Hiscript 2 one step qrt pcr probe kit

RT-qPCRs are conducted in a 25 μL reaction mixture containing 5 μL RNA/DNA, 0.5 μL forward primers, 0.5 μL reverse primers, 0.25 μL probes, 5 μL RNAse-free water, 12.5 μL 2× One Step Q Probe Mix and 1.25 μL One Step Q Probe Enzyme Mix (HiScript II One Step qRT-PCR Probe Kit, Vazyme, Nanjing, China). The mixes contain all components necessary for RT-qPCR amplifications except primers, templates and water. Thermal cycling parameters are 15 min at 50 °C, followed by 30 s at 95 °C, and a subsequent 45 cycles of 95 °C for 10 s and 60 °C for 30 s. The AGS4800 quantitative real-time PCR is performed using the quantitative PCR thermocycler instrument (Daan Gene, Guangzhou, China).

The newly developed multiplex RT‐RPA/LbCas12a/AuNP visual assay was compared with two other established methods, one‐step RT‐PCR and TaqMan RT‐qPCR. The transcribed RNA standards were 10‐fold serially diluted by DEPC‐treated water and used to evaluate the sensitivity. One‐step RT‐PCR was first performed using a PrimeScript™ One‐Step RT‐PCR Kit (Takara, China) with virus/viroid‐specific primers (Table S4), according to the manufacturer’s protocol. PCR products were analysed by gel electrophoresis to determine the LOD. Next, TaqMan‐based RT‐qPCR assays were carried out using a HiScript II One‐Step qRT‐PCR Probe Kit (Vazyme, China) with the TaqMan‐MGB probe and primer set listed in Table S6. Assays were performed using a LightCycler480 (Roche). The RT‐qPCR profile consisted of cDNA synthesis (15 min at 50 °C), an initial denaturation step (30 s at 95 °C) and 45 amplification cycles (10 s at 95 °C, 30 s at 60 °C). Fluorescence information was collected during the 60 °C annealing step per cycle. Each dilution was tested in three replicates to identify an endpoint beyond which test positivity was unattainable. Sensitivity and linearity of the RT‐qPCR assays were estimated by constructing standard curves.

A clarified supernatant of homogenized lung, brain, and spleen tissues from NiV-infected hamsters was harvested for viral load detection. RNA was extracted using the RNeasy kit (Qiagen, 52906) according to the manufacturer’s instructions, and the total RNA was eluted in 50 μL of elution buffer. RNA (2 μL) was used as a template for quantitative real-time reverse transcriptase PCR (qRT-PCR). qRT-PCR was performed on a CFX96 Real-Time System (Bio-Rad) using the HiScript II One Step qRT-PCR Probe Kit (Vazyme, Q222-01) with primers and probes specific for the NiV nucleocapsid (N) gene: forward primer (5′-AACATCAGCAGGAAGGCAAGA-3′), reverse primer (5′-GCCACTCTGTTCTATAGGTTCTTC-3′), and probe (5′-FAM-TTGCTGCAGGAGGTGTGCTC-BHQ1-3′). The standard curve was constructed with 9 points in a 20 μL reaction system (1 × 10⁹ to 1 × 10¹ copies). Samples with fewer than 10 copies were defined as negative.

The multiplex RT-PCR was conducted in a 20.0 μL reaction system with a PCR machine (Eppendorf, Germany). Multiplex RT-PCR primers were adapted as previously described for AIV, NDV, and IBV (Table 2). Multiplex RT-PCR was performed using HiScript® II One Step qRT-PCR Probe Kit (Vazyme, USA). The reaction mixture contained 2.0 μL mixed primers (IBV-F, IBV-R, NDV-F, NDV-R, M-F, M-R, H5-F, H5-R, H7-F, H7-R, H9-F, H9-R), 10.0 μL of 2× One-Step Q Probe Mix, 1.0 μL One-Step Q Probe Enzyme Mix, 6.0 μL of RNase-free water, and 1.0 μL of extracted RNA (total: 20.0 μL). The multiplex RT-PCR program was as follows: a reverse transcription step at 50 °C for 5 min, initial denaturation at 95 °C for 2 min, and finally 40 amplification cycles of 95 °C for 10 s and 54 °C for 10 s.

The 12-h germlings of the wild-type strain PH-1, Fgblm10, pkr, and Fgblm10^∆CT1566 mutant were collected to isolate RNA (two biological replicates each). The RNA sample was isolated by Eastep Super Total RNA Extraction Kit (Promega, Madison, WI, USA). Reverse transcription RNA used the HiScript II One Step qRT-PCR Probe Kit (Vazyme, Nanjing, China). The qRT-PCR test used the ChamQ SYBR qPCR Master Mix (Vazyme, China).

Total RNA from cell cultures was extracted using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. Viral RNA titer was determined by a HiScript II one Step qRT-PCR Probe Kit (Vazyme). Genes tested included interferon (IFN) type I (Ifnb) and type III (Ifnl1), IFN signaling molecules (Irf3 and Stat1), IFN-stimulated genes (ISGs; Isg15, Oas1, Mx1 and Ifitm1) and NOD-like receptor family pyrin domain-containing protein 6 (NLRP6)-inflammasome (Nlrp6, Caspase1, Il1b and Il18). Gene expression was determined by qRT-PCR using the HiScript II one Step qRT-PCR SYBR Green Kit (Vazyme). Primer sequences and probes are listed in S1 Table.

PAMs and 3D4/21 cells were infected with ASFV (MOI = 5) for 2 h at 37 °C and washed three times with PBS before incubation with fresh medium. Total RNAs were extracted from the infected cells at 8, 24 and 48 hpi using Total RNA Kit (Omega Bio-tek, USA) following the manufacturer’s instructions. ASFV CP204L mRNA was detected using HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme, China), and the detection of B646L mRNA was performed using HiScript II One Step qRT-PCR probe kit. The primers and probes used in this study are listed in Table 2. Each reaction was performed in triplicate, and results are expressed as mean ± standard deviation (SD).