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

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Romeiro, Marilia Farignoli ; de Souza, William Marciel ; Tolardo, Aline Lavado ; Vieira, Luiz Carlos ; Henriques, Dyana Alves ; de Araujo, Jansen ; Hassegawa Siqueira, Carlos Eduardo ; Colombo, Tatiana Elias ; Aquino, Victor Hugo ; Lopes da Fonseca, Benedito Antonio ; de Morais Bronzoni, Roberta Vieira ; Nogueira, Mauricio Lacerda ; Durigon, Edison Luiz ; Moraes Figueiredo, Luiz Tadeu

• Document type: Journal article
• Source: ARCHIVES OF VIROLOGY; v. 161, n. 11, p. 3171-3177, NOV 2016.
• Web of Science Citations: 1

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 A degrees C and 85.28 A degrees C. Interestingly, the assay suggested the possibility the arthritogenic alphaviruses with T-M peaks of 84.83 to 85.28 A degrees C and encephalitic alphaviruses of 83.34 A degrees C to 84.68 A degrees 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. (AU)

• FAPESP's process: 13/02256-2 - Development of Real Time RT-PCR for diagnosis of infections by Piry virus and other vesiculovirus
• Grantee: Aline Lavado Tolardo
• Support type: Scholarships in Brazil - Master
• FAPESP's process: 12/02836-6 - Development of Real Time RT-PCR Techniques for arbovirus that cause human disease and nucleotide sequencing identification of them
• Grantee: Marilia Farignoli Romeiro
• Support type: Scholarships in Brazil - Master
• FAPESP's process: 13/14929-1 - Detection of arbovirus and other RNA viruses by real time RT-PCR and study of the viroma
• Grantee: Luiz Tadeu Moraes Figueiredo
• Support type: Regular Research Grants
• FAPESP's process: 12/24150-9 - Research of virus in wild rodents, mosquitoes and ticks
• Grantee: William Marciel de Souza
• Support type: Scholarships in Brazil - Doctorate
• FAPESP's process: 14/20851-8 - Study about Alphavirus and Flavivirus infections in samples of blood bank
• Grantee: Marilia Farignoli Romeiro
• Support type: Scholarships in Brazil - Doctorate