A real-time RT-PCR for rapid detection and quantification of mosquito-borne alphaviruses

Abstract

Mosquito-borne alphaviruses are widely distributed throughout the world, causing important human illnesses. Therefore, the development of methods to enable early diagnosis of infections by alphavirus is essential. We show here the development and evaluation of a quantitative real-time RT-PCR using genus-specific primers to the nsP1 viral gene of all mosquito-borne alphaviruses. The specificity and sensitivity of the assay were tested using seven alphaviruses and RNA transcribed from Venezuelan equine encephalitis virus. The detection limits of real-time RT-PCR ranged from 10 to 76 copies per ml. The melting temperature (T M ) values for amplification of the alphavirus genomes were 83.05°C and 85.28°C. Interestingly, the assay suggested the possibility the arthritogenic alphaviruses with T M peaks of 84.83 to 85.28°C and encephalitic alphaviruses of 83.34°C to 84.68°C could be discriminated both diseases. Real-time RT-PCR may prove very useful for the screening and preliminary diagnosis in outbreaks and surveillance studies as well as for measuring the viral load in pathogenesis studies.

Venezuelan equine encephalitis complex, Madariaga and Eastern equine encephalitis viruses genome detection in human and mosquito populations

“…Over the past 40 years, various molecular assays have been developed for the detection of VEEV and VEE complex viruses [5,[11][12][13][14][15][16][17][18]25]. Many involve conventional or nested RT-PCRs that employ pan-alphavirus primers to amplify a large region of the genome, with subsequent species differentiation using specific nested primers, biotinylated probes, sequencing, or melt-curve analysis [11][12][13][14][15][16][17]. Vina-Rodriguez et al, reported an rRT-PCR that was designed from an alignment of 33 VEEV sequences [18].…”

“…The VEEV complex, MADV and EEEV viruses have been detected throughout the Americas and may account for a significant proportion of non-dengue acute febrile illness (2, 3, 5, 9). Although several assays have been developed for the molecular detection of VEEV subtypes and VEEV complex (14-20), many are laborious and time-consuming based in multiple PCR rounds or posterior genome sequencing that can be only implemented in laboratories with appropriate facilities (14)(15)(16)(17)(18)(19)(20). Co-circulation and the potential for co-infection with these viruses further complicates their detection, particularly when they present similar clinical presentations and there is a lack of available and convenient methods for detection of VEEV complex viruses and MADV (19).…”

“…Antigenbased methods are unavailable, and although serology can confirm the diagnosis, this requires paired acute and convalescent samples [2]. Current molecular tests lack optimal performance metrics characteristics necessary for routine diagnostic testing [10][11][12][13][14][15][16]. Molecular assay designs have been complicated by the genetic variability of the VEE complex viruses, which comprise of at least 14 different viral subtypes [1].…”

“…Antigen-based methods are currently unavailable, and although serology can confirm the diagnosis, this requires paired acute and convalescent samples (1,5). Current molecular tests lack optimal performance characteristics necessary for routine diagnostic testing (13)(14)(15)(16)(17)(18)(19).…”

Avaliação Da Qualidade Bacteriológica De Sucos Detox/Verdes

“…of Mayaro, and highlight the need for a comprehensive look, such as the One Health vision, for a better management of this threat in the country. O gene codificante da proteína não estrutural 1 (nsP1), é uma região do genoma bem conservada entre os Alfavírus, e por esta razão tem sido alvo de estudos para o diagnóstico de alfaviroses, bem como a identificação de Alfavírus ainda desconhecidos (ROMEIRO, M. F. et al, 2016).…”