Development of a SYBR Green based real-time PCR assay for detection and quantitation of canine parvovirus in faecal samples.
The present study describes the development of SYBR Green based real-time polymerase chain reaction (real-time PCR) for detection and quantitation of canine parvovirus type 2 (CPV 2) in faecal samples of dogs. In this assay, the primers were designed and custom-synthesized based on nucleotide sequence of VP2 gene of CPV 2. A standard curve was plotted using 10-fold serial dilution of standard plasmid DNA and Ct value. The standard curve was found to be linear over a 10(-7) dilution. The real-time PCR results were expressed as the number of DNA copies of CPV 2 per mg of faecal samples and showed range of 1.0 x 10(3) to 7.0 x 10(9) copies of viral DNA per mg of stool samples. The analytical sensitivity of the SYBR Green based real-time PCR was shown to be equivalent to 10 copies. Faecal samples (47) from dogs suspected of CPV 2 infection were analyzed by real-time PCR, haemagglutination (HA) assay and by a conventional PCR and 24, 20 and 22 samples were found positive for CPV 2, respectively. Comparison between the results of three different assays revealed that real-time PCR is more sensitive than HA and conventional PCR and allow the detection of low titers of CPV 2 in infected dogs.
Kumar
Manoj
M
Virology Laboratory, Centre for Animal Disease Research and Diagnosis (CADRAD), Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly, U.P. 243122, India.
Nandi
Sukdeb
S
D000818:Animals; D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D004285:Dogs; D005243:Feces / Q000821:virology; D009930:Organic Chemicals / Q000378:metabolism*; D010322:Parvoviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D017993:Parvovirus, Canine / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D014773:Virology / Q000379:methods*
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I