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. 2005 Dec;130(1-2):72-8.
doi: 10.1016/j.jviromet.2005.06.005. Epub 2005 Jul 15.

Genotype-specific fluorogenic RT-PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs

Nicola Decaro  1 , Vito Martella, Dominga Ricci, Gabriella Elia, Costantina Desario, Marco Campolo, Nicola Cavaliere, Livia Di Trani, Maria Tempesta, Canio Buonavoglia
Affiliations

Genotype-specific fluorogenic RT-PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs

Nicola Decaro et al. J Virol Methods. 2005 Dec.

Abstract

Two genotype-specific fluorogenic RT-PCR assays were developed for the detection and quantitation of canine coronavirus (CCoV) type I and type II RNA in the faeces of dogs with diarrhoea. Both the fluorogenic assays showed high specificity, sensitivity and reproducibility, allowing a precise quantitation of CCoV type I and type II RNA over a linear range of about eight orders of magnitude (from 10(1) to 10(8) copies of standard RNA). Comparison with genotype-specific gel-based RT-PCR assays revealed that the fluorogenic assays were more sensitive and more rapid than conventional amplifications, with a large increase in throughput. The genotype-specific fluorogenic assays were then used to detect and measure viral loads in the faecal samples collected from dogs naturally or experimentally infected with type I, type II, or both genotypes. Of 174 samples collected from naturally infected dogs, 77 were positive for CCoV type I and 46 for CCoV type II. Thirty-eight dogs were found to be infected naturally by both genotypes, with viral RNA titres generally higher for type I in comparison to type II. At the same time, dogs infected experimentally shed type I RNA with higher titres with respect to type II.

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Figures

Fig. 1
Fig. 1
Detection of CCoV type I and type II RNA in faecal samples of dogs infected naturally by conventional and fluorogenic genotype-specific RT-PCR assays. Numbers indicate the samples positive (+) or negative (−) for CCoV. Results according to both techniques are shown in bold.
Fig. 2
Fig. 2
Logarithmic distribution of the RNA amounts of CCoV type I and type II in the faeces of dogs infected naturally. Values are expressed as RNA copy numbers per μl of template. Each dot represents a faecal sample; bars indicate the median values.
Fig. 3
Fig. 3
Number of copies of viral RNA per μl of template detected in faecal samples of the dogs infected with CCoV type I (a), type II (b), or both genotypes (c) by the genotype-specific fluorogenic RT-PCR assays. Asterisks indicate the samples tested CCoV-positive by the conventional RT-PCR assays. Symbol (+) indicates days post infection in which diarrhoea was observed.
See this image and copyright information in PMC

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