Development of a one-step SYBR Green I real-time RT-PCR assay for the detection and quantitation of Araraquara and Rio Mamore hantavirus.
Hantaviruses are members of the family Bunyaviridae and are an emerging cause of disease worldwide with high lethality in the Americas. In Brazil, the diagnosis for hantaviruses is based on immunologic techniques associated with conventional RT-PCR. A novel one-step SYBR Green real-time RT-PCR was developed for the detection and quantitation of Araraquara (ARAV) and Rio Mamore hantavirus (RIOMV). The detection limit of assay was 10 copies/μL of RNA in vitro transcribed of segment S. The specificity of assay was evaluated by melting curve analysis, which showed that the Araraquara virus amplified product generated a melt peak at 80.83 ± 0.89 °C without generating primer-dimers or non-specific products. The assay was more sensitive than conventional RT-PCR and we detected two samples undetected by conventional RT-PCR. The one-step SYBR Green real-time quantitative RT-PCR is specific, sensible and reproducible, which makes it a powerful tool in both diagnostic applications and general research of ARAV and RIOMV and possibly other Brazilian hantaviruses.
Virology Research Center, School of Medicine in Ribeirão Preto, University of São Paulo, Av. Bandeirantes n.3900, Monte Alegre, Ribeirão Preto, São Paulo14049-900, Brazil. alexmmachado@yahoo.com.br
Alex Martins
AM
Machado
William Marciel
WM
de Souza
Michelly
M
de Pádua
Aline Rafaela
AR
da Silva Rodrigues Machado
Luiz Tadeu Moraes
LT
Figueiredo
D052160:Benzothiazoles; D001938:Brazil; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D003959:Diamines; D005456:Fluorescent Dyes / Q000737:chemistry; D006476:Orthohantavirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D018778:Hantavirus Infections / Q000175:diagnosis / Q000821:virology*; D006801:Humans; D009930:Organic Chemicals / Q000737:chemistry; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I