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Simultaneous detection of West Nile and Japanese encephalitis virus RNA by duplex TaqMan RT-PCR.
J Virol Methods. 2013 Nov; 193(2):554-7.JV

Abstract

West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important mosquito-borne viruses of the Flaviviridae family, associated with encephalitis, mainly in humans and horses. WNV is also pathogen for many bird species. The incidence of human and animal WNV infections in Europe has risen, mostly in recent years, and JEV was detected in 2011 in mosquitoes collected in Italy and may emerge in Europe in the same way as other flaviviruses had emerged recently (USUTU and Bagaza virus) and should be regarded as a potential threat to public health. Prompt identification and discrimination between WNV and JEV provides critical epidemiological data for prevalence studies and public and animal health management policies. Here we describe a quantitative one-step duplex TaqMan RT-PCR, targeting non-structural protein 2A gene (NS2A-qRT-PCR), based on only one primer pair and two probes for differential diagnosis of WNV and JEV. Also this assay enables the detection of both WNV lineages (WNV-1 and WNV-2). To access the specificity of NS2A-qRT-PCR a panel of different arboviruses were used. The assay was shown to be specific for both WNV lineages (WNV-1 and WNV-2), WNV related Kunjin virus and JEV, since no cross-reactions were observed with other tested arboviruses. Sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA from WNV and JEV. The duplex NS2A-qRT-PCR assay was shown to be very sensitive, being able to detect 10 copies of WNV and JEV RNA. This assay is a suitable tool for the diagnosis of WNV and JEV, and provides a valuable addition to the methods currently available for routine diagnosis of these zoonoses and for surveillance studies.

Authors+Show Affiliations

INIAV, Rua General Morais Sarmento, 1500-311 Lisboa, Portugal.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Evaluation Study
Journal Article

Language

eng

PubMed ID

23892127

Citation

Barros, Silvia C., et al. "Simultaneous Detection of West Nile and Japanese Encephalitis Virus RNA By Duplex TaqMan RT-PCR." Journal of Virological Methods, vol. 193, no. 2, 2013, pp. 554-7.
Barros SC, Ramos F, Zé-Zé L, et al. Simultaneous detection of West Nile and Japanese encephalitis virus RNA by duplex TaqMan RT-PCR. J Virol Methods. 2013;193(2):554-7.
Barros, S. C., Ramos, F., Zé-Zé, L., Alves, M. J., Fagulha, T., Duarte, M., Henriques, M., Luís, T., & Fevereiro, M. (2013). Simultaneous detection of West Nile and Japanese encephalitis virus RNA by duplex TaqMan RT-PCR. Journal of Virological Methods, 193(2), 554-7. https://doi.org/10.1016/j.jviromet.2013.07.025
Barros SC, et al. Simultaneous Detection of West Nile and Japanese Encephalitis Virus RNA By Duplex TaqMan RT-PCR. J Virol Methods. 2013;193(2):554-7. PubMed PMID: 23892127.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Simultaneous detection of West Nile and Japanese encephalitis virus RNA by duplex TaqMan RT-PCR. AU - Barros,Silvia C, AU - Ramos,Fernanda, AU - Zé-Zé,Líbia, AU - Alves,Maria J, AU - Fagulha,Teresa, AU - Duarte,Margarida, AU - Henriques,Margarida, AU - Luís,Tiago, AU - Fevereiro,Miguel, Y1 - 2013/07/25/ PY - 2013/01/23/received PY - 2013/07/05/revised PY - 2013/07/12/accepted PY - 2013/7/30/entrez PY - 2013/7/31/pubmed PY - 2014/4/20/medline KW - Diagnosis KW - Duplex RT-PCR KW - JEV KW - WNV SP - 554 EP - 7 JF - Journal of virological methods JO - J Virol Methods VL - 193 IS - 2 N2 - West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important mosquito-borne viruses of the Flaviviridae family, associated with encephalitis, mainly in humans and horses. WNV is also pathogen for many bird species. The incidence of human and animal WNV infections in Europe has risen, mostly in recent years, and JEV was detected in 2011 in mosquitoes collected in Italy and may emerge in Europe in the same way as other flaviviruses had emerged recently (USUTU and Bagaza virus) and should be regarded as a potential threat to public health. Prompt identification and discrimination between WNV and JEV provides critical epidemiological data for prevalence studies and public and animal health management policies. Here we describe a quantitative one-step duplex TaqMan RT-PCR, targeting non-structural protein 2A gene (NS2A-qRT-PCR), based on only one primer pair and two probes for differential diagnosis of WNV and JEV. Also this assay enables the detection of both WNV lineages (WNV-1 and WNV-2). To access the specificity of NS2A-qRT-PCR a panel of different arboviruses were used. The assay was shown to be specific for both WNV lineages (WNV-1 and WNV-2), WNV related Kunjin virus and JEV, since no cross-reactions were observed with other tested arboviruses. Sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA from WNV and JEV. The duplex NS2A-qRT-PCR assay was shown to be very sensitive, being able to detect 10 copies of WNV and JEV RNA. This assay is a suitable tool for the diagnosis of WNV and JEV, and provides a valuable addition to the methods currently available for routine diagnosis of these zoonoses and for surveillance studies. SN - 1879-0984 UR - https://www.unboundmedicine.com/medline/citation/23892127/Simultaneous_detection_of_West_Nile_and_Japanese_encephalitis_virus_RNA_by_duplex_TaqMan_RT_PCR_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0166-0934(13)00279-6 DB - PRIME DP - Unbound Medicine ER -