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Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction
Published on: November 1, 2018
Detection of infectious canine parvovirus type 2 by mRNA real-time RT-PCR
Gabriella Elia1, Alessandra Cavalli, Costantina Desario
1Department of Animal Health and Well-being, Faculty of Veterinary Medicine of Bari, S.p. per Casamassima km 3, 70010 Valenzano, Bari, Italy. g.elia@veterinaria.uniba.it
Abstract:
A TaqMan real-time RT-PCR assay was developed for detection of RNA transcripts produced by replicating CPV-2. A pair of primers and a TaqMan probe targeting the spliced NS2 mRNA were designed. A synthetic DNA fragment was constructed to mimic the spliced NS2 mRNA by PCR-based gene assembly and was used for generation of standard RNAs. The detection limit of the assay was 1x10(2) RNA copies and standard curve displayed a linear range from 1x10(2) to 1x10(9) copies and a good reproducibility. The assay was then applied to determine the mRNA loads in the tissues of dogs naturally infected by CPV-2. mRNA was detected in a variety of tissues, including the central nervous system.
