Development of RT-qPCR and semi-nested RT-PCR assays for molecular diagnosis of hantavirus pulmonary syndrome

by Bruno Tardelli Diniz Nunes, Maria Helena Rodrigues de Mendon ça, Darlene de Brito Simith, Adriana Freitas Moraes, Carla Conceição Cardoso, Ivy Tsuya Essashika Prazeres, Ana Alice de Aquino, Alessandra da Conceição Miranda Santos, Alice Louize Nunes Queiroz, Daniela Sueli Guerreiro Rodrigues, Regis Bruni Andriolo, Elizabeth Salbé Travassos da Rosa, Livia Carício Martins, Pedro Fernando da Costa Vasconcelos, Daniele Barbosa de Almeida Medeiros Hantavirus Pulmonary Syndrome is an, often fatal, emerging zoonotic disease in the Americas caused by hantaviruses (family:Hantaviridae). In Brazil, hantavirus routine diagnosis is based on serology (IgM-ELISA) while RT-PCR is often used to confirm acute infection. A Semi-nested RT-PCR and an internally controlled RT-qPCR assays were developed for detection and quantification of four hantaviruses strains circulating in the Brazilian Amazon: Anajatuba (ANAJV) and Castelo dos Sonhos (CASV) strains of Andes virus (ANDV) species; and Rio Mamor é (RIOMV) and Laguna Negra (LNV) strains of LNV species. A consensus region in the N gene of these hantaviruses was used to design the primer sets and a hydrolysis probe.In vitro transcribed RNA was diluted in standards with known concentration. MS2 bacteriophage RNA was detected together with hantavirus RNA as an exogenous control in a duplex reaction. RT-qPCR efficiency was around 100% and the limit of detection was 0.9 copies/ μL of RNA for RT-qPCR and 10 copies/μL of RNA for Semi-neste...
Source: PLoS Neglected Tropical Diseases - Category: Tropical Medicine Authors: Source Type: research