A real-time PCR assay for rapid detection and quantitation of canine parvovirus type 2 in the feces of dogs.
We describe a rapid, sensitive and reproducible real-time PCR assay for detecting and quantifying canine parvovirus type 2 (CPV-2) DNA in the feces of dogs with diarrhea. An exogenous internal control was added to control the assay performance from extraction to amplification. The method was demonstrated to be highly specific and sensitive, allowing a precise CPV-2 DNA quantitation over a range of eight orders of magnitude (from 10(2) to 10(9) copies of standard DNA). The reproducibility of the CPV-2 real-time PCR assay was assessed by calculating the coefficients of variation (CV) intra-assay and inter-assay for samples containing amounts of CPV-2 DNA spanning the whole range of the real-time PCR standard curve. Then, fecal specimens from diarrheic dogs were analyzed by hemagglutination (HA), conventional PCR and real-time amplification. Comparison between these different techniques revealed that real-time PCR is more sensitive than HA and conventional gel-based PCR, allowing to detect low viral titers of CPV-2 in infected dogs.
Decaro
Nicola
N
Department of Animal Health and Well-being, Faculty of Veterinary Medicine of Bari, Strada per Casamassima Km 3, 70010 Valenzano, Bari, Italy. n.decaro@veterinaria.uniba.it
Elia
Gabriella
G
Martella
Vito
V
Desario
Costantina
C
Campolo
Marco
M
Trani
Livia Di
LD
Tarsitano
Elvira
E
Tempesta
Maria
M
Buonavoglia
Canio
C
D000818:Animals; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D003967:Diarrhea / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D004283:Dog Diseases / Q000175:diagnosis / Q000821:virology*; D004285:Dogs; D005243:Feces / Q000821:virology; D006385:Hemagglutination Inhibition Tests / Q000662:veterinary; D010322:Parvoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D017993:Parvovirus, Canine / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism
D004279:DNA, Viral; D019914:Taq Polymerase