Homogeneous duplex polymerase chain reaction assay using switchable lanthanide fluorescence probes.

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Complementation probe technology enables sensitive detection by forming fluorescent complexes.
• Lanthanide chelate probes offer unique optical properties for biological assays.
• Chlamydia trachomatis (Ct) detection is crucial for public health.

Purpose of the Study:

• To develop a novel duplex PCR assay for Chlamydia trachomatis (Ct) detection.
• To synthesize new terbium(III) and europium(III) based complementation probes.
• To evaluate the diagnostic accuracy and performance of the assay with urine samples.

Main Methods:

• Developed a duplex PCR assay utilizing switchable lanthanide chelate complementation probes.
• Synthesized new terbium(III) ion carrier chelate and antenna ligand.
• Detected Ct using europium(III) probes and an internal control with terbium(III) probes.
• Concentrated Ct bacteria from urine via centrifugation.

Main Results:

• Achieved high diagnostic accuracy for Ct detection, ranging from 99-100%.
• Observed very high europium(III) signal-to-background ratios (up to 244) in positive samples.
• Demonstrated effective amplification control for Ct-negative results.
• Showcased the robustness of complementation probes with simple sample preparation.

Conclusions:

• The developed duplex PCR assay with lanthanide chelate complementation probes is highly accurate for Ct detection.
• The assay's high performance is suitable for samples with potential impurities after basic preparation.
• This method offers a sensitive and reliable tool for Chlamydia trachomatis diagnostics.