Primescript rt pcr kit perfect real time

The number of copies of viral genomes in fcwf-4 cells infected with FCoV was determined using RT-qPCR [11 (link)]. In brief, the medium was removed 20 h after infection, and RNA from the cells was prepared using Isogen (Nippongene), according to the manufacturer’s protocol. Total RNA was reverse transcribed using the PrimeScript RT-PCR Kit (Perfect Real Time; Takara Bio). Viral cDNAs were quantified using qPCR with primers specific for the gene encoding FCoV-N (forward, 5′-TGGCCACACAGGGACAAC-3′; reverse, 5′-AGAACGACCACGTCTTTTGGAA-3′) and the TaqMan probe (FAM-TTCATCTCCCCAGTTGACG-BHQ-1). Reaction mixtures were prepared according to the manufacturer’s protocol using Premix EXTaq (Takara Bio), and sequences were amplified using the 7500 Sequence Detection System (Life Technologies). The viral RNA copy number was normalized to that of the feline gene encoding feline β2-microglobulin (β2M) (GenBank accession no. NM_001009876) using qPCR with forward (5′-CGCGTTTTGTGGTCTTGGTCTTGGT-3′) and reverse (5′-AAACCTGAACCTTTGGAGAATGC-3′) primers specific for β2M. The TaqMan probe, TAMRA-CGGACTGCTCTATCTGTCCCACCTGGA-BHQ-2, was used to detect β2M mRNA.

Isogen-LS (Nippon Gene, Toyama, Japan) was used for RNA preparation from clinical specimens (whole blood, pleural fluid, ascites, pericardial effusion and cerebral fluid), and fcwf-4 cells infected with FCoV (79–1146 strain) according to the manufacturer’s protocol. Total RNA was reverse-transcribed using the PrimeScript RT-PCR kit (Perfect Real Time; Takara Bio, Shiga, Japan), as reported previously [22 (link)].

All candidate RT-qPCR assays independently tested one COVID-19-confirmed clinical sample using PrimeScript^TM RT-PCR (Perfect Real-time) kit (TaKaRa Biotechnology, Japan) by three times to screen the optimum primer-probe set. Each assay was performed in a 25 μl reaction volume containing 12.5 μl of buffer, 0.5 μl of TaKaRa Ex Taq^TM HS, 0.5 μl of enzyme mix, 0.48 μM of each primer, 0.24 μM probe, 5 μl of RNase-free water, and 5 μl of RNA template. Amplifications were performed on a 7500 Fast Real-Time PCR system (Applied Biosystems, Waltham, MA, United States) using the following conditions: 10 min at 42°C for reverse transcription, 2 min at 95°C for initial denaturation, followed by 45 cycles of 95°C for 10 s and 55°C for 30 s. The fluorescence signal was acquired at the end of each annealing step. The cycle threshold value (Ct) and fluorescence intensity of designed primer–probe sets were recorded and compared. The candidate assay yielded the lowest Ct-value, and the strongest fluorescence response was selected for further analysis.