title: A SYBR Green I based real time RT-PCR assay for specific detection and quantitation of Peste des petits ruminants virus.
abstract: BACKGROUND
  Peste des petits ruminants (PPR) is an economically important disease of small ruminants such as sheep and goats. The disease is characterized by severe pyrexia, oculo-nasal discharge, pneumonia, necrosis and ulceration of the mucous membrane and inflammation of the gastro-intestinal tract leading to severe diarrhea. A SYBR Green I based real time RT-PCR targeting the N gene of PPRV has not been established for PPRV detection. Thus, the objective of present study was to develop highly sensitive N gene target SYBR Green I real time RT-PCR for specific detection and quantification of PPRV in clinical samples. A set of primers was designed to detect the nucleocapsid (N) gene of PPRV.
  
  
  RESULTS
  The assay exhibited high specificity as all the viruses which have clinical and structural similarities to PPRV including Canine distemper virus (CDV), Measles virus (MV), Bluetongue virus (BTV) and Newcastle disease virus (NDV) failed to show an amplification signal. The lower detection limit of the assay was 5.11 copies/μl (Ct value of 33.67 ± 0.5) and 0.001 TCID₅₀/ml (Ct value of 34.7 ± 0.5) based on plasmid copy number and tissue culture infectivity titre. The assay was 3-log more sensitive than the conventional RT-PCR. The coefficient of variation (CV) values for intra- and inter-assay variability were low, ranging from 0.32%-2.31%, and 0.71%-5.32%, respectively. To evaluate the performance of the newly developed assay, a total of 36 clinical samples suspected of PPR were screened for the presence of PPRV in parallel with conventional RT-PCR. The real time RT-PCR assay detected PPRV in 30 (83.3%) of clinical samples compared to 16 (44.4%) by conventional RT-PCR.
  
  
  CONCLUSIONS
  The two-step SYBR Green I based real time RT-PCR assay reported here is highly sensitive, specific, reproducible and rapid for detection and quantification of PPRV nucleic acids.
authors.affiliation: College of Veterinary Medicine, Jigjiga University, Jijiga, Ethiopia. tsegalem.abera@gmail.com.
authors.forename: Tsegalem
authors.initials: T
authors.lastname: Abera
authors.forename: Ardhanary
authors.initials: A
authors.lastname: Thangavelu
authors.forename: Navamani Daniel
authors.initials: ND
authors.lastname: Joy Chandran
authors.forename: Angamuthu
authors.initials: A
authors.lastname: Raja
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D003959:Diamines; D015511:Goat Diseases / Q000175:diagnosis / Q000821:virology; D006041:Goats; D009930:Organic Chemicals; D029021:Peste-des-Petits-Ruminants / Q000175:diagnosis / Q000821:virology*; D018107:Peste-des-petits-ruminants virus / Q000302:isolation & purification*; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis / Q000821:virology
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I