2720 thermal cycler pcr instrument

Total RNA was extracted from whole blood samples with RNAiso Plus Reagent according to introduction of the kit. Then cDNA was synthesized by PCR instrument (2720 Thermal Cycler PCR instrument, Applied Biosystems, USA) with Prime Script™ RT Master Mix Kit. The cycling program was 37°С for 15 min, 85°С for 5 s, and 4°С for 7 min. Ultimately, semiquantitative analysis of the virus gene expression level was conducted with RT-PCR instrument (StepOnePlus™ Real Time PCR instrument, Applied Biosystems, USA) by using SYBR® Premix Ex Taq™ Kit (Tli RNaseH Plus). The primers of RHDV for the RT-PCR reaction were designed based on RHDV capsid protein VP60 sequences (GenBank No: AY269825) and primers of β-actin were designed based on Oryctolagus cuniculus actin (NM-001101683). The primer sequences were as follows: RHDV forward: 5'-CCATCATGTTCGCGTCTGTT-3'; RHDV reverse: 5'-GGGCGTACGTCAATGAGTTC-3'; β-actin forward: 5'-TGGCATCCTGACGCTCAA-3'; β-actin reverse: 5'-TCGTCCCAGTTGGTCACGAT-3'.

Total RNA was extracted from blood samples with RNAiso Plus Reagent according to the kit instructions. Then, cDNA was synthetized using a PCR machine (2720 Thermal Cycler PCR instrument, Applied Biosystems, America) with a PrimeScript^™ RT Master Mix Kit. The cycling program was 37°C for 15 min, 85°C for 5 s, and 4°C for 7 min. Finally, the semi-quantitative analysis of viral replication was conducted using a RT-PCR machine (StepOnePlus^™ Real Time PCR instrument, Applied Biosystems, America) and a SYBR^® Premix Ex Taq^™ (Tli RNaseH Plus) Kit. The primers for DHAV and β-actin were designed in our previous study [6 (link)]. The primer sequences used were as follows: DHAV forward: 5’- GCCACCCTTCCTGAGTTTGT-3’, DHAV reverse: 5’-TACCATTCCACTTCTCCTGCTT-3’; β-actin forward: 5’-CTTTCTTGGGTATGGAGTCCTG-3’, β-actin reverse: 5’-TGATTTTCATCGTGCTGGGT-3’. The reaction parameters were as follows: 95°C for 30 s, 95°C for 5 s (40 cycles) and 60°C for 30 s.

Total RNA was extracted from the samples mentioned above with the FastPure Cell/Tissue Total RNA Isolation Kit according to the kit instructions. Then, cDNA was synthetized using a PCR instrument (2720 Thermal Cycler PCR instrument, Applied Biosystems, USA) with the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) Kit. Finally, the semi-quantitative analysis of viral replication was conducted using a real-time PCR instrument (Step One Plus™ Real Time PCR instrument, Applied Biosystems) with the ChamQ™ SYBR qPCR Master Mix Kit. The primers for DHAV-1 and β-actin were designed in our previous study. The primer sequences designed in our previous research [43 ] were as follows: DHAV-1 forward, 5′-GCCACCCTTCCTGAGTTTGT-3′; DHAV-1 reverse, 5′-TACCATTCCACTTCTCCTGCTT-3′; β-actin forward, 5′-CTTTCTTGGGTATGGAGTCCTG-3′; and β-actin reverse, 5′-TGATTTTCATCGTGCTGGGT-3′. The reaction parameters were as follows: 95 °C for 30 s, 95 °C for 5 s (40 cycles) and 60 °C for 30 s.