A rapid, sensitive, and high-throughput pathogen detection method and application of identifying pathogens based on multiplex PCR technology combined with capillary electrophoresis.

Respiratory infections are one of the leading causes of death worldwide. Rapid and accurate detection of pathogens is crucial for timely treatment. This study established a detection method based on multiplex PCR combined with capillary electrophoresis, capable of simultaneously detecting 28 common respiratory pathogens. We used specially designed homo-tag primers, significantly reducing the formation of primer dimers and improving the specificity and sensitivity of amplification. After two rounds of PCR amplification, the products were subjected to fragment analysis using the ABI 3500XL Genetic Analyzer, enabling automated result interpretation. The detection limit of this method reaches 2.77 × 10 copies/ml, with some pathogens reaching as low as 2.77 × 10 copies/ml. The detection of 147 clinical specimens showed that the overall consistency of this method with culture, colloidal gold, fluorescent quantitative PCR, and NGS was 75.5 %. The multiplex PCR detection method established in this study is rapid, sensitive, and high-throughput, can be used for routine screening of common pathogens in respiratory infections, providing an important basis for clinical diagnosis and treatment.