Method Description

DNA from clinical specimens is extracted using the Roche MagNA Pure system. Toxoplasma gondii DNA is then detected by using real-time polymerase chain reaction (PCR) to amplify the target sequence. The LightCycler amplifies and monitors fluorescent development of target nucleic acid after each cycle. The continuous monitoring is derived from the fluorescence resonance energy transfer (FRET) principle: a hybridization probe with a donor fluorophore on the 3' end is excited by an external light source and emits light that is absorbed by a second hybridization probe with an acceptor fluorophore at the 5' end. The acceptor fluorophore emits light of a different wavelength that can be measured with a signal that is proportional to the amount of specific PCR product. Melting temperature analysis is used following amplification for sensitive and specific detection of amplified target DNA.(Cockerill FR, Uhl FR. Applications and challenges of real-time PCR for the clinical microbiology laboratory. In: Reischl U, Wittwer C, Cockerill F, eds. Rapid Cycle Real-Time PCR, 2002; Nolte FS. Target amplification techniques. In: Carroll KC, Pfaller MA, Landry ML, et al, eds. Manual of Clinical Microbiology. 12th ed. ASM Press; 2019)