Premix ex taq probe qpcr kit

Total RNA was extracted from cell lysates using RNeasy (QIAGEN), and an aliquot of 100 ng was reverse-transcribed with PrimeScript RT Reagent Kit with gDNA Eraser (Takara) and the included primer mix. An aliquot of 10 ng cDNA was used as a template for amplifying sequences of human GAPDH, and LRP1 and VSV with corresponding QuantiTect primers (QIAGEN) and specific primers (Table S3), respectively, and the SYBR Premix Ex Taq (Tli RNaseH Plus) kit (Takara). RRN18S was amplified in a similar manner, but with 2 ng cDNA as a template. RNA levels of EMCV, LACV, RVFV, SARS-CoV-2, and SFSV were detected using specific primers and TaqMan probes (Table S3) (Bird et al, 2007 (link); Weidmann et al, 2008 (link); Qin et al, 2018 (link); Corman et al, 2020 (link)), and the Premix Ex Taq (probe qPCR) kit (Takara). All PCRs were performed in a StepOne Plus instrument (Applied Biosystems). The values obtained for each gene were normalized against GAPDH mRNA levels (or RRN18S mRNA levels in the case of the VSV RNA) using the threshold cycle (ΔΔCT) method (Livak & Schmittgen, 2001 (link)).

Table S3. Primer and probe list for detection of virus RNA by qPCR.

The WSSV load was determined in the muscle of 50 shrimp at 96 hpi and 228 hpi each, using the TIANamp marine animals DNA kit (Tiangen Biotech Co., Ltd., Beijing, China) for DNA extraction. Subsequent TaqMan real-time PCR quantification [24 (link)], performed with an ABI 7500 fluorescence quantitative PCR system (Applied Biosystems, Foster City, CA, USA) and Premix ExTaq™ (Probe qPCR) kit (TaKaRa, Dalian, China), calculated viral load by the WSSV to 18S rRNA gene ratio, facilitating selection of high and low WSSV load shrimp for transcriptome sequencing. Primer and probe details are provided in Table 1.

In house qPCR was performed using Premix Ex Taq™ (Probe qPCR) Kit (Takara Bio Inc. Shiga, Japan) in a CFX96 Touch Real-Time PCR detection system (Bio-Rad Laboratories). The primer sequences used in the qPCR assay have been described previously (Leung et al., 2001 (link)). The 20 µL qPCR mixture contained 10 µL of 2 × Premix, 1 µL of 10 µM BKV forward primer (5′- AGTGGATGGGCAGCCTATGTA-3′), 1 µL of 10 µM BKV reverse primer (5′-TCATATCTGGGTCCCCTGGA-3′), 0.5 µL of 10 µM hydrolysis probe (5′-FAM- AGGTAGAAGAGGTTAGGGTGTTTGATGGCACAG-BHQ1-3′), and 5 µL of template solution. The thermal cycling protocol was 1 min at 95 °C for initial denaturation followed by 40 cycles of 5 s at 95 °C for denaturation and 15 s at 60 °C for annealing and extension.

Aedes importin β3 expression was evaluated by RT–qPCR in mock-infected and DENV-2 acutely infected C6/36-HT cells and mock-infected C6-L55 cells from the time course assays. Specific gene primers and TaqMan probes are shown in Table S1.4. The mRNA levels were normalized against Aedes S7 rRNA.
The Premix Ex Taq Probe qPCR kit (Takara RR390 L, Kusatsu, Japan) was used according to the manufacturer’s recommendations. Briefly, the reaction mixture consisted of 2 µL of cDNA, 10.0 µL of Premix Ex Taq (Probe qPCR), forward primer (10 µM), reverse primer (10 µM), 2 µL TaqMan probe, and 6 µL water. Control reactions without cDNA were carried out to confirm the absence of contamination in the qPCR. The qPCR conditions for importin β3 were 95 °C for 35 s, 45 cycles of 95 °C for 21 s, 54 °C for 35 s and 60 °C for 30 s, and final extension at 4 °C for 10 min. qPCR conditions for S7 rRNA were 95 °C for 35 s, 45 cycles of 95 °C for 21 s and 60 °C for 30 s, and final extension at 4 °C for 10 min. All assays were performed in triplicate. The StepOne™ Real-Time PCR System (Applied Biosystems™, Waltham, MA, USA) was used. The relative transcript levels were determined by the ΔΔCt method [14 (link)]. Data are presented as 2^−ΔΔCt ± S.D. (p < 0.05) determined by two-way ANOVA.

Total DNA was extracted from blood and tissue samples from the experimental mice with an Axygen^® AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Axygen^®, USA). According to the protocol, the primers used for q-RT-PCR were designed to be specific for the gG sequences in the HSV2 genome (Table 1). q-RT-PCR was performed using the Takara Premix Ex Taq^™ (Probe qPCR) Kit (TaKaRa, China).

To quantify the microbial biomass in the sediment samples, TaqMan qPCR with the primer set 349F (AGGCAGCAGTDRGGAAT) and 806R (GGACTACYVGGGTATCTAAT), and a TaqMan probe (FAM-TGCCAGCAGCCGCGGTAATACRDAG-TAMRA) were used to detect the V3 & V4 regions of the 16S rRNA gene. PCR reactions were carried out with 2 ng/μL template DNA using Premix Ex Taq™ Probe qPCR Kit (Takara, RR390B). The bacterial biomass of each sediment sample was calculated as 16S-copies per gram of dry biosolids (determined based on the sediment water loss after drying at 105 ℃ for 8 h).