A novel TaqMan probe-based qPCR method for rapid detection of the bacteria-associated amoeba Heterostelium pallidum

Dictyostelid cellular slime molds (dictyostelids) exhibit complex interactions with bacteria and have become an important research tool for studies of the relationships that exist between the ecological environment and pathogenic microorganisms. Heterostelium pallidum, a species of dictyostelid, is the type species of the genus Heterostelium. Traditional methods for isolating H. pallidum are time-consuming and ineffective. In the present study, a pair of specific primers and probes based on the genome of H. pallidum was designed and screened, optimized reaction conditions were determined, and a real-time fluorescence quantitative PCR detection method was developed. A standard curve was constructed with concentrations ranging from 1 ng/μL to 10 fg/μL. These yielded a correlation coefficient of 0.996 and an amplification efficiency of 103.354%. This method was tested and found to work for three random environmental soil samples. An effective CT value was detected in a soil sample where only a single clone of H. pallidum was isolated. The TaqMan probe-based fluorescence quantitative PCR method demonstrated high sensitivity and accuracy. The results obtained in this study have improved the limitations of traditional isolation methods used for H. pallidum, and these can be used for the rapid detection and determination of H. pallidum in different environments, providing reference data for research on the soil environment, microbial assemblages, and public health.IMPORTANCEHeterostelium pallidum has been widely used in studies of chemotaxis and signaling in organisms as well as in other aspects of cell biology. However, its ecological relationships within diverse soil bacterial communities and its broader environmental roles remain poorly characterized, unlike the extensively studied model organism Dictyostelium discoideum. The question of how to enhance the value of H. pallidum in ecology through the application of specific technological approaches is a seriously understudied topic. Based on this situation, the present study developed a quantitative detection method for H. pallidum. Efficient detection of this organism in soil samples validated the applicability of this method for complex environmental samples. The specific probe developed in this study for H. pallidum changes the situation of time-consuming and low sensitivity associated with traditional methods of isolation, thus providing technical references for the application of H. pallidum in a wide range of ecological and environmental research.