Development of a reliable dual-gene amplification RT-PCR assay for the detection of Turkey Meningoencephalitis virus in Turkey brain tissues

Bagaza virus and Israel turkey meningoencephalomyelitis virus are a single virus species

“…AF013363). Moreover, molecular diagnostic assays designed for ITV-infected turkeys targeting the NS5 gene (Davidson et al, 1998(Davidson et al, , 2000 or amplifying NS5 and E genes simultaneously (Davidson et al, 2012) 5isolate 105520 obtained in 2010 from a 10-week-old turkey (these five ITV isolates will be named, respectively, ITV-1 to ITV-5 hereafter). The sequencing strategy basically followed the original strategy employed for the BAGV full genome sequencing (Agüero et al, 2011), although some additional primers had to be specifically designed to complete the sequencing (primer and sequencing information available upon request).…”

Development of duplex dual-gene and DIVA real-time RT-PCR assays and use of feathers as a non-invasive sampling method for diagnosis ofTurkey Meningoencephalitis Virus

“…The NS5 gene is considered a conserved gene, because it is involved in virus replication, and the env gene, which is exposed on the virus external surface as the immunodominant protein, is more evolving genetically. Therefore, similar to our previous end-point dual-gene amplification (Davidson et al, 2012), we now designed the real-time dual-gene amplification to detect the stable gene, as a marker for the TMEV detection, and the evolving gene, the env gene, to indicate the appearance of a new virus variant TMEV.…”

“…End-point RT-PCR (epRT-PCR) was performed as described before (Davidson et al, 2012), with the following modifications that were included to match dual-gene duplex amplification by the rtRT-PCR, and to enable sensitivity comparison. Briefly, RT-PCR was performed with Thermo Scientific Verso 1-step RT-PCR ReddyMix kit, (ABgene Ltd., Surrey, UK) according to the manufacturer's instructions.…”

“…Since the env gene encodes for externally exposed and selection-prone immunodominant proteins, the diagnostic PCR was based on the relatively conserved NS replication flavivirus NS5 gene . To provide a detection assay for isolates in which possible genomic changes might have occurred in the env gene, a dual-gene end-point amplification RT-PCR, based on the NS5 and the TMEV envelope gene, was developed (Davidson et al, 2012).…”

A Duplex Quantitative Real-Time Reverse Transcription-PCR for Simultaneous Detection and Differentiation of Flaviviruses of the Japanese Encephalitis and Ntaya Serocomplexes in Birds

“…In 2010, BAGV emerged in Southern Spain (20) in an area where WNV and USUV were co-circulating in the same avian population (21). Its synonymous virus, ITV, also re-emerged in Israel in the same time period (22). Likewise, in other areas of the world, similar patterns of flavivirus emergence are being observed, particularly involving those belonging to the JE and Ntaya groups (7,23,24).…”