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Published on: March 13, 2018
Development and evaluation of a rapid duplex qPCR assay for Treponema pallidum using the GeneSoC microfluidic
Takuya Kawahata1, Fumiya Banno1, Minami Hama1
1Division of Microbiology Virology Section, Osaka Institute of Public Health, 1-3-3, Nakamichi, Higashinari-Ku, Osaka, Osaka, 537-0025, Japan.
Background:
Early diagnosis of primary syphilis is essential for timely treatment and prevention of transmission. Although nucleic acid amplification tests are useful for detecting Treponema pallidum (TP), the development of rapid duplex molecular assays suitable for point-of-care testing remains limited.
Methods:
We developed a rapid duplex quantitative PCR (qPCR) assay using the GeneSoC microfluidic platform targeting the polA gene and the TpN47 (tp0574) region. The assay was evaluated using TP DNA standards, the Nichols reference strain, and clinical specimens, including lesion swabs and urine samples. Direct testing without DNA extraction was also evaluated. Performance was compared with a comparator qPCR assay performed on the ABI StepOnePlus system.
Results:
The assay showed good linearity over a range of 6.25 to 200 copies/µL, with correlation coefficients (R2) of 0.9927 for polA and 0.9867 for TpN47. Amplification efficiencies were 92.8% for polA and 85.3% for TpN47. TP DNA was detected within 15 minutes from both extracted DNA samples and direct clinical specimens without DNA extraction. For comparison, the ABI StepOnePlus assay required approximately 41 minutes to complete 45 amplification cycles under the conditions used in this study. The assay showed qualitative agreement with the comparator qPCR and showed no cross-reactivity with other pathogens. Detection was also achieved in a sample collected during the seronegative window period.
Conclusions:
This rapid duplex qPCR assay enables the detection of TP within 15 minutes from clinical specimens without DNA extraction, although DNA extraction may improve detection in specimens containing low amounts of target DNA. The microfluidic thermal cycling platform contributes to this rapid turnaround by enabling faster temperature transitions than conventional thermal cyclers.
