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- 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.
- Authors
Tian, C.; Lin, Z.; He, X.; Luo, Q.; Luo, C.; Yu, H.; Chen, R.; Wu, X.; Zhu, D.; Ren, Z.; Bi, Y.; Ji, J.
- 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.
- Subjects
FLUORESCENT dyes; BIOLOGICAL assay; JAPANESE B encephalitis; DISEASE outbreaks; LABORATORY swine; CROSS reactions (Immunology); SYMPTOMS; COMPARATIVE studies
- Publication
Archives of Virology, 2012, Vol 157, Issue 8, p1481
- ISSN
0304-8608
- Publication type
Article
- DOI
10.1007/s00705-012-1330-y