Application of droplet digital PCR in the diagnosis of talaromycosis

ZHANG Hua; PAN Yanna; RAO Ji; RUAN Yiting; WEI Xuantong; LAN Jihui; HU Min; LUO Lanbo; DU Baiyinzi; DU Mingwei; PAN Weihua

doi:10.3969/j.issn.1673-3827.2025.01.004

Abstract：Objective To develop a droplet digital polymerase chain reaction(ddPCR)-based nucleic acid detection method for Talaromyces marneffei and verify its sensitivity in detecting trace samples,thereby broadening the precision range of molecular detection techniques for Talaromyces marneffei.Methods The primer and probe sequences that have been successfully used in real-time fluorescent quantitative PCR for Talaromyces marneffei detection were screened,and a ddPCR detection system was established based on these sequences.Plasmids carrying the target sequence region were used to simulate clinical samples of Talaromyces marneffei,and other common clinical pathogenic fungal were used as no-template controls to evaluate the specificity and detection limit of the ddPCR detection method in this study.Results The ddPCR detection system in this study exhibited high specificity and no cross-reactivity with various no-template controls.When the template concentration was higher than 7.78 copies/μL,the detection system could detect positive results with 100%accuracy.The entire quantitative detection process was simple to operate,and compared with qPCR,it did not require the preparation of a standard curve to obtain quantitative results from trace samples.Conclusion ddPCR is a novel,sensitive,and easy-to-operate nucleic acid detection method with high specificity and extremely sensitive detection limits.Its quantitative results are helpful for operators to interpret the validity of detection data,and it has the potential for further clinical application.