title: Development of multiplex PCR-ligase detection reaction assay for detection of West Nile virus.
abstract: We have developed a novel multiplex reverse transcription-PCR ligase detection reaction (RT-PCR/LDR) assay for the detection of West Nile virus (WNV) in both clinical and mosquito pool samples. The method relies on the amplification of three different genomic regions, one in the coding sequence of nonstructural protein NS2a and two in nonstructural protein NS5, to minimize the risk of detection failure due to genetic variation. The sensitivity of the PCR is complemented by the high specificity of the LDR step, and the detection of the LDR products can be achieved with capillary electrophoresis (CE) or a universal DNA microarray. We evaluated the limit of detection by both one-step and two-step multiplex RT-PCR/LDR/CE approaches, which reached, respectively, 0.005 and 0.017 PFU. The assay demonstrated 99% sensitivity when mosquito pool samples were tested and 100% sensitivity with clinical samples when the one-step approach was used. The broad strain coverage was confirmed by testing 34 WNV isolates belonging to lineages 1 and 2, and the high specificity of the assay was determined by testing other flaviviruses, as well as negative mosquito pool and clinical samples. In summary, the multiplex RT-PCR/LDR assay could represent a valuable complement to WNV serological diagnosis, especially in early symptomatic patients. In addition, the multiplexing capacity of the technique, which can be coupled to universal DNA microarray detection, makes it an amenable tool to develop a more comprehensive assay for viral pathogens.
authors.affiliation: Department of Medicine, Division of International Medicine and Infectious Diseases, Weill Medical College of Cornell University, New York, New York 10021, USA.
authors.forename: S
authors.initials: S
authors.lastname: Rondini
authors.forename: M R
authors.initials: MR
authors.lastname: Pingle
authors.forename: S
authors.initials: S
authors.lastname: Das
authors.forename: R
authors.initials: R
authors.lastname: Tesh
authors.forename: M S
authors.initials: MS
authors.lastname: Rundell
authors.forename: J
authors.initials: J
authors.lastname: Hom
authors.forename: S
authors.initials: S
authors.lastname: Stramer
authors.forename: K
authors.initials: K
authors.lastname: Turner
authors.forename: S N
authors.initials: SN
authors.lastname: Rossmann
authors.forename: R
authors.initials: R
authors.lastname: Lanciotti
authors.forename: E G
authors.initials: EG
authors.lastname: Spier
authors.forename: J
authors.initials: J
authors.lastname: Muñoz-Jordán
authors.forename: D
authors.initials: D
authors.lastname: Larone
authors.forename: E
authors.initials: E
authors.lastname: Spitzer
authors.forename: F
authors.initials: F
authors.lastname: Barany
authors.forename: L M
authors.initials: LM
authors.lastname: Golightly
mesh_terms: D000818:Animals; D009033:Culicidae / Q000821:virology; D011088:DNA Ligases / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D019075:Electrophoresis, Capillary; D006801:Humans; D046228:Microarray Analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014901:West Nile Fever / Q000175:diagnosis* / Q000821:virology; D014902:West Nile virus / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D017361:Viral Nonstructural Proteins; D011088:DNA Ligases