title: Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India.
abstract: Bluetongue virus (BTV) infects all ruminants, including cattle, goats and camelids, causing bluetongue disease (BT) that is often severe in naïve deer and sheep. Reverse-transcription-loop-mediated-isothermal-amplification (RT-LAMP) assays were developed to detect eastern or western topotype of BTV strains circulating in India. Each assay uses four primers recognizing six distinct sequences of BTV genome-segment 1 (Seg-1). The eastern (e)RT-LAMP and western (w)RT-LAMP assay detected BTV RNA in all positive isolates that were tested (n=52, including Indian BTV-1, -2, -3, -5, -9, -10, -16, -21 -23, and -24 strains) with high specificity and efficiency. The analytical sensitivity of the RT-LAMP assays is comparable to real-time RT-PCR, but higher than conventional RT-PCR. The accelerated eRT-LAMP and wRT-LAMP assays generated detectable levels of amplified DNA, down to 0.216 fg of BTV RNA template or 108 fg of BTV RNA template within 60-90min respectively. The assays gave negative results with RNA from foot-and-mouth-disease virus (FMDV), peste des petits ruminants virus (PPRV), or DNA from Capripox viruses and Orf virus (n=10), all of which can cause clinical signs similar to BT. Both RT-LAMP assays did not show any cross-reaction among themselves. The assays are rapid, easy to perform, could be adapted as a 'penside' test making them suitable for 'front-line' diagnosis, helping to identify and contain field outbreaks of BTV.
authors.affiliation: Department of Animal Biotechnology, College of Veterinary Sciences, LLR University of Veterinary and Animal Sciences, Hisar, Haryana, India. Electronic address: sushilamaan105@gmail.com.
authors.forename: S
authors.initials: S
authors.lastname: Maan
authors.affiliation: Department of Animal Biotechnology, College of Veterinary Sciences, LLR University of Veterinary and Animal Sciences, Hisar, Haryana, India; Resource Faculty, Department of Animal Biotechnology, College of Veterinary Sciences, LLR University of Veterinary and Animal Sciences, Hisar, Haryana, India.
authors.forename: N S
authors.initials: NS
authors.lastname: Maan
authors.affiliation: Department of Animal Biotechnology, College of Veterinary Sciences, LLR University of Veterinary and Animal Sciences, Hisar, Haryana, India.
authors.forename: K
authors.initials: K
authors.lastname: Batra
authors.affiliation: Department of Animal Biotechnology, College of Veterinary Sciences, LLR University of Veterinary and Animal Sciences, Hisar, Haryana, India.
authors.forename: A
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Animal Biotechnology, College of Veterinary Sciences, LLR University of Veterinary and Animal Sciences, Hisar, Haryana, India.
authors.forename: A
authors.initials: A
authors.lastname: Gupta
authors.affiliation: Ella Foundation, Genome Valley Hyderabad, 500 078 T.S, India.
authors.forename: Panduranga P
authors.initials: PP
authors.lastname: Rao
authors.affiliation: National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Hebbal, Bengaluru 560024 K.A, India.
authors.forename: Divakar
authors.initials: D
authors.lastname: Hemadri
authors.affiliation: College of Veterinary Science, Acharya N.G. Ranga Agricultural University, Rajendra Nagar, Hyderabad 500 030, T.S, India.
authors.forename: Yella Narasimha
authors.initials: YN
authors.lastname: Reddy
authors.affiliation: The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking GU24 0NF Surrey, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Guimera
authors.affiliation: The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking GU24 0NF Surrey, United Kingdom.
authors.forename: M N
authors.initials: MN
authors.lastname: Belaganahalli
authors.affiliation: The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking GU24 0NF Surrey, United Kingdom.
authors.forename: P P C
authors.initials: PP
authors.lastname: Mertens
mesh_terms: D000818:Animals; D001819:Bluetongue / Q000175:diagnosis* / Q000821:virology; D001820:Bluetongue virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D017931:DNA Primers; D016679:Genome, Viral; D015511:Goat Diseases / Q000175:diagnosis* / Q000821:virology; D006041:Goats / Q000821:virology; D007194:India; D021141:Nucleic Acid Amplification Techniques / Q000379:methods* / Q000592:standards; D029021:Peste-des-Petits-Ruminants / Q000175:diagnosis / Q000821:virology; D018107:Peste-des-petits-ruminants virus / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D012756:Sheep / Q000821:virology; D013696:Temperature
chemical_list: D017931:DNA Primers
keywords: BTV; Eastern; Indian strains; Isothermal amplification; LAMP; Seg-1; Western