Optimization of TaqMan-based quantitative PCR diagnosis for Entamoeba histolytica using droplet digital PCR.

ABSTRACT:

Background

TaqMan-probed quantitative PCR (qPCR) is highly valued for diagnosing Entamoeba histolytica infections (amebiasis). However, unclear cycle threshold (Ct) values often yield low-titer positive results, complicating interpretation. This study aimed to optimize qPCR primer-probe sets with logically determined cut-off Ct value using droplet digital PCR (ddPCR).

Methodology/principal findings

Amplification efficacy was evaluated using ddPCR by measuring absolute positive droplet counts (APD) and mean fluorescence intensity at different PCR cycles and annealing temperatures (AT). A primer-probe specific cut-off Ct value was determined from a standard curve by correlating Ct values with APD. Twenty primer-probe sets targeting small subunit rRNA gene regions (X64142) were

SUBMITTER: Kawashima A 

PROVIDER: S-EPMC12165414 | biostudies-literature | 2025 Jun

REPOSITORIES: biostudies-literature