Visual Detection of Murray Valley Encephalitis Virus by Reverse Transcription Loop-Mediated Isothermal Amplification *

3,# A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of Murray valley encephalitis virus (MVEV) infection. The reaction was performed in one step in a single tube at 63 °C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was 100 copies per reaction based on 10-fold dilutions of in vitro transcribed RNA derived from a synthetic MVEV DNA template. No cross-reaction was observed with other encephalitis-associated viruses. The assay was further evaluated using spiked cerebrospinal fluid sample with pseudotype virus containing the NS5 gene of MVEV. Murray valley encephalitis virus (MVEV) is a mosquito-borne Flavivirus (Flaviviridae: Flavivirus) which is closely related to Japanese encephalitis virus, West Nile virus and St. Louis encephalitis virus. MVEV is the most serious of the endemic arboviruses in Australia [1] . In 2010-2011, there were 16 confirmed human cases of Murray Valley encephalitis acquired in Australia [1-2] . The activity of MVEV in Australia is monitored by detection of seroconversions in flocks of sentinel chickens. The availability of the RT-PCR assay for the detection of MVEV provides additional opportunities to confirm the presence of this virus in clinical samples [3] . However, these methods might not be suitable in local ports of Entry-Exit Inspection and Quarantine or for field use because the methods are time-consuming and require the sophisticated instrumentation. There is a growing demand for a simple, rapid and sensitive molecular test for the inspection and quarantine testing in the local ports in China. Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method firstly described in 2000 [4] . It is a powerful gene amplification tool due to its simplicity, speed, specificity and cost-effectiveness and nowadays, this technique is being used increasingly for rapid detection and typing of emerging viruses [5-9] . In the present report, a sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of the MVEV infection. The NS5 gene of MVEV was used to distinguish the members of the genus Flavivirus and all the NS5 nucleotide sequences of MVEV available in GenBank were downloaded and compared. The most conserved segment within the NS5 gene of MVEV (corresponding to the nucleotide positions at 10504-10908, numbering based on the MVEV strain MVE-1-51, GenBank accession no. AF161266.1) was selected as the target. All primers were designed by a software program for LAMP primer design (Eiken Chemical Co. Ltd., Tokyo, Japan) and then subsequently validated by BLAST (http://www.ncbi. nlm.nih.gov/BLAST). All the primers were HPLC purified, as shown in Table 1.