RESUMO
Nitroxide-labeled nucleic acids are used as a molecular size sensor to identify as few as one genome under polymerase chain reaction (PCR) conditions by electron paramagnetic resonance (EPR) spectroscopy. DNA identification is based on differences in the EPR spectra of mononitroxide-labeled nucleic acids. The experimental data imply that rapid DNA identification can be achieved in many systems by EPR at the molecular level.
Assuntos
DNA de Protozoário/análise , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Encephalitozoon/genética , Genoma de Protozoário , Óxidos de Nitrogênio/química , Sondas de Oligonucleotídeos/química , Marcadores de Spin , Animais , Dicroísmo Circular , Óxidos N-Cíclicos/química , Primers do DNA/química , Encephalitozoon/química , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , Espectrofotometria UltravioletaRESUMO
Cyclospora cayetanensis, a coccidian parasite, with a fecal-oral life cycle, has become recognized worldwide as an emerging human pathogen. Clinical manifestations include prolonged gastroenteritis. While most cases of infection with C. cayetanensis in the United States have been associated with foodborne transmission, waterborne transmission has also been implicated. We report on the development and application of a real-time, quantitative polymerase chain reaction assay for the detection of C. cayetanensis oocysts, which is the first reported use of this technique for this organism. Both a species-specific primer set and dual fluorescent-labeled C. cayetanensis hybridization probe were designed using the inherent genetic uniqueness of the 18S ribosomal gene sequence of C. cayetanensis. The real-time polymerase chain reaction assay has been optimized to specifically detect the DNA from as few as 1 oocyst of C. cayetanensis per 5 microl reaction volume.
Assuntos
Cyclospora/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , Cyclospora/genética , Cyclospora/crescimento & desenvolvimento , Ciclosporíase/diagnóstico , Fezes/parasitologia , Citometria de Fluxo/métodos , Humanos , Oocistos/isolamento & purificação , Testes de Sensibilidade ParasitáriaRESUMO
This is the first report of blue autofluorescence as a useful characteristic in the microscopic detection of Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Neospora caninum, Besnoitia darlingi, and Sarcocystis neurona oocysts or sporocysts. This autofluorescence is of sufficient intensity and duration to allow identification of these oocysts from complex microscopic sample backgrounds. As with the autofluorescence of related coccidia, the oocysts glow pale blue when illuminated with an ultraviolet (UV) light source and viewed with the correct UV excitation and emission filter set.