Method for detecting various microorganisms by integrated multiple nest type PCR one-step method

Abstract

The invention discloses a method for detecting various microorganisms by an integrated multiple nest type PCR one-step method. The method comprises the steps of performing differentiation designing of a universal primer and a specific primer of NM-PCR based on the characteristics of DNA bar code genes of microorganisms to be detected, establishing an NM-PCR one-step detection system, namely performing parameter optimization by using the genomic DNA of the microorganisms to be detected as a PCR reaction formwork, performing quick DNA extraction on microorganism genomes in samples to be detected, performing a NM-PCR amplification reaction by using the genomic DNA in the samples as a formwork, and performing gel electrophoresis detection on PCR reaction products. The method disclosed by the invention concurrently has specificity of the nest type PCR and high flux of multiplex PCR, two-time PCR processes are completed only through one-step PCR reaction, traditional PCR multi-step operations are avoided, the microbial pollution situations of clinical samples such as environment water sources and foods can be quickly and massively detected, the specificity is high, the flus is high, the detection time can be greatly shortened, and the cost can be greatly reduced.

Description

technical field [0001] The invention relates to the technical field of microorganism detection, in particular to a method for detecting multiple microorganisms in one step by integrating multiple nested PCR, which is used for rapid PCR detection of pathogenic bacteria in clinical and food fields. Background technique [0002] Microbial detection, especially pathogenic bacteria detection, plays a pivotal role in the fields of clinical microbiological detection, infectious disease diagnosis, and food hygiene and safety monitoring. Traditional detection techniques such as conventional culture, biochemical identification, or serological typing have certain limitations, such as extremely cumbersome operations, time-consuming and labor-intensive operations, and the inability to accurately identify the genotype of microorganisms. In recent years, methods based on nucleic acid amplification include Nucleic Acid Sequence-Based Amplification (NASBA), Self-sustained Sequence Replicatio...

AI Technical Summary

Problems solved by technology

[0004] The technical problem solved by the present invention is to overcome the defects between the high throughput and specificity of the existing PCR amplification technology, and provide a method for integrating multiple nested PCR into one-step PCR method to detect multiple microorganisms, that is, Nested-multiplexPCR, referred to as NM-PCR, which combines the specificity of nested PCR and the high throughput of multiplex PCR, avoids the cumbersome process of the traditional two-step PCR method and the possibility of increased pollution, and can quickly detect the microbial contamination of clinical samples such as environmental water sources and food , strong specificity, high throughput, greatly reducing detection time and cost

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