Development of one-step quantitative reverse transcription PCR for the rapid detection of flaviviruses

Abstract

BackgroundThe genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses.MethodsA Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively.ResultsTwo degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10–100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays.ConclusionThe assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis.

Potential for Zika virus transmission through blood transfusion demonstrated during an outbreak in French Polynesia, November 2013 to February 2014

“…PCR assays for dengue would not detect the Zika viral genome. Pan-flavivirus assays and subsequent sequencing analysis can be used as an alternative screening test for possible ZIKAV infection [43,44]. Virus isolation is carried out for research purposes.…”

Japanese Encephalitis, Recent Perspectives on Virus Genome, Transmission, Epidemiology, Diagnosis and Prophylactic Interventions

“…The Pan-Flavi assay (TaqMan probe-based qRT-PCR) has been developed recently by targeting conserved NS5 gene region for a concurrent finding of different flaviviruses including YFV, WNV, TBEV, DENV, and JEV in a single-tube (Patel et al, 2013). JEV RT-LAMP assay has improved sensitivity compare to routine RT-PCR with equal sensitivity to qRT -PCR assay with the detection limits ranging between 0.1 and 10 plaque forming units (PFUs) of virus (Toriniwa & Komiya, 2006).…”

Development of multiplex suspension microarrays for the detection of alphaviruses and flaviviruses of public health significance

“…Additionally, the region has been the focus of previous studies to develop universal primers for amplification of flaviviruses and probes for use in the molecular assays (Pierre et al, 1994, Maher-Sturgess et al, 2008, Figueiredo et al, 1998, Tanaka, 1993, Pyke et al, 2004. A loss of sensitivity can be seen in assays using universal primers compared to specific primers, although this is not always the case (Domingo et al, 2011, Patel et al, 2013. However, utilising universal primers has the added advantage of the ability to test for multiple viruses in the one geographical region that cause very similar clinical signs; and can also to increase the size of the panel by inclusion of related viruses in the future.…”

“…For example, further studies to determine the effect the extraction technique has on assay sensitivity for detecting virus in mosquito pools could be conducted, as discussed in Section 2.4.3, or the effect of mosquito pool size could be investigated. PCR efficiency is a more difficult issue to address, as it is accepted in general that concensus primers will have a lower sensitivity than virus-specific primers (Moreau et al, 2007, Patel et al, 2013. However, virus-specific primers could be designed and multiplexed with the consensus primers in a manner similar to Patel et al (Patel et al, 2013), if further validation indicates that improved sensitivity for certain viruses should be investigated.…”

“…PCR efficiency is a more difficult issue to address, as it is accepted in general that concensus primers will have a lower sensitivity than virus-specific primers (Moreau et al, 2007, Patel et al, 2013. However, virus-specific primers could be designed and multiplexed with the consensus primers in a manner similar to Patel et al (Patel et al, 2013), if further validation indicates that improved sensitivity for certain viruses should be investigated. Hybridisation efficiency may be influenced by linker design, an avenue which was not explored in this study.…”