Quantitative one-step real-time RT-PCR for the fast detection of the four genotypes of PPRV.
A one-step real-time Taqman RT-PCR assay (RRT-PCR) for peste des petits ruminants virus (PPRV) was developed to detect the four lineages of PPRV by targeting the nucleoprotein (N) gene of the virus. This new assay was compared to a conventional RT-PCR on reference strains and field materials. Quantitation was performed against a standard based on a synthetic transcript of the NPPR gene for which a minimum of 32 copies per reaction were detected with a corresponding C(t) value of 39. Depending on the lineage involved, the detection limit of RRT-PCR was decreased by one to three log copies relative to the conventional method. The lower stringency occurred with lineage III because of minor nucleotide mismatches within the probe region. The assay did not detect phylogenetically or symptomatically related viruses of ruminants (such as rinderpest, bluetongue, and bovine viral diarrhea viruses). However, it was capable of detecting 20% more positive field samples with low viral RNA loads compared to the conventional PCR method. When compared on a proficiency panel to the method developed by Bao et al. (2008), the sensitivity of the in-house assay was slightly improved on lineage II. It proved significantly faster to perform and hence better adapted for monitoring large numbers of at risk or diseased animals.
Kwiatek
Olivier
O
CIRAD, UMR CIRAD/INRA Contrôle des Maladies, F-34398 Montpellier Cedex 5, France.
Keita
Djénéba
D
Gil
Patricia
P
Fernández-Pinero
Jovita
J
Jimenez Clavero
Miguel Angel
MA
Albina
Emmanuel
E
Libeau
Genevieve
G
D000818:Animals; D002162:Camelus / Q000821:virology; D003937:Diagnosis, Differential; D015511:Goat Diseases / Q000175:diagnosis* / Q000821:virology; D006041:Goats; D019590:Nucleocapsid Proteins / Q000235:genetics*; D029021:Peste-des-Petits-Ruminants / Q000175:diagnosis* / Q000821:virology; D018107:Peste-des-petits-ruminants virus / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000821:virology; D019914:Taq Polymerase; D019562:Viral Load / Q000379:methods*
D019590:Nucleocapsid Proteins; D019914:Taq Polymerase