Method for preparing live bacteria internal standard based on gene substitution technique

Abstract

The invention discloses a method for preparing internal standard of viable bacteria based on the gene substitution technology, which pertains to the field of biotechnology and comprises the steps: (1) the selection of homologous recombination sequences, amplification internal standard sequences and kanamycin-resistant gene sequences of double-exchange DNA and the design of corresponding enzyme-cutting sites are carried out between the upstream and the downstream and target genes in the gene substitution process; (2) homologous recombination plasmids pKSV7-UIKD containing the upstream and downstream homologous sequences, the internal standard sequences and the kanamycin-resistant gene are prepared; (3) the screening of electroporation-competent listeria monocytogenes of the homologous recombination plasmids and recipient cell transformant having donator properties after the corresponding electroporation is carried out; (4) the screening of viable listeria monocytogene internal standard which carries out gene substitution is carried out; and (5) the prepared amplification internal standard is verified by adding the viable bacteria internal standard in the samples to be detected and the like. The method avoids the occurrence of false negative phenomenon caused by the inhibiting ingredients, thus greatly improving the accuracy of microbial fluorescence quantitative PCR detection.

Description

technical field [0001] The invention relates to a method in the field of biotechnology, in particular to a method for preparing an internal standard of viable bacteria based on gene replacement technology that can be widely used in fluorescence quantification. Background technique [0002] Fluorescent quantitative PCR (abbreviation for real-time quantitative polymerase chain reaction) refers to: fluorescent quantitative polymerase chain reaction. Fluorescence quantitative PCR technology is a nucleic acid quantitative technology developed on the basis of ordinary PCR technology. It is a double-labeled fluorescent probe complementary to the target fragment sequence added to the ordinary PCR system. The 5' of the probe The end is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group. When the probe is intact, the reporter group cannot produce fluorescence due to the effect of the quencher group; in PCR amplification, due to the 5...

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Problems solved by technology

So far there is no report about the application of live bacteria internal standard in microbial PCR detection

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