Development and validation of the 6-dye lyophilized fluorescent multiplex PCR system.

Abstract

Aiming to address the issues associated with traditional liquid STR reagents-such as dependence on cold-chain storage, cumbersome operation, and unsuitability for on-site rapid detection-this study has successfully developed a six-color fluorescent lyophilized multiplex amplification reagent. The reagent includes 29 autosomal STR loci and 1 gender identification locus. By screening high-tolerance Taq polymerase, optimizing lyoprotectant formulations, and developing a lyophilization process based on thermodynamic analysis, stable ambient-temperature storage of key reagent components has been achieved. Performance validation demonstrates that the lyophilized reagent exhibits high sensitivity (100% detection at 0.125 ng DNA), remains stable for 60 days at room temperature, rehydrates rapidly (<1 second), and shows some tolerance to common inhibitors. Benefiting from the "whole-volume loading" advantage, its detection capability for trace samples surpasses that of traditional liquid reagents. In parallel comparison with similar liquid reagents, it achieves completely consistent locus detection rates (100%), fully meeting the requirements for individual identification. With simple operation and a ready-to-use, disposable design, this reagent effectively overcomes the reliance on cold-chain logistics. It provides robust technical and reagent support for rapid on-site forensic testing, emergency response, grassroots applications, and DNA database construction, demonstrating significant application prospects.