Taqman fast virus 1 step master mix kit

Viral RNA was extracted with QIAamp Viral RNA Mini Kit (Qiagen¯, Germany),
according to the manufacturer’s protocol and amplified by real-time RT-PCR, in a
RealPlex 4 Thermocycler (Eppendorf¯, Germany), using TaqMan¯ Fast Virus 1-Step
Master Mix kit (ThermoFisher¯, USA) and five different pairs of primers with
their respective probe, referred throughout this article as ZIKV A to ZIKV E
(Supplementary Table S1). The amount of each primer and probe used individually
per reaction was 125 nM, 2.5 µL of master mix, 3 µL of RNA and the final volume
was set to 10 µL. The cycling condition for all protocols was 10 min at 50°C to
ensure cDNA synthesis, 1 minute at 95°C to activate DNA polymerase and
inactivate the reverse transcriptase, and 45 cycles at 95°C for 15 s to denature
cDNA and 60°C for 45 s to primer annealing and extension. The ten-fold serial
dilution of plaque forming units (PFU) equivalents was used to test specificity
and sensitivity of primer and probe sets. Table S1 shows each primer and probe
sequence with its genome region and position, amplicon size, and their
respective reference.

Brain tissue was collected on day 13 after birth. The brains were lysed with sterile 1 × PBS and homogenized with a homogenizer work center (IKA^® T10 basic). Homogenates were cleared by centrifugation, and total RNA was extracted. According to the manufacturer's instructions, total RNA from culture, extract-containing organs in 1 × PBS was extracted using QIAamp Viral RNA (Qiagen^®). Quantitative RT-PCR was performed using the TaqMan^® Fast Virus 1-Step Master Mix kit (ThermoFisher^®) in an ABI PRISM 7300 Sequence Detection System (Applied Biosystems). Amplifications were carried out in 10 µL reaction mixtures containing 2 × reaction mix buffer, 20 µM of each primer, 10 µM of the probe, and 5 µL of RNA template. Primers specific to ZIKV were used: Primer F (5′-CAG CTG GCA TCA AGA AYC-3′) and Primer R (5′-CAC YTG TCC CAT CTT YTT CTCC-3′). The standard curve method was employed for virus quantification. The Ct values for this target were compared for calibration, and the brain weight was used for normalization.

To evaluate the protection efficacy of vaccine candidates against Delta and Omicron variants, the immunized mice were challenged with 6 × 10⁵ TCID₅₀ of Delta variant (CCPM-B-V-049-2105-8) or Omicron variant (BA.1, CCPM-B-V-049-2112-18) via the i.n. route. For Delta variant challenge experiments, the BALB/c mice were transduced intranasally with 8 × 10⁹ vp of Ad5-hACE five days before the SARS-CoV-2 infection. Three days post challenge, all mice were euthanized and necropsied, and lung tissues were collected for virus titration and pathological examination. The mice experiments with Delta and Omicron variants challenge were conducted under ABSL3 facilities in IMBCAMS. SARS-CoV-2-specific qRT-PCR assays were performed using TaqMan Fast Virus 1-Step Master Mix kit (Thermo Fisher Scientific, USA) on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad, USA) according to the manufacturer’s protocol. The sequences of primers and probes used in the qRT-PCR assays were same as the above description, except for detecting Omicron variant gRNA with the probe sequence of ACTCCGCATTACGTTTGGTGGACC. Mice lung tissues were stained with H&E for pathological examination.

We detected two mixed infection cases from birth cohort [93 (link)] and family cohort studies conducted in Lima, Peru. By following up those two cases, we successfully observed 20-days or 29-days prolonged shedding of mixed infection with two different genotypes: one child with GII.3 and GII.4, and another with GII.4 and GII.NA2, respectively. In addition to NGS and subsequent clonal population analysis, we performed genotype-specific qPCR to quantify the viral load of each genotype during the shedding. Briefly, viral RNA was quantified by duplex one-step real-time PCR with GII-specific primers and genotype-specific probes (S6 Table) using TaqMan Fast Virus 1-Step Master Mix kit (Applied Biosystems). The genotype-specific control plasmids were generated in house with corresponding partial viral genomes inserted into pCI vectors (Promega). In addition, with the 10% PBS suspension of the stool samples, we detected human IgA response with corresponding VLPs as antigens by ELISA. The VLPs were produced as described elsewhere using baculovirus expression system [8 (link),16 (link),114 (link)] with VP1-encoding sequences from GII.3 (Maizuru010524; accession number EF547399), GII.4 (RockvilleD1; KY424328), and GII.NA2 (PNV06929; MG706448) viruses.

Viral RNA was extracted from ZIKV C6/36 cell culture supernatants and DENV stocks by using TRIzol^® LS reagent according to the manufacturer’s instructions (Thermo Fisher Scientific). The assay is based on the amplification of a ZIKV envelope gene region20 (link), and a DENV capsid region46 (link), using ZIKV and DENV-2 specific primers and probes, listed in Table S1. The reactions were performed using the TaqMan^® Fast Virus 1-Step Master Mix kit (Applied Biosystems). The concentration of both ZIKV and DENV viral RNA (copies/milliliter) was estimated by using the standard curve generated from DENV2 transcripts46 (link). This standard curve was later compared to ZIKV RNA measured by spectrophotometry, to confirm their direct correlation.

Viral RNA was extracted from the supernatant of C6/36 cell culture infected with ZIKV and from dengue serotype 2 (DENV2) strain K0049 (SE Asian genotype, C6/36 passage 3) by using TRIzol LS reagent according to the manufacturer’s instructions (Thermo Fisher Scientific). Quantitative RT-PCR assays were based on the amplification of a ZIKV envelope gene region 3, and a DENV2 capsid region 1, using the ZIKV and DENV2-specific primers and probes, listed in Table 1. The reactions were performed using the TaqMan Fast Virus 1-Step Master Mix kit (Applied Biosystems), on a StepOnePlus Real-Time PCR system (Applied Biosystems). The concentrations of both ZIKV and DENV2 viral RNA (copies/mL) were estimated by using the standard curve generated from DENV2 transcripts. This standard curve was later compared to ZIKV RNA measured by spectrophotometry, to confirm their direct correlation.