title: Genetic detection of peste des petits ruminants virus under field conditions: a step forward towards disease eradication.
abstract: BACKGROUND
  The devastating viral disease of small ruminants namely Peste des petits ruminants (PPR) declared as target for "Global Eradication" in 2015 by the Food and Agriculture Organization (FAO) and the World Organization for Animal Health (OIE). For a successful eradication campaign, molecular diagnostic tools are preferred for their specificity, efficacy and robustness to compliment prophylactic measures and surveillance methods. However, molecular tools have a few limitations including, costly equipment, multi-step template preparation protocols, target amplification and analysis that restrict their use to the sophisticated laboratory settings. As reverse transcription-loop mediated isothermal amplification assay (RT-LAMP) has such an intrinsic potential for point of care diagnosis, this study focused on the genetic detection of causative PPR virus (PPRV) in field conditions. It involves the use of a sample buffer that can precipitate out virus envelope and capsid proteins through ammonium sulphate precipitation and exposes viral RNA, present in the clinical sample, to the LAMP reaction mixture.
  
  
  RESULTS
  The test was evaluated using 11 PPRV cultures, and a total of 46 nasal swabs (n = 32 collected in the field outbreaks, n = 14 collected from experimentally inoculated animals). The RT-LAMP was compared with the reverse transcription-PCR (RT-PCR) and real-time quantitative RT-PCR (RT-qPCR) for its relative specificity, sensitivity and robustness. RT-LAMP detected PPRV in all PPRV cultures in or less than 30 min. Its detection limit was of 0.0001TCID50(tissue culture infective dose-50) per ml with 10-fold higher sensitivity than that of RT-PCR. In 59.4% of the field samples, RT-LAMP detected PPRV within 35-55 min. The analytical sensitivity and specificity of the RT-LAMP were equivalent to that of the RT-qPCR. The time of detection of PPRV decreased by at least forty minutes or 3-4 h in case of in the RT-LAMP as compared with the RT-qPCR and the RT-PCR, respectively.
  
  
  CONCLUSIONS
  The sensitive and specific RT-LAMP test developed in this study targeting a small fragment of the N gene of PPRV is a rapid, reliable and applicable molecular diagnostic test of choice under the field conditions. RT-LAMP requiring minimal training offers a very useful tool for PPR diagnosis especially during the "Global PPR Eradication Campaign".
authors.affiliation: National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. waqasasahrafnibge@gmail.com.|Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan. waqasasahrafnibge@gmail.com.
authors.forename: Waqas
authors.initials: W
authors.lastname: Ashraf
authors.affiliation: Animal Production and Health Section, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna, Austria.
authors.forename: Hermann
authors.initials: H
authors.lastname: Unger
authors.affiliation: National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.|Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan.
authors.forename: Sunaina
authors.initials: S
authors.lastname: Haris
authors.affiliation: National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.|Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan.
authors.forename: Ameena
authors.initials: A
authors.lastname: Mobeen
authors.affiliation: National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.|Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Farooq
authors.affiliation: National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.|Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Asif
authors.affiliation: National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. qk_5@yahoo.com.|Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan. qk_5@yahoo.com.
authors.forename: Qaiser Mahmood
authors.initials: QM
authors.lastname: Khan
mesh_terms: D000818:Animals; D003124:Colorimetry; D004196:Disease Outbreaks / Q000662:veterinary; D005470:Fluorometry; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D015511:Goat Diseases / Q000175:diagnosis / Q000453:epidemiology / Q000821:virology*; D006041:Goats; D007194:India / Q000453:epidemiology; D029021:Peste-des-Petits-Ruminants / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D018107:Peste-des-petits-ruminants virus / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis / Q000821:virology*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014776:Virus Cultivation
chemical_list: D014764:Viral Proteins
keywords: Nucleocapsid gene (N); Peste des petits ruminants virus (PPRV); Reverse-transcription loop mediated isothermal amplification (RT-LAMP); Robust diagnosis