A Dekonenko1, M S Ibrahim, C S Schmaljohn. 1. Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702-5011, USA.
Abstract
BACKGROUND: Hantaviruses cause two serious human diseases: hantavirus pulmonary syndrome and hemorrhagic fever with renal syndrome. At least nine hantaviruses are known to be pathogenic for humans and numerous others, with unknown disease potential, have been detected in rodents. Assays to quickly identify specific hantaviruses would be useful both for clinical diagnosis and in risk assessment studies. OBJECTIVES: The goal of our study was to develop and test a specific and sensitive PCR-based assay for identification and differentiation of hantaviruses. STUDY DESIGN: We developed an assay that combined RNA-PCR amplification and colorimetric enzymatic detection to identify representative European, Asian, and north American hantaviruses. RNAs from 18 hantavirus strains of nine species were amplified in the presence of digoxigenin-dUTP by using a single pair of oligonucleotide primers and polymerase chain reaction (PCR) performed by using rTth DNA polymerase. Digoxigenin-labeled PCR products were hybridized in solution to virus type-specific biotinilated probes, captured onto streptavidin-coated microtiter plates and detected by horseradish peroxidase-labeled anti-digoxigenin antibodies and a chromogenic substrate. RESULTS AND CONCLUSIONS: The assay correctly identified each homologous virus type tested. The detection limit of the assay was approximately 15 PFU or at least 50 copies of the viral genome. The assay is simple and strain-specific and is adaptable for automation, making it more practical than other available techniques for accurate and reliable diagnosis and typing of hantaviruses.
BACKGROUND: Hantaviruses cause two serious human diseases: hantavirus pulmonary syndrome and hemorrhagic fever with renal syndrome. At least nine hantaviruses are known to be pathogenic for humans and numerous others, with unknown disease potential, have been detected in rodents. Assays to quickly identify specific hantaviruses would be useful both for clinical diagnosis and in risk assessment studies. OBJECTIVES: The goal of our study was to develop and test a specific and sensitive PCR-based assay for identification and differentiation of hantaviruses. STUDY DESIGN: We developed an assay that combined RNA-PCR amplification and colorimetric enzymatic detection to identify representative European, Asian, and north American hantaviruses. RNAs from 18 hantavirus strains of nine species were amplified in the presence of digoxigenin-dUTP by using a single pair of oligonucleotide primers and polymerase chain reaction (PCR) performed by using rTth DNA polymerase. Digoxigenin-labeled PCR products were hybridized in solution to virus type-specific biotinilated probes, captured onto streptavidin-coated microtiter plates and detected by horseradish peroxidase-labeled anti-digoxigenin antibodies and a chromogenic substrate. RESULTS AND CONCLUSIONS: The assay correctly identified each homologous virus type tested. The detection limit of the assay was approximately 15 PFU or at least 50 copies of the viral genome. The assay is simple and strain-specific and is adaptable for automation, making it more practical than other available techniques for accurate and reliable diagnosis and typing of hantaviruses.