Kit for rapidly detecting shigellae in food

Abstract

The invention provides a PCR (polymerase chain reaction) kit with internal amplification control and for detecting shigellae in food, relating to the technical field of biology. A method is convenient to use, has the advantages of good reliability, short detection period, high sensitivity, strong specificity, low cost and simple operating steps, is suitable for high throughput operation and standardized operation, can avoid misjudgment caused by false negative results obtained because DNA (deoxyribonucleic acid) polymerase inhibitors exist in samples, and can be used for detecting shigellae in various food. The method comprises the following steps of: (1) utilizing a pair of specific primers ipaHF / ipaHR which are designed by primer premier 6.0 software and aim at shigellae, then adding primers 27F / 1492R amplifying a prokaryote 16S rDNA conserved sequence as the internal amplification control and carrying out amplification by the two pairs of primers jointly; (2) carrying out enrichment culture on a sample to be detected for 6-8 hours and extracting genomic DNA from enrichment fluid obtained after culture as a template by adopting a boiling and freeze-thaw method to carry out amplification; and (3) preparing 25mu l of reaction system with PCR reaction premix of the kit to carry out amplification and observing the detection results under an ultraviolet lamp after agarose gel electrophoresis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a PCR kit with amplified internal standard for detecting Shigella in food. Background technique [0002] Shigella is a kind of Gram-negative bacillus, which is the most common pathogen of human bacillary dysentery, commonly known as Shigella. Bacillary dysentery caused by Shigella is the most common intestinal infectious disease, with the most patients in summer and autumn. Shigella is also one of the most common foodborne pathogens. The source of infection is mainly patients and carriers, through oral infection through food and drinking water contaminated with Shigella. Humans are mainly infected by eating food contaminated by Shigella. The main clinical features are systemic poisoning symptoms, ulcers, and purulent inflammation of the large intestine. After eating food contaminated by Shigella, the general situation will be within 6 to 24 hours. The following symptoms appear in ...

AI Technical Summary

Problems solved by technology

The disadvantage of this laboratory standard method is that the inspection steps are cumbersome, time-consuming, and the accuracy rate is low, and the number of samples completed in one inspection is small, which cannot meet the requirements of the fast and accurate inspection method demanded by the market.

The reagents and instruments used in the automatic microbial biochemical identification system are relatively expensive, and the requirements for operators are high, especially for the detection of large quantities of samples. The cost is very high, and it is not suitable for general laboratories.

The LAMP method cannot identify false negative results that may be caused by the presence of substances that inhibit enzyme activity in the sample; the LAMP method is very sensitive, and it is easy to form aerosol pollution, resulting in false positives, resulting in wrong test results; ELISA Immunological methods are complicated to operate, time-consuming and labor-intensive, and inconvenient for standardization and high-throughput operations

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