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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Development of a Droplet Digital PCR Assay Targeting the Internal Transcribed Spacer Gene for Rapid Detection of
Yingyin Yang1,2, Qinzhi Zhang1,2, Pengle Guo1,2
1Infectious Disease Center, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou 510440, China.
Abstract:
Talaromycosis caused by Talaromyces marneffei is a life-threatening mycosis in patients with acquired immunodeficiency syndrome (AIDS). The gold-standard diagnostic method relies on time-consuming cultures, which delay treatment and increase mortality. In this study, we developed a rapid and sensitive droplet digital PCR (ddPCR) assay targeting the internal transcribed spacer (ITS) gene for detecting T. marneffei and compared its performance with blood culture and quantitative PCR (qPCR) assays. The ddPCR assay had a detection limit of one copy/reaction, making it 10-fold more sensitive than qPCR. It demonstrated 100% specificity for T. marneffei, with no cross-reactivity to 15 other fungal pathogens, six bacterial pathogens, and plasma from 119 AIDS patients without talaromycosis. In 119 AIDS patients with talaromycosis, ddPCR exhibited better overall sensitivity (92.44%) than blood culture (86.55%) and qPCR (87.29%). The sensitivity of ddPCR was 97.8% (89/91) and 75% (21/28) in plasma collected before and after antifungal therapy, respectively. Moreover, fungal load measured by ddPCR negatively correlated with the time to blood culture positivity. Fungal loads in patients receiving antifungal therapy were significantly lower than those in untreated patients. These findings indicate that ddPCR facilitates rapid diagnosis of T. marneffei infection in AIDS patients and can assist clinicians in evaluating treatment efficacy by quantifying fungal load.
