Hiscript 2 u one step qrt pcr probe kit

As we used commercial kit (HiScript II U⁺ One Step qRT-PCR Probe Kit, Vazyme, Nanjing, China) in RRT-PCR, the PCR reaction system and programs were provided. So optimization consists of primer design, probe design and concentration adjustment. First, primers and probes were specifically chosen through gene alignments and primer specificity analysis. Then we determined the optimal concentrations of the two probes. Various concentrations of NP and H5-HA probes, i.e., 200nM, 250nM, 300nM and 350nM, were used in every single detection (Figure A and B in S1 Fig). Finally we confirmed the concentrations of HA and NP primers in multiple detection. In this part, amplification curves and dissociation curves (Figure C and D in S1 Fig) were performed at different concentrations of HA and NP primer pairs (350-450nM) with SYBR Premix Ex Taq II kit (Takara, Shiga, Japan). The optimal primer and probe concentrations of H5-HA primer pairs, NP primer pairs, H5-HA probe, NP probe in 20 μL RRT-PCR system were 400nM, 400nM, 350nM, 350nM, respectively. A single peak around 82.5°C was obtained in melt curves (Figure D in S1 Fig), suggesting no primer-dimer was formed.

RNA was extracted from the clinical samples, and cDNA was synthesized using the kit from Thermo Fisher (Catalog Number: 18091050) according to the manufacturer’s manual. Quantitative real-time PCR of clinical samples was performed using HiScript II U + One Step qRT-PCR Probe Kit (Vazyme Biotech Co., Ltd.) according to the manufacture’s manual. The probe and primers for quantitative real-time PCR were synthesized by Genscript, and sequences were listed in Supplementary Table 5. Further, an NMPA-granted commercial kit (Liferiver, China) was used for these clinical samples’ nucleic acid detection assay. The assay was performed according to the manufacturer’s manual. In brief, extracted RNA was subjected to a two-step quantitative real-time PCR, the fluorescent signal of FAM and TexasRed was collected and Ct value < 43 was treated as positive.

Total RNA was extracted from the lung of the animals using QIAamp Viral RNA Mini Kit (QIAGEN, Germany) and examined using HiScript II U⁺ One Step qRT-PCR Probe Kit (Vazyme, China) with indicated primers (Table 1).

Single real-time PCR was performed using the HiScript II U⁺ One Step qRT-PCR Probe Kit from Vazyme. The total volume of the PCR system was set as 10 µL, including 2× One Step U⁺ MIX, One Step U⁺ Enzyme MIX, and 50× ROX Reference Dye 2, the amount of the primer addition was optimized from 0.1 to 0.8 µL, while the amount of the probe addition was optimized from 0.1 to 0.6 µL, which were both at a final concentration of 10 µM. Amplification was carried out using the following program: 55°C 15 min, 95°C, 30 s, 45 cycles of 95°C 10 s, annealing temperature was optimized between 54°C and 60°C. Amplification with Quant Studio™ Design & Analysis Software. Signals were automatically collected at the end of each cycle.
Multiplex real-time PCR was established based on three single methods by adjusting the reaction system and conditions to reduce interference among three different fluorescent molecules and to increase amplification efficiency. The final concentration for the primer and probe was both set as 10 µM, and the amount of primer additions was optimized between 0.1 µL and 0.5 µL, while the amount of probe additions was optimized from 0.1 µL to 0.4 µL. The reaction temperature was optimized between 55°C and 65°C.

Multiplex real-time PCR was as performed using the HiScript II U⁺ One Step qRT-PCR Probe Kit from Vazyme. A 10-fold gradient dilution of 10⁸−10¹ copies/µL of the standard plasmids served as the template. Multiplex real-time PCR was conducted in tube, with three replicates assigned to each reaction to examine the detection limits of multiplex reactions. The primers and probes used in this study were tested for specificity. To avoid false positives caused by other pathogens that may be present in the assay, the multiplex real-time PCR detection assay was used to detect BVDV, BEV, BPV, S. enterica, and C. perfringens and RNA-free H₂O as negative control.

We performed qRT-PCR to evaluate the viral load and the level of cytokine gene expression at the mRNA level using a QuantGene 9600 real-time PCR system (Bioer Technology, Hangzhou, China). For viral load quantification, viral RNA in the virus supernatant was extracted using the TIANamp Virus RNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. One-step qRT-PCR was conducted with HiScript II U+ One Step qRT-PCR Probe Kit (Vazyme Biotech, Nanjing, China) using the primers/probe set targeting PRRSV ORF6 (Table 1). For quantification of the mRNA level of viral and cellular genes, total cellular RNA was extracted from cultured cells using FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme Biotech, Nanjing, China). The cDNA synthesized with a Hiscript II 1st Strand cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China) was used as the template for qPCR with AceQ U+ Probe Master Mix or AceQ qPCR SYBR Green Master Mix (Vazyme Biotech, Nanjing, China). Expression levels of PRRSV nsp9, IFN-β, IL-6, IL-8, and TNF-α were determined using the oligos listed in Table 1. The expression level of β-actin was also measured as an internal control. The relative RNA levels were calculated using the 2^−∆∆Ct method.