method based thereon, in particular to a dry powdered LAMP rapid detection kit for Pseudomonas aeruginosa and a rapid detection method based thereon. Background technique [0002] Pseudomonas aeruginosa is a common water-borne pathogen that widely exists in various types of water and has strong resistance to physical and chemical factors such as disinfectants, drying, ultraviolet rays, and adverse environments. Pseudomonas aeruginosa Bacteria are used as pollution indicator bacteria. When Pseudomonas aeruginosa spreads to pipelines and equipment in the whole process, cross-contamination will be hard to prevent. According to regulations, Pseudomonas aeruginosa should not be detected in packaged drinking water sources. Therefore, water production enterprises should pass inspections. It is very important to determine the source of pollution and carry out targeted co...

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 detection kits for Pseudomonas aeruginosa based on the loop-mediated isothermal 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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