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Rapid Detection and Quantification of Japanese Encephalitis Virus by Real‐Time Reverse Transcription Loop‐Mediated Isothermal Amplification
Author(s) -
Toriniwa Hiroko,
Komiya Tomoyoshi
Publication year - 2006
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.2006.tb03804.x
Subject(s) - japanese encephalitis , reverse transcription loop mediated isothermal amplification , virus , virology , loop mediated isothermal amplification , biology , reverse transcriptase , reverse transcription polymerase chain reaction , tick borne encephalitis virus , real time polymerase chain reaction , flavivirus , dengue virus , encephalitis , titer , microbiology and biotechnology , polymerase chain reaction , dna , gene , messenger rna , genetics , biochemistry
We established a rapid, quantitative real‐time reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) assay targeting the envelope gene of Japanese encephalitis virus. The RT‐LAMP enabled us to detect the target product within 1 hr by only reacting reverse transcriptase and Bst DNA polymerase in a single tube at an isothermal temperature. The detection sensitivity of the RT‐LAMP for Japanese encephalitis virus was 1 PFU, similar to that of conventional reverse transcription‐polymerase chain reaction (RT‐PCR). Flaviviruses of the Japanese encephalitis virus group, such as Dengue virus and West Nile virus, could not be detected. This confirmed the specificity of the RT‐LAMP assay for Japanese encephalitis virus. A standard curve was constructed by plotting viral titer against the time for virus detection by the RT‐LAMP, validating the quantitative accuracy of the assay. In addition, the amount of virus estimated by RT‐LAMP was strongly correlated ( r =0.902) with that determined by plaque assay, a conventional method for virus quantification. These results indicate that the RT‐LAMP assay established in this study is specific for Japanese encephalitis virus, and allows more rapid detection and quantification of the virus.