Primer YFall-F Probe YFall-P Primer YFall-R

Transcription

1 Primer YFall-F Probe YFall-P Primer YFall-R FIG S1. Sequence alignment for the selection of the PCR primers and probe. The amplification primers (YFallF, YFallR) and probe (YFallP) in the new assays were designed to target regions of local high conservation among known strains. For this, available sequences for the 5 -non-coding region of the viral genome in the GenBank public database were compared in a multiple alignment. Non-conserved positions are shaded in colour. Degenerated positions in the primer sequences, shown at the top, are shaded grey.

2 Sample ID #2 #9 #12 #4 #14 #10 #5 #13 #1 #6 #11 #3 #8 #7 Flaviviruses Brazil viral load (GE/sample) Reference for the assay RT-PCR technique Target virus This article YFall RT-qPCR YFall RT-tHDA weak weak + ( ) TaqMan RT-PCR ( ) TaqMan RT-PCR (+) TaqMan RT-PCR ( ) (+) (1) TaqMan RT-PCR ( ) ( ) (+) TaqMan RT-PCR ( ) ( ) ( ) (+) TaqMan RT-PCR ( ) ( ) (+) TaqMan RT-PCR + + ND + + ( ) ( ) + ( ) ( ) (+) TaqMan RT-PCR ( ) ( ) + ( ) ( ) ( ) ( ) (+) (2) TaqMan RT-PCR ( ) ( ) PANFLAV SYBR-RT-PCR ( ) ( ) + ( ) ( ) + ( ) (+) (+) (+) PANFLAV Heminest RT-PCR (+)* PANFLAV (3) Heminest RT-PCR ( ) (+)* PANFLAV Heminest RT-PCR (+)* (+)* (+) PANFLAV (4) TaqMan RT-PCR (+) TaqMan RT-PCR (+) TaqMan RT-PCR ( ) ( ) ( ) ( ) ( ) (+) (5) TaqMan RT-PCR ( ) ( ) ( ) ( ) ( ) (+) TaqMan RT-PCR ( ) ( ) ( ) ( ) ( ) (+) TaqMan RT-PCR + + ND + + ( ) ( ) ( ) ( ) ( ) (+) TaqMan RT-PCR ( ) ( ) ( ) ( ) ( ) ( ) ( ) (+) TaqMan RT-PCR ( ) (+) In-house RT-nested PCR ( ) ( ) + ( ) ( ) ( ) ( ) (+) RT-nested PCR + ( ) ( ) + ( ) ( ) + ( ) + + (+) (+) (+) (+) (6) RT-nested PCR ( ) ( ) + ( ) + ( ) ( ) PANFLAV RT-nested PCR + + ND + ( ) + + ( ) ( ) + PANFLAV (7) RT-PCR ( ) ( ) ( ) ( ) ( ) (+) (8) TaqMan RT-PCR ( ) ( ) ( ) + (+) (+) (+) (9) RT-PCR ( ) ( ) + ( ) ( ) ( ) ( ) (+) PANFLAV (10) RT-nested PCR ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) RT-nested PCR + + ( ) ( ) ( ) ( ) PANFLAV Not provided TaqMan RT-PCR ( ) ( ) ( ) ( ) ( ) (+) RT-nested PCR + ( ) ( ) ( ) ( ) ND ( ) ( ) ND ( ) Brazil JEV, WNV SLEV, TBEV DENV 1 4 none none 2

3 3 TABLE S1 Comparison of assay performance between the RT-qPCR and RT-tHDA in this work and the protocols employed during a previous molecular diagnosis EQA, based on a common set of samples. The 32 sets of results from the 28 EQA participating laboratories are from reference (11). Each row represents the results from an individual laboratory applying the methods referenced in the first column. Positive samples involved varying amounts of RNA from strain, a wild-type Brazilian strain, and a wild-type strain. Two negative controls (#7 and #8) and two samples containing RNA from other flaviviruses (#3 and #11) were also included. Incorrect results are shaded grey. +, positive result; (+), false positive result;, correct negative result; ( ), false negative result; *, identified by sequencing; ND, not done; PANFLAV, panflavivirus;, yellow fever virus; SLEV, St. Louis encephalitis virus; JEV, Japanese encephalitis virus; WNV, West Nile virus; TBEV, tick borne encephalitis virus; DENV1 4, dengue virus strains 1 to 4. REFERENCES 1. Drosten, C., S. Gottig, S. Schilling, M. Asper, M. Panning, H. Schmitz, and S. Gunther Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-pcr. Journal of clinical microbiology 40:

4 4 2. Moureau, G., S. Temmam, J. P. Gonzalez, R. N. Charrel, G. Grard, and X. de Lamballerie A real-time RT-PCR method for the universal detection and identification of flaviviruses. Vector borne and zoonotic diseases 7: Scaramozzino, N., J. M. Crance, A. Jouan, D. A. DeBriel, F. Stoll, and D. Garin Comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-pcr assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences. Journal of clinical microbiology 39: Charlier, N., R. Molenkamp, P. Leyssen, J. Paeshuyse, C. Drosten, M. Panning, E. De Clercq, P. J. Bredenbeek, and J. Neyts Exchanging the yellow fever virus envelope proteins with Modoc virus prm and E proteins results in a chimeric virus that is neuroinvasive in SCID mice. Journal of virology 78: Bae, H. G., A. Nitsche, A. Teichmann, S. S. Biel, and M. Niedrig Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay. Journal of virological methods 110: Sanchez-Seco, M. P., D. Rosario, C. Domingo, L. Hernandez, K. Valdes, M. G. Guzman, and A. Tenorio Generic RT-nested-PCR for detection of flaviviruses using degenerated primers and internal control followed by sequencing for specific identification. Journal of virological methods 126:

5 5 7. Brown, T. M., G. J. Chang, C. B. Cropp, K. E. Robbins, and T. F. Tsai Detection of yellow fever virus by polymerase chain reaction. Clinical and diagnostic virology 2: Mantel, N., M. Aguirre, S. Gulia, Y. Girerd-Chambaz, S. Colombani, C. Moste, and V. Barban Standardized quantitative RT-PCR assays for quantitation of yellow fever and chimeric yellow fever-dengue vaccines. Journal of virological methods 151: Kuno, G Universal diagnostic RT-PCR protocol for arboviruses. Journal of virological methods 72: Heraud, J. M., D. Hommel, A. Hulin, V. Deubel, J. D. Poveda, J. L. Sarthou, and A. Talarmin First case of yellow fever in French Guiana since Emerging infectious diseases 5: Domingo, C., C. Escadafal, L. Rumer, J. A. Mendez, P. Garcia, A. A. Sall, A. Teichmann, O. Donoso-Mantke, and M. Niedrig First international external quality assessment study on molecular and serological methods for yellow Fever diagnosis. PloS one 7:e36291.