title: Development of a fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification assay for rapid detection of seasonal Japanese B encephalitis outbreaks in pigs.
abstract: The standardization and validation of a one-step, single-tube, accelerated fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the NS3 gene of Japanese B encephalitis virus (JEV) is described for rapid, simple, and high-throughput detection of JEV. The amplification can be completed in 35 min under isothermal conditions at 63°C by employing a set of six primers targeting the NS3 gene of JEV. The RT-LAMP assay described demonstrated high sensitivity for detecting JEV, with a detection limit in swine samples of 8.13 PFU/ml. The specificity of the selected primer sets was established by cross-reactivity studies with pathogens that exhibit similar clinical signs and testing of samples from healthy animals. The clinical applicability of the RT-LAMP assay was validated using either spiked samples or samples from seasonal outbreaks. The comparative evaluation of the RT-LAMP assay revealed 79.59 % concordance with conventional RT-PCR targeting the E gene of JEV. The RT-LAMP assay reported here is a valuable tool for rapid real-time and high-throughput seasonal infection surveillance and quarantine after outbreak through blood sampling by using ordinary real-time PCR thermocyclers without purchasing an expensive Loopamp real-time turbidimeter.
authors.affiliation: Guangdong Inspection and Quarantine Technology Center, Guangzhou, Guangdong 510623, China. gzvettian@163.com
authors.forename: C J
authors.initials: CJ
authors.lastname: Tian
authors.forename: Z X
authors.initials: ZX
authors.lastname: Lin
authors.forename: X M
authors.initials: XM
authors.lastname: He
authors.forename: Q
authors.initials: Q
authors.lastname: Luo
authors.forename: C B
authors.initials: CB
authors.lastname: Luo
authors.forename: H Q
authors.initials: HQ
authors.lastname: Yu
authors.forename: R
authors.initials: R
authors.lastname: Chen
authors.forename: X W
authors.initials: XW
authors.lastname: Wu
authors.forename: D Z
authors.initials: DZ
authors.lastname: Zhu
authors.forename: Z J
authors.initials: ZJ
authors.lastname: Ren
authors.forename: Y Z
authors.initials: YZ
authors.lastname: Bi
authors.forename: J
authors.initials: J
authors.lastname: Ji
mesh_terms: D000818:Animals; D002681:China / Q000453:epidemiology; D017931:DNA Primers; D004196:Disease Outbreaks / Q000662:veterinary*; D004664:Encephalitis Virus, Japanese / Q000235:genetics* / Q000302:isolation & purification*; D004672:Encephalitis, Japanese / Q000175:diagnosis / Q000453:epidemiology / Q000662:veterinary* / Q000821:virology; D005456:Fluorescent Dyes; D025202:Molecular Diagnostic Techniques / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020365:RNA Helicases / Q000235:genetics; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000235:genetics; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases