ET-PCR (endonuclease restriction-mediated real-time polymerase chain reaction) nucleic acid testing technology

Abstract

The invention discloses a PCR (polymerase chain reaction) amplification method. In the method, a restriction endonuclease sequence is added to a 5'end of an upstream primer or a downstream primer for amplification, a fluorophore is marked at the 5'end of the primer, a quenching group capable of specifically quenching the fluorophore at the 5'end is marked in the middle of the primer, the restriction endonuclease is added to a PCR amplification system and subjected to a PCR. The method has the advantages that the amplification speed is high, the reaction is sensitive, the specificity is high, and real-time, rapid and multiplex detection and analysis of nucleic acid can be realized.

Description

technical field [0001] The invention relates to a nucleic acid amplification method, which belongs to the field of microorganisms and molecular biology. Background technique [0002] In the field of modern biology and medicine, nucleic acid amplification is an indispensable technology, widely used in clinical testing, basic research, archaeological research, epidemiological research, transgenic research and other fields. Among the nucleic acid amplification technologies that have been developed and designed, PCR amplification technology is the first established in vitro nucleic acid amplification technology, which has epoch-making significance. This technology has been widely used in medical, biological and chemical related fields. [0003] When PCR technology is used for nucleic acid amplification, three thermal cycle steps need to be repeated: nucleic acid melting (95°C), primer annealing (45-65°C), and extension amplification (72°C). After the PCR amplification is comple...

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Problems solved by technology

At present, the diagnosis of Streptococcus pneumoniae, Staphylococcus aureus and Enterococcus faecalis mainly relies on traditional enrichment culture and biochemical identification. This method takes about 5 to 7 days, including enrichment, selective culture and subsequent biochemical identification. Its disadvantage is time-consuming and labor-intensive, and the interpretation of biochemical results depends on human subjective judgment, resulting in poor repeatability of results and easy misjudgment

With the rapid development of nucleic acid diagnostic technology, PCR technology has been used to diagnose pathogens in clinical and basic laboratories. However, this technology requires subsequent electrophoresis operations, which cannot meet the needs of rapid and convenient detection.

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