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

Abstract

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 Mamoré (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-nested RT-PCR. There was no amplification of either negative samples or samples positive to other pathogens. To assess the protocol for clinical sensitivity, specificity and general accuracy values, both assays were used to test two groups of samples: one comprising patients with disease (n = 50) and other containing samples from healthy individuals (n = 50), according to IgM-ELISA results. A third group of samples (n = 27) infected with other pathogens were tested for specificity analysis. RT-qPCR was more sensitive than semi-nested RT-PCR, being able to detect three samples undetected by conventional RT-PCR. RT-qPCR clinical sensitivity, specificity and general accuracy values were 92.5%, 100% and 97.63%, respectively. Thus, the assays developed in this study were able to detect the four Brazilian Amazon hantaviruses with good specificity and sensitivity, and may become powerful tools in diagnostic, surveillance and research applications of these and possibly other hantaviruses.

Fig 1. Comparison between amplification efficiencies of hantavirus RT-qPCR in singleplex and multiplex with MS2 EIC formats.

Amplification plot and Standard Curve for each assay format are depicted. Threshold was fixed where the amplification efficiency was higher, inside the exponential phase of each amplification plot in logarithmic scale, with 0.3481 ΔRn in singleplex (A) and 0.4492 ΔRn in multiplex (B). Ct variation between singleplex and multiplex was within 1 Ct. Standard curves were constructed with seven 1:10 dilutions ranging from 10⁷ copies to 10¹ copies of in vitro RNA. Amplification efficiency was 97.6% for singleplex (R²:0.999) (A) and 97.7% for multiplex (R²:0.998) (B). Ct: Cycle threshold, HTN M: hantavirus RT-qPCR multiplex format, HTN S: hantavirus RT-qPCR singleplex format.

Fig 2. Agarose gel electrophoresis of hantavirus Semi-nested RT-PCR amplification products from clinical samples positive for IgM-ELISA.

Agarose gel (1.5%) stained with SYBR safe dye were run for 50 min at 120V in TBE buffer. Each lane was loaded with 5μL of sample diluted in BlueJuice^™ Gel Loading Buffer (Invitrogen). NTC: No template Control, S1-6: sample 1–6, PC: Positive Control, MW: Molecular weight (100bp DNA Ladder—Invitrogen).