Advanced yellow fever virus genome detection in point-of-care facilities and reference laboratories

Abstract

Reported methods for the detection of the yellow fever viral genome are beset by limitations in sensitivity, specificity, strain detection spectra, and suitability to laboratories with simple infrastructure in areas of endemicity. We describe the development of two different approaches affording sensitive and specific detection of the yellow fever genome: a real-time reverse transcription-quantitative PCR (RT-qPCR) and an isothermal protocol employing the same primer-probe set but based on helicase-dependent amplification technology (RT-tHDA). Both assays were evaluated using yellow fever cell culture supernatants as well as spiked and clinical samples. We demonstrate reliable detection by both assays of different strains of yellow fever virus with improved sensitivity and specificity. The RT-qPCR assay is a powerful tool for reference or diagnostic laboratories with real-time PCR capability, while the isothermal RT-tHDA assay represents a useful alternative to earlier amplification techniques for the molecular diagnosis of yellow fever by field or point-of-care laboratories.

Fig 1

Performance of the isothermal YF RT-tHDA assay. Dilutions of plasma samples representing different strains of YFV (samples labeled 2, 9, 12, 4, 14, 10, 5, 13, 1, and 6 at the top of the image), other related flaviviruses (samples 11 and 3), and negative controls (samples 8 and 7) were subjected to the YFV RT-tHDA assay. The correct performance of the lateral-flow detection method is confirmed by the presence of the control line, C, in all test stripes. Positives samples are identified by the presence of the additional detection line, D.

Fig 2

Parallel detection of the YFV genome in clinical samples by the new RT-qPCR and RT-tHDA assays. The sample set consisted of urine samples from healthy YF vaccinees (samples 1 to 17, YFV strain 17D), a negative (water) control (sample 18), a negative urine control (sample 20), and one positive control (spiked urine, sample 19). In the RT-tHDA assay, a positive detection line was observed on the lateral-flow strip with 8 of the 9 samples that were positive by the more sensitive YF RT-qPCR assay. GE, genome equivalents; rxn, reaction.