| Abstract | | A fluorescence resonance energy transfer (FRET)-based real-time PCR (RT-PCR) was developed for very sensitive and specific detection of Molluscum contagiosum virus (MCV), as well as reliable differentiation of the two MCV subtype genetic lineages, MCV1 and MCV2, in a single reaction. The assay employs modified primers specific for the viral MC021L gene and uses two novel FRET hybridization probes to detect polymorphisms specific for each of the two subtypes. The sensitivity of the assay at a 95% detection level for both MCV subtypes was 3.3 DNA copies/reaction and the dynamic range was nine orders of magnitude, discriminating 10-10(9) viral genome equivalents/reaction. Post-amplification probe-specific dissociation analysis differentiated the two viral subtypes reliably in all tested concentrations. Testing of 43 tissue specimens clinically diagnosed as MCV lesions showed complete agreement with the results obtained with previously described MCV specific MC080R Taqman RT-PCR and MC021L whole gene sequencing. The novel assay is simple, robust and easy to perform, and may be of great value for clinical and epidemiological studies of MCV infections and related conditions.
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