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Development and Preliminary Evaluation of a New Real‐Time RT ‐ PCR Assay For Detection of Peste des petits Ruminants Virus Genome
Author(s) -
Polci A.,
Cosseddu G. M.,
Ancora M.,
Pii C.,
El Harrak M.,
Sebhatu T. T.,
Ghebremeskel E.,
Sghaier S.,
Lelli R.,
Monaco F.
Publication year - 2015
Publication title -
transboundary and emerging diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.392
H-Index - 63
eISSN - 1865-1682
pISSN - 1865-1674
DOI - 10.1111/tbed.12117
Subject(s) - taqman , virology , real time polymerase chain reaction , peste des petits ruminants , biology , peste des petits ruminants virus , reverse transcription polymerase chain reaction , heterologous , rna extraction , microbiology and biotechnology , duplex (building) , polymerase chain reaction , virus , rna , gene , dna , genetics , messenger rna
Summary A duplex real‐time reverse transcription‐polymerase chain reaction (q RT ‐ PCR ) assay was developed for a simple and rapid diagnosis of P este des petits ruminants ( PPR ). q RT ‐ PCR primers and T aq M an probe were designed on a conserved region of nucleocapsid protein ( N p) of PPR virus ( PPRV ) genome. An in vitro transcript of the target region was constructed and tested to determine analytical sensitivity. Commercial heterologous Armored RNA ® was used as an internal positive control ( IPC ) for either RNA isolation or RT ‐ PCR steps. The detection limit of the newly designed duplex real‐time RT ‐ PCR (q RT ‐ PCR PPR _ N p) was approximately 20 copies/ μ l with a 95% probability. No amplification signals were recorded when the q RT ‐ PCR PPR _ N p was applied to viruses closely related or clinically similar to PPRV ‐ or to PPR ‐negative blood samples. A preliminary evaluation of the diagnostic performance was carried out by testing a group of 43 clinical specimens collected from distinct geographic areas of A frica and M iddle E ast. q RT ‐ PCR PPR _ N p showed higher sensitivity than the conventional gel‐based RT ‐ PCR assays, which have been used as reference standards. Internal positive control made it possible to identify the occurrence of 5 false‐negative results caused by the amplification failure, thus improving the accuracy of PPRV detection.