PCR method and kit for detecting internal amplification control of salmonella in foods

Abstract

The invention provides a PCR method and a kit for detecting internal amplification control of salmonella in foods and relates to the biotechnical field. The method is convenient to use, good in reliability, short in detection period, high in sensitivity, strong in specificity, low in cost, simple in operating step and suitable for high throughput operation and standard operation. The method comprises the following steps: (1) designing a pair of specific primers for salmonella by using primer primier6.0 software, then adding a 27F / 1492R primer for amplifying a prokaryote 16srDNA conserved sequence as internal amplification control and amplifying by using two pairs of primers jointly; (2) carrying out enrichment culture on a to-be-detected sample for 6-8 hours and extracting a genome DNA from the cultured enrichment liquid by using a boiling and freezing method as a template for amplification; and (3) preparing a 25mu L reaction system by using a PCR reaction premixed solution of the kit for amplification, and after agarose gel electrophoresis, observing the detection result under an ultraviolet lamp.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a PCR method and a kit for detecting Salmonella in food with an amplified internal standard. Background technique [0002] Salmonellosis caused by Salmonella is one of the important zoonotic diseases in public health, and it is a common, frequent and harmful bacterial foodborne disease. Foodborne diseases caused by Salmonella account for 40%-60% of the total number of bacterial foodborne diseases in some countries. Every year, many foodborne diseases caused by Salmonella occur in our country, seriously endangering the food safety and health of the people. In addition to infecting humans, Salmonella can also infect many animals. Humans and animals can be asymptomatic carriers after infection, and can also manifest as fatal diseases with clinical symptoms, which may aggravate morbidity or mortality, or reduce animal reproductive productivity, causing great economic losses. Therefore...

AI Technical Summary

Problems solved by technology

At present, the national standard detection method for Salmonella (GB4789.4-2010) is the culture method. After the samples are collected by the culture method, the bacteria are first enriched for 8-18 hours, and then different culture media are used for the enrichment culture, and then suspicious colonies are picked for further analysis. Biochemical identification or identification using a fully automatic microbial biochemical identification system; its disadvantages are: long cultivation period, requiring more than 60 hours, time-consuming and laborious, and for the "living non-culturable state" (Viable But Non-Culturable state) that is still pathogenic , VBNC) have low bacterial sensitivity, which can lead to false negative test results, especially for frozen foods, which have a very high false negative rate and cause great food safety hazards

The reagents and instruments used in the Real-time PCR method and the automatic microbial biochemical identification system are relatively expensive, and have high requirements for operators, especially for the detection of large quantities of samples. The cost is very high, and it is difficult for conventional laboratories to accept; and Real-time PCR and LAMP methods may cause false negative results due to the presence of substances that inhibit enzyme activity in the sample; LAMP method is highly sensitive, but it is very easy to cause aerosol pollution, resulting in false positives, resulting in wrong test results; ELISA immunological method The operation is complicated, time-consuming and laborious, and it is not convenient for standardization and high-throughput operation

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