Droplet digital PCR detection method of Listeria monocytogenes and application thereof

Abstract

The invention provides a droplet digital PCR detection method of Listeria monocytogenes and application thereof, and relates to the technical field of biological detection. The droplet digital PCR detection method of the Listeria monocytogenes includes the steps that firstly, DNA of a listeria monocytogenes standard substance, DNA of a sample to be detected and DNA of a non-target strain are extracted respectively; then the obtained DNA is subjected to microdroplet digital PCR amplification to obtain reaction liquid; and finally the reaction liquid is detected and analyzed. A probe for microdroplet digital PCR amplification has a nucleotide sequence as shown in SEQ ID NO.3, and a primer is provided with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2. The primer has better specificity on the amplification of the listeria monocytogenes and can be used for accurately measuring the content of the listeria monocytogenes in food. The droplet digital PCR detection method of the Listeria monocytogenes is simple, short in period, high in sensitivity, high in precision and good in stability, and has no cross reaction with escherichia coli and the like.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a droplet digital PCR detection method for Listeria monocytogenes and an application thereof. Background technique [0002] Droplet digital PCR (Droplet digital PCR, ddPCR) is a new nucleic acid quantitative detection technology. Different from traditional quantitative PCR (qPCR) technology, droplet digital PCR adopts an absolute quantitative method and does not rely on standard curves and reference samples to directly detect targets. The copy number of the sequence, the detection limit can reach a single copy, and it has better sensitivity, specificity and accuracy than traditional RT-PCR. Droplet digital PCR technology has great advantages in the detection of trace nucleic acid samples, the detection of rare mutations in complex backgrounds, and the identification of small differences in expression. [0003] Listeria monocytogenes (Listeria monocytogenes), referre...

AI Technical Summary

Problems solved by technology

[0004] At present, the commonly used detection methods include traditional biological detection, including EB enrichment method and LB enrichment method, through the isolation and culture of microorganisms, biochemical reactions, hemolysis tests, cooperative hemolysis tests, animal tests, etc. These experimental equipment are not demanding and can be operated Strong, but the inspection cycle is long, it takes 6d-7d, this traditional detection method can no longer meet the requirements of on-line detection in the food production process, rapid detection of food before marketing and consumption; traditional immunological detection methods, such as ELISA As a detection method, there is a commercial Listeria monocytogenes detection kit produced by Germany's Baifa Company. The ELISA method is relatively simple to operate, depends on the quality of the antibody, and has strong specificity, but the detection limit is relatively high. Due to the expensive preparation of monoclonal antibodies, the detection cost of various food samples with ELISA kits is relatively high; there is also a commonly used RT-PCR method, although it has the advantages of high sensitivity, short detection time, and quantitative detection, but its Quantification depends on the standard curve, the detection limit cannot detect a single copy and has high requirements for operation, which is prone to false positives and false negatives

Therefore, the establishment of a droplet digital PCR (ddPCR) detection method for Listeria monocytogenes in food can effectively solve the problems of insufficient sensitivity, low detection accuracy, long detection cycle, and cumbersome operation of traditional methods in food safety detection.

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