Method Description

This Heartland virus (HRTV) real-time reverse transcription polymerase chain reaction (RT-PCR) laboratory-developed test is based on primer and probe sequences reported by Savage et al. (CDC assay, 1). It provides qualitative detection of HRTV RNA from serum and CSF of patients with suspected infection. HRTV RNA in clinical specimens is first extracted using the Roche MagNA Pure 96 instrument. RT-PCR testing is then performed on the Roche LightCycler 480 II (LC480).

This RT-PCR assay employs oligonucleotide forward and reverse primers and a TaqMan hydrolysis probe specific to the small segment of the non-structural protein region of Heartland virus. A reverse transcription step is first performed to convert RNA to complementary DNA (cDNA). The primers then bind to the target cDNA sequence and facilitate amplification of an 86 base pair amplicon product during PCR. Using the LC480 software, analysis of the resultant amplification curves is performed to allow for detection of HRTV RNA. Generation of an amplification curve and a crossing point (Cp) value indicates the presence of HRTV RNA in the specimen and may be used to support the diagnosis of acute HRTV infection in the appropriate clinical and epidemiologic setting.(Unpublished Mayo method.)