Method for detecting fluorescent quantitative PCR of 12 pathogenic bacteria in real time at the same time

Abstract

The invention provides a method for detecting fluorescent quantitative PCR of 12 pathogenic bacteria in real time at the same time, including the steps: collecting specific pathogenic gene or toxin gene of the target pathogen and using it as a target gene to design primers and probes so as to make the reaction conditions consistent; extracting a genome template of a sample to be detected; adding the template respectively into tubules equipped with different specific upstream and downstream primers and probes, and then adding the corresponding fluorescent quantitative PCR reagents; under the same cycle of fluorescent quantitative PCR, the corresponding primers and probes are used to detect the samples simultaneously, quickly and quantitatively in their respective reaction tubes. Easier, Quick and efficient, Twelve common pathogenic bacteria (Escherichia coli O157: H7, Listeria monocytogenes, Salmonella, Vibrio parahaemolyticus, Streptococcus betae, Yersinia enterocolitica, Streptococcusfaecalis, Shigella, Proteus mirabilis, Vibrio fluvialis, Campylobacter jejuni, Staphylococcus aureus) can be detected simultaneously in drinking water and food economically.

Description

technical field [0001] The invention relates to the technical field of pathogenic bacteria detection, in particular to a method for simultaneously detecting 12 kinds of pathogenic bacteria by adopting a simple, fast, efficient and economical high-throughput real-time fluorescent quantitative PCR technology. Background technique [0002] Human production, domestic wastewater, human and animal feces, and natural disasters will cause some pathogenic bacteria to enter the water body, causing pollution of the water environment and the spread of various diseases, such as Shigella and Salmonella. At the same time, at this stage, my country's food safety is already an issue that cannot be ignored, which is related to the livelihood of the people in our country. In recent years, the food safety issue has also received the attention of the country and people. my country also urgently needs efficient, accurate, time-saving and labor-saving detection methods for common pathogenic bacter...

AI Technical Summary

Problems solved by technology

Ordinary PCR can realize the rapid detection of pathogenic bacteria, but it has low sensitivity and cannot quantify pathogenic bacteria; compared with ordinary PCR, multiplex PCR can detect multiple target bacteria in the same system, but it is easy to detect multiple sets of primers. mutual interference, and higher requirements for primers; LAMP technology has the advantages of small reaction system, high sensitivity, and good specificity, but during amplification, primers may complement each other to amplify non-specific bands and cause false positives; gene chip technology Fast and comprehensive, but high repeatability, poor stability, and complicated operation and post-processing; although multiplex PCR, LAMP, and gene chips have the ability to detect several pathogenic bacteria at one time, the biggest deficiency is that they cannot quantify pathogenic bacteria

Although fluorescent quantitative PCR can quantitatively detect pathogenic bacteria, due to the limitation of primer probe design, only one kind of pathogenic bacteria may be detected in one experiment, and the present invention designs primers and probes for specific pathogenic genes , and make the reaction conditions consistent, so a high-throughput real-time fluorescent quantitative PCR method for simultaneous detection of 12 pathogenic bacteria is proposed

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