Total RNA was extracted from homogenized mouse tissues using a TRIzol reagent (ThermoFisher Scientific, America). SARS-CoV-2 RNAs were detected by one-step RT-PCR using a THUNDERBIRD Probe One-Step qRT-PCR (TOYOBO, Japan) following the manufacturer's protocols. The total viral genomic RNA (gRNA) gene primers targeting the N protein described in our previous studies (Xu et al., 2020 (link); Zeng et al., 2022 ) were used, including 5′-GGGGAACTTCTCCTGCTAGAAT-3′, 5′-CAGACATTTTGCTCTCAAGCTG-3′, probe 5′-FAM-TTGCTGCTGCTTGACAGATT-TRMRA-3′. The viral sub-genomic RNA (sgRNA) gene primers targeting the E protein were used, including 5′- CGATCTCTTGTAGATCTGTTCTC-3′, 5′- ATATTGCAGCAGTACGCACACA-3′, probe 5′- FAM-ACACTAGCCATCCTTACTGCGCTTCG-TAMRA-3′. Serial dilutions of the SARS-CoV-2 RNA reference standard (National Institute of Metrology, China) were used in each run, in parallel to calculate copy numbers in each sample on ViiA 7 Real-Time PCR System (Applied Biosystems).
Thunderbird probe one step qrt pcr
Manufactured by Toyobo
Sourced in Japan
THUNDERBIRD Probe One-Step qRT-PCR is a laboratory equipment product designed for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. It enables the detection and quantification of RNA targets in a single-step process.
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2 protocols using thunderbird probe one step qrt pcr
1
SARS-CoV-2 Detection by One-Step RT-PCR
2
SARS-CoV-2 RNA Detection and Inflammation Markers
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Total RNA was extracted from homogenized mouse tissues using a TRIzol reagent (Thermo Fisher Scientific). SARS-CoV-2 RNAs were detected by one-step RT-PCR using a THUNDERBIRD Probe One-Step qRT-PCR (TOYOBO, Japan) following the manufacturer's protocols. The primers targeting the N protein described in our previous study43 (link) were used, including 5’-GGGGAACTTCTCCTGCTAGAAT-3’, 5’-CAGACATTTTGCTCTCAAGCTG-3’, probe 5’-FAM-TTGCTGCTGCTTGACAGATT-TRMRA-3’. Serial dilutions of the SARS-CoV-2 RNA reference standard (National Institute of Metrology, China) were used in each run, in parallel to calculate copy numbers in each sample. To detect RNA transcript of inflammatory mediators, the target transcripts were determined by quantitative PCR using qScriptTM One-Step qRT-PCR kit (Quanta Biosciences, #95057-050) on CFX96 real-time PCR system (Bio-Rad). The primer sequences are listed in Table S2.
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