Thunderbird probe one step qrt pcr kit

Viral RNA was extracted with a QIAamp Viral RNA Mini Kit (QIAGEN), RNeasy Mini Kit (QIAGEN) or MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit (Thermo Fisher Scientific) and quantified by real time RT-PCR analysis with a one-step qRT-PCR kit (THUNDERBIRD Probe One-step qRT-PCR kit, TOYOBO) using 5’-ACAGGTACGTTAATAGTTAATAGCGT-3’, 5’- ATATTGCAGCAGTACGCACACA-3’, and 5’-FAM-ACACTAGCCATCCTTACTGCGCTTCG-TAMRA-3’ (SARS-CoV-2 and SARS-CoV relative quantification) [52 ]; 5’-AAATTTTGGGGAVVAGGAAC-3’, 5’-TGGCAGCTGTGTAGGTCAAC-3’, and 5’-FAM-ATGTCGCGCATTGGCATGGA-TAMRA-3’ (SARS-CoV-2 absolute quantification); 5’-GAACAAACCCAAGGAAATTT-3’, 5’-GGATCTTTGTCATCCAATTT-3’, and 5’-FAM-ATGTCGCGCATTGGCATGGA-TAMRA-3’ (SARS-CoV absolute quantification).

Progeny vRNA was extracted from the culture supernatant of infected cells using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) and measured by quantitative real-time RT-PCR (THUNDERBIRD® Probe One-step qRT-PCR Kit, Toyobo, Osaka, Japan) using primers targeting the M gene as described previously [40 (link)]. The primer sequences were as follows: 5’-CCMAGGTCGAAACGTAYGTTCTCTCTATC-3’ (forward), 5’-TGACAGRATYGGTCTTGTCTTTAGCCAYTCCA-3’ (reverse). A TaqMan probe, which is an oligonucleotide with a fluorescent reporter dye attached to the 5’ end and a non-fluorescent quencher (NFQ) attached to the 3’ end, was used for the assay. The oligonucleotide sequence was FAM-ATYTCGGCTTTGAGGGGGCCTG-MGB-NFQ. The sequences of the primers and TaqMan probes were derived from “The Diagnosis Manual for Influenza Viruses, 3rd Edition” by the National Institute of Infectious Disease in Japan. We conducted one-step real-time RT-PCR using the CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA). The cycling conditions were as follows: a reverse transcription step at 50 °C for 10 min prior to an initial denaturation step at 95 °C for 1 min, followed by amplification for 40 cycles (denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 45 s).

Cells were incubated with SARS-CoV-1 or SARS-CoV-2 at a multiplicity of infections (MOI) of 1 in the presence of the cathepsin inhibitor, 25 μM E-64d (Abcam, Cambridge, MA, USA) or DMSO as control. After 1 h absorption, cells were washed with PBS and cultured in maintenance medium. At 4 h post-infection (hpi), total RNAs were extracted from inoculated cells using ISOGEN (Nippon Gene, Tokyo, Japan) and a Direct-zol RNA MiniPrep Kit (Zymo Research, Orange, CA, USA). Extracted RNAs were subjected to qRT-PCR analysis with the THUNDERBIRD Probe One-step qRT-PCR Kit (TOYOBO, Osaka, Japan). The sequences of primers and probes targeting the N gene of SARS-CoV-1 and SARS-CoV-2 have been described previously [33 (link),34 (link)]. Human ACTB (Beta Actin) Endogenous Control (Applied Biosystems, Foster City, CZ, USA) and nonhuman primate β-actin were employed as endogenous controls [35 (link)].

All animal experiments were performed following the Regulations on Animal Experimentation of Hokkaido University, and the protocol was approved by the Institutional Animal Care and Use Committee of Hokkaido University (approval no. 20-0026). Litters of 3-day-old BALB/c mice were inoculated orally with 1.0 × 10⁵ FFU of RVA strain SA11, 16-06, or MpR12 by gavage (n = 7 in each group). Control mice were treated with PBS as a mock-infected group (n = 7). The conditions of feces were monitored by palpation of the abdomen every day from 0 to 7 dpi. The state of the stool was classified into four categories based on the color, texture, and amount of feces according to the criteria used in a previous study: 0, normal feces; 1, exceptionally loose feces; 2, loose yellow feces; and 3, liquid feces (47 (link)). Stools with a score of ≥1 were considered diarrheal stools. Feces, small intestines, and large intestines were collected and suspended in PBS following RNA extraction using TRIzol LS (Thermo Fisher Scientific) and the Direct-zol RNA miniprep kit (Zymo Research, Irvine, CA). Extracted RNAs were subjected to qRT-PCR analysis using a Thunderbird probe one-step qRT-PCR kit (TOYOBO, Osaka, Japan). The primer and probe sequences are listed in Table S3 (61 (link)). Sera for FRNT were collected from suckling mice (n = 3 in each group) at 15 dpi.

Viral RNA copy numbers were quantified using a Thunderbird Probe one-step qRT-PCR kit (Toyobo) and TaqMan probe/primer sets specifically targeting RABV CVS N (F, 5′-TCG AAT GCT GTC GGT CAT GT-3′; R, 5′-CCG AAG AAT TCC TCT CCC AAA TA-3′; probe, 5′-FAM-CAA TCT CAT TCA CTT TGT TG-MGB-3′). The mRNA abundance of VPS4A and VPS4B were quantified using the one-step TB green PrimeScript PLUS RT-PCR kit (TaKaRa Bio) and the primer sets human VPS4A (F, 5′-TTC TCA GCC TCC TGT GAC CAC T-3′; R, 5′-ACG TCG TTC CAC CGT ATG TTG G-3′) and VPS4B (F, 5′-GGT TCT GGA TTC TGC CAT TAG GC-3′; R, 5′-CCG AAA GTC TGC TTC CGT GAG A-3′). The expression levels of housekeeping genes were quantified using predeveloped TaqMan Assay reagent human ACTB (Applied Biosystems).

To detect swine enteric pathogens, real-time reverse transcription polymerase chain reaction (qRT–PCR) was performed for 10 viral pathogens [PDCoV, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine enteric alphacoronavirus (PEAV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine torovirus (PToV), porcine sapelovirus (PSV), group A rotavirus (RVA), group B rotavirus (RVB), group C rotavirus (RVC)] using a commercial one-step qRT–PCR kit (THUNDERBIRD™ Probe One-step qRT–PCR kit, TOYOBO, Osaka, Japan). The previously reported 10 qRT-PCR assays were performed using the target gene-specific primer and probe sets of each viral pathogen, as described in their studies [7 (link),25 (link),26 (link),27 (link),28 (link),29 (link),30 (link)]. The reaction composition and conditions were performed according to the manufacturer’s instructions.