Primer probe combination and kit for plasmid quantification

Abstract

The invention belongs to the technical field of biology, and particularly relates to a primer probe combination and a kit for plasmid quantification, the primer probe combination comprises a pair of ampicillin resistance gene specific primers with nucleotide sequences as shown in SEQ ID: 1-2 and an AmpR P probe with a nucleotide sequence as shown in SEQ ID: 3; the kit further comprises a pair of kanamycin resistance gene specific primers with the nucleotide sequence shown as SEQ ID: 4-5 and a Kan P probe with the nucleotide sequence shown as SEQ ID: 6. 5'ends of the two probes are marked with fluorophores, and 3 'ends of the two probes are marked with quenching groups. The primer probe combination is used for the kit, multiple PCR reactions can be carried out, the purpose of quantifying plasmids containing AmpR or Kan is achieved, universality is high, all plasmids containing one of the two resistance gene sequences can be quantified, the method is a universal method, data credibility is high, and authenticity and traceability of quantitative results are guaranteed.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer-probe combination and a kit for plasmid quantification. Background technique [0002] Accurate quantification of templates is critical in molecular diagnostics development. Whether it is a qPCR platform or other platforms such as LAMP and NGS, template quantification should be the preparatory work before the development of the reaction system. [0003] At present, the templates used in research and development work include plasmids, pseudoviruses, etc. For the quantification of plasmids, the current mainstream methods include the following methods: ultraviolet spectrophotometry (Thermo Nanodrop) or Qubit fluorescence instrument, qPCR method and digital PCR method. Ultraviolet spectrophotometry and Qubit method require expensive instruments, and the minimum concentration limit of nucleic acid detection by this method is still high, which cannot meet the needs of ...

AI Technical Summary

Problems solved by technology

Ultraviolet spectrophotometry and Qubit method require expensive instruments, and the minimum concentration limit of nucleic acid detection by this method is still high, which cannot meet the needs of quantification of low-copy templates; digital PCR method can quantify templates as low as 1 copy, but its The operation is cumbersome, and the required instrument and reagent consumables are expensive. Generally, it is used as the initial quantification to assign values ​​to the standard. As an experimental method with relatively high accuracy and relatively low cost, qPCR method is favored by R&D workers.

However, the qPCR method also has certain defects. The qPCR method needs to be quantified by the primers designed on the template insertion sequence every time. The operation is more complicated, and the efficiency of the primers needs to be verified before use. Therefore, a universal primer sequence detection Methods for Quantification of Plasmids

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