Maxima sybr green qpcr kit

HPV16 titers were measured using qPCR-based DNA encapsidation assay as previously described [41 (link)]. To detect endonuclease-resistant genomes in CV stocks and Optiprep fractions the following method was used. Briefly, viral genomes were released from 10 μL benzonase-treated CV stock or 20 μL Optiprep fraction by re-suspension in 200 μL HIRT DNA extraction buffer (400 mM NaCl/10 mM Tris-HCl (pH 7.4)/10 mM EDTA (pH 8.0)), 2 μL 20 mg/mL proteinase K, and 10 μL 10% SDS, for 2–4 h at 37 °C. Following digestion, the DNA was extracted twice using phenol-choloroform-isoamyl alcohol (25:24:1), followed by extraction in an equal amount of chloroform. DNA was ethanol precipitated overnight at −20 °C. Samples were centrifuged, and the DNA pellet was washed with 70% ethanol and resuspended in 20 μL of Tris-EDTA overnight. To quantify viral genomes, a Thermo Scientific Maxima SYBR Green qPCR kit was utilized. Amplification of the HPV16 E2 open reading frame (ORF) was performed using 0.3 μM of forward primer HPV16E2-5′ and HPV16E2-3′ (Table S1). Amplification of the E2 ORF of serially diluted pBSHPV16 DNA, ranging from 10⁸–10⁴ copies/μL, was used to generate a standard curve. A Bio-Rad iQ5 Multicolor Real-Time qPCR machine and software were utilized for PCR amplifications and subsequent data analysis.

Quantification of intracellular viral RNA was performed based on a SYBR-Green system using the Maxima SYBR-Green qPCR Kit (Thermo-Scientific, Braunschweig, Germany) with primers flanking HEV ORF2 (FWD: 5′-GGT GGT TTC TGG GGT GAC-3′; REV: 5′-AGG GGT TGG TTG GAT GA-3′) and RPL27 as a housekeeping gene (FWD: 5′-AAA GCT GTC ATC GTG AAG AAC -3′; REV: 5′-GCT GCT ACT TTG CGG GGG TAG-3′). In order to be able to normalize intracellular measurements, SYBR-Green based assays were used for this experimental setup. Quantification of extracellular viral RNA was performed based on a hydrolysis-probe system using the LightCycler Multiplex RNA Master Mix (Roche Diagnostics, Mannheim, Germany) and LightMix Modular Hepatitis E Virus Kit (quantitative kit, TIB Molbio, Berlin, Germany). Light mix modular HEV kit is a CE marked based real time PCR kit with a detection limit of 200 IU/mL for HEV genotypes 1–4 (containing HEV specific primers and a respective hydrolysis probe). All of the procedures were performed according to the manufacturer’s protocols and were analyzed with either LightCycler 2.0 or LightCycler 480 Instrument II (Roche, Mannheim, Germany).

The virus titer in different transgenic plants with amplicon V2 RNAi was determined through Thermo Scientific Maxima SYBR Green qPCR kit (cat# K0241). The DNA of transgenic and non-transformed plants was diluted 10× before using as a template. CLCuKoV-Bur plasmid construct and CLCuMB plasmid construct were used as the standard for absolute quantification, while virus-infected plants were used as positive control. The master mix contained 10 µl SYBER green, 0.35 µl of Forward primer (10 pmol, Table 1), 0.35 µl of Reverse primer (10 pmol, Table 1), and 8.3 µl of template DNA (100/reaction). The qPCR reaction was started with an initial denaturation at 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 59 °C for 30 s, and 72 °C for 30 s. A final extension was given at 72 °C for 10 min.