Veriti thermal cycler

The 1st round PCR reaction (Figure 1) was a total of 30 μL reaction. The reaction mix contained 6 μL of Red Load Taq, 13.4 μL of RNase free water, 0.3 μL of primers 224 and 222, and 10 μL of cDNA. Thermal cycling was done in a Veriti thermal cycler (Applied Biosystems, California, USA) as follows: 94°C for 3 minutes, then 45 cycles of 94°C for 30 seconds, 42°C for 30 seconds, and 60°C for 60 seconds, with ramp of 40% from 42°C to 60°C. This was then followed by 72°C for 7 minutes and held at 4°C until the reaction was terminated.
Four (PE-VP1-PCR, EA-VP1-PCR, EB-VP1-PCR, and EC-VP1-PCR [9 ]) different second-round PCR assays were run in this study (Figure 1). The 2nd-round PCR assay was also a 30 μL reaction. The PCR reaction mix contained 6 μL of Red Load Taq, 18.4 μL of RNase free water, 0.3 μL of forward and reverse primers, and 5 μL of the first-round PCR product. Thermal cycling was done in a Veriti thermal cycler (Applied Biosystems, California, USA). The cycling conditions were 94°C for 3 minutes followed by 45 cycles of 94°C for 30 seconds, 42°C for 30 seconds, and extension at 60°C for 30 seconds, with ramp of 40% from 42°C to 60°C. This was then followed by 72°C for 7 minutes and subsequently held at 4°C until the reaction was terminated. The PCR products were resolved in a 2% agarose gels stained with ethidium bromide and viewed using a UV transilluminator.

For routine testing, viral RNA was extracted using the High Pure Viral Nucleic Acid kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions. Two RT–PCR primer sets were used, a universal IBV RT–PCR generating a fragment of approximately 350 base pairs of the S1 gene with primers XCE1 + and XCE3- (Cavanagh et al., 1999 (link)) and a genotype-specific RT–PCR for D1466-like strains using forward primer 5′-TACRggMAATTTTACTgATgg-3′ and reverse primer 5′-CTgACTgCTTACAAgAACC-3′. Both RT-PCRs were performed using a VeritiTM Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA) in combination with the Qiagen one-step RT–PCR kit (Qiagen, Hilden, Germany) under the following conditions: reverse transcription reaction for 30 min at 50°C; denaturation 15 min at 95°C, followed by 45 cycles with 30 s at 95°C, 30 s at 50°C and 60 s at 72°C. The S1 amplicons were separated on a 1% agarose gel, and visualized with ethidium bromide staining and an ultraviolet light transilluminator. The purified amplicons were sequenced bidirectionally using Sanger sequencing with both the XCE1+ and XCE3-primers (BaseClear, Leiden, the Netherlands). Consensus sequences were constructed using MEGA7.0 (Kumar et al., 2016 (link)).

Total RNA was extracted from isolated murine PCs/5TGM1 cells and reverse transcribed as described above. Separate PCRs for Glipr1 and Actb (see primer sequences above) was performed using AmpliTaq Gold^TM DNA Polymerase (Thermo Fisher Scientific) on a VeritiTM Thermal Cycler (Thermo Fisher Scientific). The PCR products from the cDNA were then visualised by agarose gel electrophoresis using a 2% (w/v) agarose gel containing 1:10,000 GelRed® (Biotium).

The DNA was amplified by polymerase chain reaction (PCR) using specific primers for rs7975232, rs1544410, rs731236, and rs2228570. Primer sequence descriptions of the four single nucleotide polymorphisms (SNPs) are shown in Table S1. The PCR reaction system included 0.5 µL primer, 2.5 µL PCR buffer, 0.75 µL MgCl₂, 2.0 µL dNTP, 0.2 µL Taq polymerase, 1.0 µL DNA, and 17.55 µL water to a final reaction volume of 25 µL. PCR was carried out at Thermo Fisher Scientific Veriti^TM Thermal Cycler(Thermo Fisher Scientific Inc., Wilmington, DE, USA) using the following procedures: 1 cycle of 95 °C denaturation for 10 min, 35 cycles of 95 °C denaturation for 15 s, annealing temperature adjusted by primer for 30 s, and 72 °C extension for 1 min 30 s. Thereafter, this PCR product went through sequencing reaction using BigDye^TM Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc., Wilmington, DE, USA) in ABI PRISM 3130 Genetic Analyzer (Thermo Fisher Scientific Inc., Wilmington, DE, USA). The produced nucleotide sequences were analyzed with the Sequencing Analysis Software v5.2 (Thermo Fisher Scientific Inc., Wilmington, DE, USA).

PCR amplification was performed according to the DNA barcoding protocol in the Chinese Pharmacopoeia on an Applied Biosystems Veriti^TM Thermal Cycler (Thermo Fisher Scientific). Individual amplifications of ITS2 were carried out in 25 uL total volumes, including 10 ng template DNA and 2× Master Mix (AmpliTaq gold fast PCR MM, Thermo Fisher Scientific). The PCR annealing temperature was increased to 58 °C, and 25 cycles were run.
The PCR products were purified with Agencount Ampure XP beads (Beckman) and the PCR size and concentrations were measured on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Folsom, CA, USA) with the Qubit platform.

To perform SSR analysis, 39 microsatellite markers were used under optimized conditions (Table 4). Molecular markers were amplified using multiplex (Table 5) PCR in an automated thermocycler Veriti^TM Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The 10 μL reaction mixture contained 2× QIAGEN Multiplex PCR Master Mix (QIAGEN, Hilden, Germany), 5 μM of each primer, and 100 ng of DNA. The amplification profile consisted of the initial denaturation of the template DNA and HotStarTaq DNA polymerase activation at 95 °C for 15 min, followed by 35 cycles of denaturation at 94 °C for 30 s, primer annealing at 55–60 °C (depending on the primer) for 90 s, and product extension at 72 °C for 60 s. The final extension of the amplicons was performed at 60 °C for 30 min. The PCR products were checked by electrophoresis in an agarose gel (2%), and then separated using capillary electrophoresis in an ABI PRISM 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The SSR data were processed using GeneMapper software v6 (Applied Biosystems, Foster City, CA, USA).