Rna ultrasense one step quantitative rt pcr system

Blood collection for serum isolation for viral load analysis by qRT-PCR and plaque assays was performed, as described in Section 2.2 and Figure 1a. Tissues were collected at scheduled study termination, unscheduled euthanasia as well as when animals were found dead in their cage; tissues collected for viral load analysis by qRT-PCR and plaque assay were lung (lower left lobe), spleen, liver, axillary lymph node from the virus-inoculated arm, adrenal gland, and brain. Viral loads in serum and tissues were determined via plaque assay, as previously described [40 (link)]. Viral loads were determined by qRT-PCR as described here. Viral RNA was quantified in serum and tissue samples via qRT-PCR. One-step qRT-PCR was performed using RNA UltraSense One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA, USA), with primers and probe designed to detect a region of the MARV glycoprotein (GP) gene. The primer and probe sequences were as follows: Marburg marburgvirus Forward Primer: 5′ GGC CTT CAG GGC AGG TGT A 3′; Marburg marburgvirus Reverse Primer: 5′ CCT GTG CAT GAG GGT TTT GA 3′; Marburg marburgvirus Probe: 6-FAM 5′ CCT TGC TGT TAG ATC CTC CTA CCA A MGB-NFQ-3′.

HIV-1 RNA was extracted from plasma with QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol with on-column DNase I treatment. HIV-1 plasma viral load was quantified with RNA UltraSense One-Step Quantitative RT-PCR System (Invitrogen, Waltham, MA) using the following conditions: 50°C for 15 min; 95°C for 2 min; 40 cycles at 95°C for 15 sec and 60°C for 1 min. The primers and probe target the HIV-1 gag as previously described [17 (link)]. AcroMetrix HIV-1 panel (ThermoFisher Scientific, Fremont, CA) was used for standard curve.

Mengovirus detection was used to validate the process; samples with negative mengovirus amplification needed to be repeated from RNA extraction.
RT Real-time PCR was performed using primers and TaqMan probe shown in Table 2, to confirm the process effectiveness.
The reaction was performed using RNA UltraSense™ One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA, USA) in a total volume of 20 μL containing 5 μL of Ultrasense reaction mix (5×), 1 μL of each primer (12.5 μM and 22.5 μM, Forward and Reverse, respectively), 1 μL of probe (6.25 μM), 0.5 μL of Rox reference dye (50×), 1.25 μL of RNA Ultrasense enzyme mix and 10.25 μL of DNAse-RNase-free water (Sigma–Aldrich, St. Louis, MO, USA). Five μL of RNA template were added to the reaction mix, and positivity was detected when Ct ≤ 40.
The reaction was performed in a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Reverse transcription was performed for 1 h at 55 °C; samples were then incubated at 95 °C for 5 min and amplified for 45 cycles of 15 s at 95 °C, 1 min at 60 °C and 1 min at 65 °C. Negative and positive amplification controls were included in each run.

Viral copy number in culture supernatants and cell lysates was measured by RT-qPCR as previously described (Chagan-Yasutan et al., 2013 (link)) using the RNA UltraSense One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA, USA) and Thermal Cycler Dice Real Time System (Takara Bio, Otsu, Japan) according to the manufacturers’ protocols. Primers and hydrolysis probes specific to the 3′ untranslated region of each of the four DENV genotypes have been previously published (Chagan-Yasutan et al., 2013 (link)). The forward and reverse primers were as follows 5′-AAGGACTAGAGGTTAGAGGAGACCC-3′ and 5′-CGTTCTGTGCCTGGAATGATG-3′ (Warrilow et al., 2002 (link)). The TaqMan probe was labeled at the 5′ and 3′ ends with a 6-carboxyfluorescein (FAM) reporter and Black Hole quencher (BHQ)-1 (i.e., 5′[FAM]–TGGGARAGACCAGAGATCCTGCTGTCT–[BHQ1]3′). The copy numbers obtained by DENV RT-qPCR were linearly associated with plaque numbers (data not shown).

Viral RNA extraction was carried out on 150 μl of viral suspension using a NucleoSpin^® RNA virus kit (Macherey-Nagel GmbH & Co.) according to the manufacturer’s instructions. Primers, probes and RT-qPCR conditions used in this study are listed in Table 1 for HEV and in the ISO 15216:2017 for HAV. Modified-probe included in assay A (Schlosser et al., 2014 (link)) contains a ZEN internal quencher. Modification of assay C (adapted from Mansuy et al., 2004 (link)) consists of an RT reaction held at 45°C for 60 min. RT-qPCRs were carried out in 96-well plates using the LightCycler 480 instrument (Roche Diagnostics) and a half-scale modification of the RNA UltraSense One-Step quantitative RT-PCR system (Invitrogen SA), by using half volumes of all reagents.
Quality control of the RT-qPCR process included negative (nuclease-free water) and positive (RNA) controls added to each PCR plate. Each viral RNA was analyzed in duplicate. HEV and HAV quantification was calculated by plotting the quantification cycles (Cqs) to an external standard curve built with the International Standard WHO HEV RNA (250,000 IU/ml) and HAV reference material (code RM000HAV, Public Health England), respectively.

Vero cells were pretreated for 2 hours with equivalent volumes of either DMSO (control), 17-AAG (10 µM), BAPTA (10 µM), or KNK437 (10 µM), prior to infection with the MP-12 strain of RVFV at an MOI of 0.1. Following infection, cells were washed with PBS and media containing either DMSO or compound was added back to the cells. Twenty-four hours later, supernatants were collected for analysis of viral RNA. Viral RNA was extracted using Ambion’s (Applied Biosystems/Ambion, Austin, TX) MagMAX viral RNA extraction kit and quantitated using q-RT-PCR with primers and probe for G2, originally described by Drosten et al[31] (link). Q-RT-PCR assays were performed using Invitrogen’s (Carlsbad, CA) RNA UltraSense One-Step Quantitative RT-PCR System on an ABI 7000 sequence detection system. Absolute quantitation of RVFV RNA was performed using a standard curve where the RNA concentration is expressed in genomic copies/reaction. Currently, our assay detects 10² copies per reaction 100% of the time and 10¹ copies 50% of the time, which is in agreement with other published methods of PCR detection [32] (link). Cell viability assays were performed using CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer’s instructions. DTX 880 multimode detector (Beckman Coulter) was used for detection of Luminescence.