Rapid nucleic acid sequencing method based on fluorescence PCR

Abstract

The invention relates to a rapid nucleic acid sequencing method based on fluorescence PCR, is successfully applied in sequencing of bacterial genes, and can be used for identification of pathogenic bacteria. An FTA card nucleic acid is used for saving pathogenic bacteria nucleic acid, a real-time fluorescence PCR technology is used for amplifying a target fragment, and machine loading is performed for double deoxidization sequencing. A developed fluorescence PCR rapid sequencing reagent kit includes the following reagents: 1) one piece of FTA card; 2) one tube of PCR reaction liquid internally filled with an SYBR GREEN fluorescent PCR reaction solution; 3) one tube of an upstream primer and one tube of a downstream primer which are internally filled with target sequence amplification and sequencing primers; 4) a set of double deoxidization sequencing reagents; 5) a set of sequencing product purification reagents including one tube of a XTerminator solution and one tube of an SAM solution; and 6) one tube of deionized water internally filled with DNA enzyme-free sterile deionized water. Because the FTA card is used for saving the nucleic acid, after the card is dried naturally, the card can be stored for a long time at room temperature, is easy to save, has no need for nucleic acid extraction during PCR amplification, and avoids biological security risks of a bacterial suspension treating operation process during common bacteria gene sequencing.

Description

technical field [0001] The invention relates to a rapid nucleic acid sequencing method based on fluorescent PCR, which is successfully applied in bacterial gene sequencing and can be used for identification of pathogenic bacteria. Background technique [0002] In 1977, Sanger invented the dideoxy DNA sequencing method. So far, the two classic traditional sequencing methods, the dideoxy chain termination method sequencing and the chemical degradation method sequencing, are the most widely used. Completed core technology. The most widely used in this method is the BigDye terminator method of Applied Biosystems. The main steps include DNA extraction, PCR amplification, qualitative detection of PCR products, purification of PCR products, sequencing reaction, purification of sequencing products, capillary electrophoresis, data analysis, sequence splicing (for long fragments), etc. In recent years, due to the improvement and popularization of sequencing technology, as well as th...

AI Technical Summary

Problems solved by technology

Although the second-generation and third-generation sequencing technologies have been commercialized, the cost is still high, and they are not suitable for daily testing by grass-roots testing institutions

In addition, for the bacterial sequencing identification method, the lack of systematic basic research on related genetic markers is a major reason hindering the large-scale application of this method

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