Introduction -The feline coronaviruses (FCoVs), single-stranded RNA viruses beloging to the Coronaviridae family, cause an inapparent or mild enteric infection but also cause a rare, fatal immune-mediated disease, feline infectious peritonitis (FIP). FCoVs are ubiquitous viruses with a prevalence of 55-60% above all where large number of cats are housed closely, but FIP is uncommon manifestation of this infection. The aim of this work was to set up a Real Time PCR for the detection and quantification of FCoVs. Methods - Viral RNA of strain 420/02 liver was extracted from tissue and a fragment of ORF7b was amplified by RT-PCR One Tube. The PCR product of 102 bp has been cloned and serial diluitions of the recombinant plasmid were tested with a rotor-gene SYBER Green Real Time system to obtain a standard curve. Results - The Real Time assay was able to detect 10-20 copies of DNA/_l with a reaction efficiency from 0.766 to 0.786 and a R^2 Value over 0.99. Discussion - Real-Time PCR is resulted a sensitive and fast technique and its application to cats' organs or feces and comparation with other diagnostic tests are necessary to evaluate sensitivity and diagnostic employ.
Battilani M., Balboni A., Bassani M., Prosperi S. (2006). Quantification of feline coronaviruses (FCoVs) by Real Time PCR.. s.l : s.n.
Quantification of feline coronaviruses (FCoVs) by Real Time PCR.
BATTILANI, MARA;BALBONI, ANDREA;BASSANI, MILENA;PROSPERI, SANTINO
2006
Abstract
Introduction -The feline coronaviruses (FCoVs), single-stranded RNA viruses beloging to the Coronaviridae family, cause an inapparent or mild enteric infection but also cause a rare, fatal immune-mediated disease, feline infectious peritonitis (FIP). FCoVs are ubiquitous viruses with a prevalence of 55-60% above all where large number of cats are housed closely, but FIP is uncommon manifestation of this infection. The aim of this work was to set up a Real Time PCR for the detection and quantification of FCoVs. Methods - Viral RNA of strain 420/02 liver was extracted from tissue and a fragment of ORF7b was amplified by RT-PCR One Tube. The PCR product of 102 bp has been cloned and serial diluitions of the recombinant plasmid were tested with a rotor-gene SYBER Green Real Time system to obtain a standard curve. Results - The Real Time assay was able to detect 10-20 copies of DNA/_l with a reaction efficiency from 0.766 to 0.786 and a R^2 Value over 0.99. Discussion - Real-Time PCR is resulted a sensitive and fast technique and its application to cats' organs or feces and comparation with other diagnostic tests are necessary to evaluate sensitivity and diagnostic employ.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
