Quantitect rt pcr kit

RNA was extracted from mosquitoes 5 days post-infection using a QIAshredder kit to homogenize tissues and a RNeasy kit to extract RNA (Qiagen). For analysis of viral RNA in mosquitoes by qRT-PCR, qRT-PCR were performed in the same closed tube with 100ng of total RNA per reaction using the Quantitect RT-PCR Kit (Qiagen) on an Eco Real-Time PCR System (Illumina) or a QuantStudio 3 (ThermoFisher) with a total reaction volume of 10 μL. For patients Z08 and Z09, RNA from 500 µL of urine was isolated with ZR Urine RNA Isolation kit (Zymo) following manufacturer’s protocol. ZIKV primers used were F 5′-AATGGGAAGGAAAGAAGAGG-3′ and R 5′-GCTGGGGTGATGAGAGTTGT-3′. Ribosomal protein s17 primers used were F 5′-CGGAGACCAAGGAGATGTTG-3′ and R 5′-CTGTAGCCCTGTGCCGATG-3′. Cycling conditions were 50 °C for 30 min and 95 °C for 15 min, followed by 45 cycles of 94 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s. Relative quantities of target cDNA were determined using the Pfaffl method and a single data point from the control group was set to 1.0^{26 (link)}. Ct values were reported as averages ± standard deviation.

To assess the expression of serotonin related genes, the qRT-PCR was performed. The genes investigated were Tryptophan hydroxylase 1 (TpH1), responsible for 5-HT synthesis in the gut district; serotonin transporter (SLC6A4), which regulates the recaption of 5-HT at luminal level; HTR3A and HTR4, coding for the intestinal receptors of serotonin. qRT-PCR reactions were conducted in 96-well plates using the Applied Biosystems’s StepOnePlus Real Time PCR System (Applied Biosystem, CA, USA). Specific primers for the selected serotonin-related genes were provided by Qiagen (QuantiNova LNA PCR assay) and the Quantitect RT-PCR kit (QIAGEN, Hilden, Germany), with a Master Mix containing SYBR Green as fluorescent dye, was used for the qPCR reaction. Parameters set for the qRT-PCR were 95 °C for 2 min, then 40 cycles at 95 °C for 10 s and finally at 60 °C for 30 s. Each reaction was repeated in triplicate and the absence of contaminants was assessed substituting cDNA with Ultrapure water (negative control). The relative expression of each gene was determined via the ∆∆CT method [43 (link)]. qRT-PCR results were graphed using the statistical program “GraphPad Prism 8.0.1”.

Mice deficient in receptors for type I and type II interferons, (IFNAGR^-/-, AGB6) were bred under specific pathogen-free conditions. Groups of 4-5-week-old, age-matched, mixed-sex AGB6 mice were inoculated subcutaneously into both rear footpads with 20 μl containing either 10⁷ genome equivalents (GE) of either DENV2 alone or DENV2 plus recombinant D7 protein. DENV2 alone samples contained purified S2 cell supernatant without D7 protein as a vehicle control. Forty-eight hours post-infection, mice were euthanized and left and right foot pads and left and right popliteal draining lymph nodes (DLN) were collected independently. Total RNA was extracted using an RNeasy kit (Qiagen). qRT-PCR was performed by adding equal amounts of RNA into each reaction and data were normalized to the actin reference gene. Mouse actin RNA was amplified using Forward: 5’ GGC TGT ATT CCC CTC CAT CG 3’ and Reverse: 5’ CCA GTT GGT AAC AAT GCC ATG T 3’ primers. Amplification of targets were performed using a duplex format in 0.2 ml, 96-well PCR plates (BIO-RAD) with a total reaction volume of 25 μl. Reverse transcription and quantitative PCR were performed in the same closed tube with 100 ng of total RNA per reaction using the Quantitect RT-PCR Kit (Qiagen).

Viral RNA was extracted from allantoic fluids using the QIAamp Viral RNA mini kit (Qiagen, Hilden, Germany) as per the manufacturer’s recommendations. The RNA concentration was measured with a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific< Waltham, MA, USA). The forward primer IBV (IBV-F: 5′-ATGCTCAACCTTGTCCCTAGCA-3′), reverse primer (IBV-R: 5′-TCAA-ACTGCGGATCA-TCACGT-3′), and TaqMan^® probe (IBV-TM: FAM-TTGGAAGTAGAGTGACGCC-CAAACTTCA-BHQ1) specific to IBV N gene were used to determine IBV copy numbers in one step RT-qPCR [7 (link)]. The master mix was utilized in a total volume of 25 µL containing 12.5 µL of QuantiTect RT-PCR kit (Qiagen, Hilden, Germany), 0.5 µL of each primer (50 pmol), 0.125 µL (30 pmol) of the probe, 0.25 µL of RT-enzyme, 8.125 µL of RNase-free water and 3 µL of RNA template [45 (link)]. The reaction was performed and analyzed using a Stratagene MX3005P real-time PCR machine (Agilent technologies, Santa Clara, CA, USA).

A total of 100 RNA samples collected between February and March 2015 were selected at random from the RNA sample library in DNH. rRT-PCR was carried out with a QuantiTect RT-PCR kit (Qiagen) and LightMix Modular Ebola Virus Zaire (2014) (TIB Molbiol, Berlin, Germany) as a reference test. Aliquots of 5 μl of RNA samples were added to 25-μl reaction mixtures. Each reaction was performed in a SmartCycler II system (Cepheid, Sunnyvale, CA) with a thermal cycle profile consisting of 42°C for 15 minutes, 95°C for 15 minutes, followed by 40 cycles of 95°C for 15 s and 55°C for 45 s. To quantify viral RNA, the standard curve described with 10-fold serially diluted synthesized RNA with partial sequences of Mayinga strains was used. The RT-LAMP test with Genie III was performed as an index test by the Nagasaki University team in DNH as described above. The Nagasaki University team was blinded to all sample information, including clinical status and reference diagnostic test results. At the end of this trial, the sample information was disclosed to the Nagasaki University team, and the results of reference rRT-PCR tests were compared to those of RT-LAMP.