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. 2004 Aug;119(2):145-50.
doi: 10.1016/j.jviromet.2004.03.012.

Quantitation of canine coronavirus RNA in the faeces of dogs by TaqMan RT-PCR

Nicola Decaro  1 , Annamaria Pratelli, Marco Campolo, Gabriella Elia, Vito Martella, Maria Tempesta, Canio Buonavoglia
Affiliations

Quantitation of canine coronavirus RNA in the faeces of dogs by TaqMan RT-PCR

Nicola Decaro et al. J Virol Methods. 2004 Aug.

Abstract

A TaqMan fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed for the detection and quantitation of canine coronavirus (CCoV) RNA in the faeces of naturally or experimentally infected dogs. The CCoV fluorogenic RT-PCR assay, which targeted the ORF5 (M gene), was more sensitive than a conventional RT-PCR assay targeting the same gene, showing a detection limit of 10 copies of CCoV standard RNA, and was linear from 10 to 10(8) copies, allowing quantitation of samples with a wide range of CCoV RNA loads. A total of 78 faecal samples of diarrhoeic dogs were tested simultaneously by conventional and fluorogenic RT-PCR: 29 were negative by both techniques, whereas 27 tested positive by conventional RT-PCR and 48 by the established CCoV fluorogenic assay. One sample, which was positive by conventional RT-PCR, gave no signal in the fluorogenic assay. In addition, by the fluorogenic assay CCoV shedding in the faecal samples of an experimentally infected dog was monitored for 28 days. The high sensitivity, simplicity and reproducibility of the CCoV fluorogenic RT-PCR assay, combined with its wide dynamic range and high throughput, make this method especially suitable for efficacy trials on CCoV vaccines.

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Figures

Fig. 1
Fig. 1
Standard curve of the CCoV fluorogenic RT-PCR assay. Ten-fold dilutions of standard RNA prior to amplification were used, as indicated on the x-axis, whereas the corresponding cycle threshold (CT) values are presented on the y-axis. Each dot represents the result of triplicate amplifications of each dilution. The coefficient of determination (R2) and the slope value (s) of the regression curve were calculated and are indicated.
Fig. 2
Fig. 2
Detection of CCoV in faecal samples of naturally infected dogs: comparison between conventional and fluorogenic RT-PCR. Numbers indicate the samples positive (+) or negative (−) for CCoV. Results according to both techniques are shown in bold.
Fig. 3
Fig. 3
Number of copies of CCoV genomic RNA in the faecal samples of the experimentally infected dog by fluorogenic RT-PCR. Asterisks indicate the CCoV positive samples by conventional RT-PCR. Symbol (+) indicates days post-infection in which a mild diarrhoea was observed.
See this image and copyright information in PMC

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