Taqman fast virus 1 step master mix rt qpcr kit

ZIKV RNA was isolated from both fluid and tissue samples as previously described [39 (link)]. Viral RNA was then quantified using a highly sensitive RT-qPCR assay based on the one developed by Lanciotti et al. [57 (link)], though the primers were modified with degenerate bases at select sites to accommodate African-lineage Zika viruses. RNA was reverse-transcribed and amplified using the TaqMan Fast Virus 1-Step Master Mix RT-qPCR kit (Life Technologies, Carlsbad, CA, USA) on the LC96 instrument (Roche, Indianapolis, IN, USA), and quantified by interpolation onto a standard curve made up of serial 10-fold dilutions of in vitro transcribed RNA. RNA for this standard curve was transcribed from a plasmid containing an 800 bp region of the Zika virus genome that is targeted by the RT-qPCR assay. The final reaction mixtures contained 150 ng random primers (Promega, Madison, WI), 600 nM each primer and 100 nM probe. Primer and probe sequences are as follows: forward primer: 5’- CGYTGCCCAACACAAGG-3’, reverse primer: 5′-CCACYAAYGTTCTTTTGCABACAT-3′ and probe: 5′-6-carboxyfluorescein-AGCCTACCTTGAYAAGCARTCAGACACYCAA. The limit of detection for fluids (plasma, extraembryonic coelomic fluid, amniotic fluid, CSF) with this assay is 150 copies/ml. The theoretical limit of detection for the tissues is 3 copies/mg [23 (link)].

ZIKV RNA was isolated from both fluid and tissue samples as previously described [17 (link)]. Viral RNA was then quantified using a highly sensitive RT-qPCR assay based on the one developed by Lanciotti et al. [25 (link)], though the primers were modified with degenerate bases at select sites to accommodate African-lineage Zika viruses. RNA was reverse-transcribed and amplified using the TaqMan Fast Virus 1-Step Master Mix RT-qPCR kit (Life Technologies, Carlsbad, CA) on the LightCycler 480 or LC96 instrument (Roche, Indianapolis, IN), and quantified by interpolation onto a standard curve made up of serial tenfold dilutions of in vitro transcribed RNA. RNA for this standard curve was transcribed from a plasmid containing an 800 bp region of the Zika virus genome that is targeted by the RT-qPCR assay. The final reaction mixtures contained 150 ng random primers (Promega, Madison, WI), 600 nM each primer and 100 nM probe. Primer and probe sequences are as follows: forward primer: 5’- CGYTGCCCAACACAAGG-3’, reverse primer: 5′-CCACYAAYGTTCTTTTGCABACAT-3′ and probe: 5′-6-carboxyfluorescein-AGCCTACCTTGAYAAGCARTCAGACACYCAA. The limit of detection for fluids (plasma, amniotic fluid, CSF) with this assay is 150 copies/ml. The theoretical limit of detection for the tissues is 3 copies/mg [18 (link)].

Viral RNA was isolated from plasma using the Maxwell Viral Total Nucleic Acid Purification kit on the Maxwell 48 RSC instrument (Promega). Viral RNA was then quantified using a highly sensitive Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay based on the one developed by Lanciotti et al. (2008), though the primers were modified to accommodate both Asian- and African-lineage Zika viruses. RNA was reverse transcribed and amplified using the TaqMan Fast Virus 1-Step Master Mix RT-qPCR kit (LifeTechnologies) on the LightCycler 480 (Roche) and quantified by interpolation onto a standard curve made up of serial 10-fold dilutions of in vitro-transcribed RNA. RNA for this standard curve was transcribed from a plasmid containing an 800-bp region of the Zika virus genome that is targeted by the RT-qPCR assay. The final reaction mixtures contained 150 ng random primers (Promega), 600 nM each primer, and 100 nM probe. Primer and probe sequences are as follows: forward primer: 5′-CGYTGCCCAACACAAGG-3′, reverse primer: 5′-CCACYAAYGTTCTTTTGCABACAT-3′ and probe: 5′−6-carboxyfluorescein-AGCCTACCTTGAYAAGCARTCAGACACYCAA-BHQ1-3′. The reactions cycled with the following conditions: 50°C for 5 minutes, 95°C for 20 seconds followed by 50 cycles of 95°C for 15 seconds, and 60°C for 1 min. The limit of detection of this assay is 150 copies/mL.