Sars cov 2 direct detection rt qpcr kit

Manufactured by Takara Bio

Sourced in Japan

The SARS-CoV-2 direct detection RT-qPCR kit is a laboratory tool designed for the detection of the SARS-CoV-2 virus. It utilizes reverse transcription and quantitative PCR (RT-qPCR) technology to identify the presence of SARS-CoV-2 genetic material in samples.

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RT-qPCR kit (72 citations) QPCR kits (5 citations) QPCR detection kit (2 citations) RT kits (2 citations)

16 protocols using sars cov 2 direct detection rt qpcr kit

Quantifying SARS-CoV-2 RNA via RT-qPCR Assays

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To quantify SARS-CoV-2 RNA, two RT-qPCR assays, NIID_2019-nCoV_N (hereafter, NIID_N2) (Shirato et al. 2020 (link)), and a combination of CDC 2019-nCoV_N1 and CDC 2019-nCoV_N2 (CDC_N1N2) (Centers for Disease Control and Prevention 2020 ), were performed. Primers and probes, with sequences that are listed in Table S1, were purchased from Takara Bio (Kusatsu, Japan). Reaction mixtures were prepared using the One Step PrimeScript III RT-qPCR Mix (Takara Bio) for the NIID_N2 assay, and the SARS-CoV-2 Direct Detection RT-qPCR Kit (Takara Bio) for the CDC_N1N2 assay. Thermal cycling was performed with an ABI 7500 Fast thermal cycler (Thermo Fisher Scientific, MA, USA), and the thermal cycling conditions for both RT-qPCR assays were as follows: initial incubation at 50 °C for 30 min and initial denaturation at 95 °C for 5 min, followed by 45 cycles of denaturation at 95 °C for 15 s, and the primer annealing and extension reaction at 60 °C for 60 s.
The previously isolated strain, JPN/TY/WK-521 (provided by Dr. Nagata, National Institute of Infectious Diseases) (Matsuyama et al. 2020 (link)), was used to compare the two RT-qPCR assays. RNA was extracted from the stocked isolate using the QIAamp Virus RNA kit (Qiagen) and analyzed via RT-qPCR assays using NIID_N2 and CDC_N1N2 sets.

Kitamura K., Sadamasu K., Muramatsu M, & Yoshida H. (2020). Efficient detection of SARS-CoV-2 RNA in the solid fraction of wastewater. The Science of the Total Environment, 763, 144587.

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Real-time RT-PCR for SARS-CoV-2 Detection

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Real-time RT–PCR was performed as previously described^{31 ,32}. In brief, 5 μl of culture supernatant was mixed with 5 μl of 2× RNA lysis buffer (2% Triton X-100, 50 mM KCl, 100 mM Tris HCl (pH 7.4), 40% glycerol, 0.8 U μl⁻¹ recombinant RNase inhibitor (Takara, catalogue no. 2313B)) and incubated at room temperature for 10 min. RNase-free water (90 μl) was added, and the diluted sample (2.5 μl) was used as the template for real-time RT–PCR performed according to the manufacturer’s protocol using the One Step TB Green PrimeScript PLUS RT–PCR kit (Takara, catalogue no. RR096A) and the following primers: forward N, 5′-AGCCTCTTCTCGTTCCTCATC AC-3′; and reverse N, 5′-CCGCCATTGCCAGCCATT C-3′. The copy number of viral RNA was standardized with a SARS-CoV-2 direct detection RT–qPCR kit (Takara, catalogue no. RC300A). The fluorescent signal was acquired using a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific), a CFX Connect Real-Time PCR Detection System (Bio-Rad) or a 7500 Real Time PCR System (Applied Biosystems).

Mlcochova P., Kemp S.A., Dhar M.S., Papa G., Meng B., Ferreira I.A., Datir R., Collier D.A., Albecka A., Singh S., Pandey R., Brown J., Zhou J., Goonawardane N., Mishra S., Whittaker C., Mellan T., Marwal R., Datta M., Sengupta S., Ponnusamy K., Radhakrishnan V.S., Abdullahi A., Charles O., Chattopadhyay P., Devi P., Caputo D., Peacock T., Wattal C., Goel N., Satwik A., Vaishya R., Agarwal M., Mavousian A., Lee J.H., Bassi J., Silacci-Fegni C., Saliba C., Pinto D., Irie T., Yoshida I., Hamilton W.L., Sato K., Bhatt S., Flaxman S., James L.C., Corti D., Piccoli L., Barclay W.S., Rakshit P., Agrawal A, & Gupta R.K. (2021). SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion. Nature, 599(7883), 114-119.

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SARS-CoV-2 Quantification in Infected Cells

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For quantification of SARS-CoV-2 in the supernatant of infected cells, RNA extraction and RT-qPCR were performed using the SARS-CoV-2 Direct Detection RT-qPCR kit according to the manufacturer's instructions (Takara Bio Inc., Kusatsu, Japan); Eight µl of the supernatant was used in a 20 µl-reaction. The RNA genome quantity was determined using the new coronavirus positive control RNA (Nihon Gene Research Laboratory, Sendai, Japan) as a standard. Cellular RNA in the infected VeroE6/TMPRSS2 cells was prepared using the Maxwell RSC instrument (Promega Corp., Madison, WI) according to the manufacturer’s protocol. RT-qPCR for specific amplification of the N gene of SARS-CoV-2 was performed using One Step PrimeScript III RT-qPCR mix (Takara Bio Inc.) according to the manufacturer's protocol. The Primer/Probe Set (2019-n) (Takara Bio Inc.) contains two primer sets, N and N2, both annealing to the N gene of SARS-CoV-2. Thermal cycling was carried out as follows: reverse transcription at 52 °C for 5 min, initial denaturation at 95 °C for 10 s, 45 cycles of denaturation at 95 °C for 5 s, and a final annealing/extension at 60 °C for 30 s. LightCycler 480 System II (Roche Diagnostics K. K., Basel, Switzerland) was used as the instrument for the PCR reaction.

Yamamotoya T., Nakatsu Y., Kanna M., Hasei S., Ohata Y., Encinas J., Ito H., Okabe T., Asano T, & Sakaguchi T. (2021). Prolyl isomerase Pin1 plays an essential role in SARS-CoV-2 proliferation, indicating its possibility as a novel therapeutic target. Scientific Reports, 11, 18581.

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Viral Isolation from SARS-CoV-2 Positive Specimens

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We performed viral isolation for all RT-qPCR-positive cases with the available residual specimens as described previously^{17 (link)}. Briefly, VeroE6/TMPRSS2 cells were seeded in 96-well flat-bottom plates; 100 μL of respiratory and air samples were mixed with Dulbecco’s Modified Eagle Medium supplemented with 2% bovine fetal serum and an antibiotic–antimycotic solution (Thermo Fisher Scientific); they were inoculated in duplicates. The culture supernatant was changed to a fresh medium 1 day post-infection (d.p.i.) and incubated at 37 °C with 5% CO₂. On 5 d.p.i., we observed a cytopathic effect. Following 5 d.p.i., the supernatant was collected, and RT-qPCR was performed using the SARS-CoV-2 direct detection RT-qPCR kit (Takara Bio, Shiga, Japan) to confirm the propagation of SARS-CoV-2.

Suzuki T., Morioka S., Yamamoto K., Saito S., Iida S., Teruya K., Takasaki J., Hojo M., Hayakawa K., Kutsuna S., Miyamoto S., Ozono S., Suzuki T., Kodama E.N, & Ohmagari N. (2023). Nasopharyngeal SARS-CoV-2 may not be dispersed by a high-flow nasal cannula. Scientific Reports, 13, 2669.

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SARS-CoV-2 Viral Isolation and Quantification

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Viral isolation was attempted for all RT-qPCR positive cases with available residual respiratory specimens, as described previously.⁴⁹ (link) Briefly, VeroE6/TMPRSS2 cells⁵⁰ (link) (JCRB1819, JCRB Cell Bank) were seeded in 96-well flat-bottom plates, respiratory specimens mixed with DMEM supplemented with 2% FBS and Antibiotic-Antimycotic Solution (Thermo Fisher Scientific) were inoculated in duplicate, and the culture supernatant was changed to fresh medium one day post-infection (d.p.i.) and was incubated at 37°C and supplied with 5% CO₂. On one and five d.p.i., a cytopathic effect was observed. After five days, the supernatant was collected and RT-qPCR using the SARS-CoV-2 direct detection RT-qPCR kit (Takara) was performed in order to confirm the propagation of SARS-CoV-2. The median tissue culture infectious dose (TCID₅₀) in the residual specimens was determined for all viral isolation positive cases.

Miyamoto S., Arashiro T., Ueno A., Kanno T., Saito S., Katano H., Iida S., Ainai A., Ozono S., Hemmi T., Hirata Y., Moriyama S., Kotaki R., Kinoshita H., Yamada S., Shinkai M., Fukushi S., Takahashi Y, & Suzuki T. (2023). Non-Omicron breakthrough infection with higher viral load and longer vaccination-infection interval improves SARS-CoV-2 BA.4/5 neutralization. iScience, 26(2), 105969.

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Real-time RT-PCR for SARS-CoV-2 Quantification

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Real-time RT-PCR was performed as previously described^{3 (link),53 (link)}. Briefly, 5 μl of culture supernatant was mixed with 5 μl of 2 × RNA lysis buffer [2% Triton X-100, 50 mM KCl, 100 mM Tris-HCl (pH 7.4), 40% glycerol, 0.8 U/μl recombinant RNase inhibitor (Takara, Cat# 2313B)] and incubated at room temperature for 10 min. RNase-free water (90 μl) was added, and the diluted sample (2.5 μl) was used as the template for real-time RT-PCR performed according to the manufacturer’s protocol using the One Step TB Green PrimeScript PLUS RT-PCR kit (Takara, Cat# RR096A) and the following primers: Forward N, 5’-AGC CTC TTC TCG TTC CTC ATC AC-3’; and Reverse N, 5’-CCG CCA TTG CCA GCC ATT C-3’. The viral RNA copy number was standardized with a SARS-CoV-2 direct detection RT-qPCR kit (Takara, Cat# RC300A). Fluorescent signals were acquired using a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific), a CFX Connect Real-Time PCR Detection system (Bio-Rad), an Eco Real-Time PCR System (Illumina) or a 7500 Real Time PCR System (Applied Biosystems).

, & Gupta R. (2022). SARS-CoV-2 Omicron spike mediated immune escape and tropism shift. Research Square.

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SARS-CoV-2 Detection via Direct RT-qPCR

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A total of 10 PCR testing sessions (364 samples), which included precamp screening and in- and postcamp inspection, were performed. Saliva samples were examined by a SARS-CoV-2 Direct Detection RT-qPCR Kit (Takara Bio, Otsu, Shiga, Japan) using a LightCycler 96 (Roche Molecular Systems, Branchburg, NJ, USA) for the detection of SARS-CoV-2 according to the methods described by the manufacturers. The specificity (100% = 10/10 positive samples detected) and the sensitivity (100% = 15/15 negative samples detected) of the SARS-CoV-2 Direct Detection RT-qPCR Kit were confirmed by the National Institute of Infectious Diseases (NIID), Japan [https://www.niid.go.jp/niid/images/lab-manual/2019-nCoV-17-current.pdf (Japanese document) accessed on 28 August 2021].

Ogasawara I., Hamaguchi S., Hasegawa R., Akeda Y., Ota N., Revankar G.S., Konda S., Taguchi T., Takanouchi T., Imoto K., Okimoto N., Sakuma K., Uchiyama A., Yamasaki K., Higashino T., Tomono K, & Nakata K. (2021). Successful Reboot of High-Performance Sporting Activities by Japanese National Women’s Handball Team in Tokyo, 2020 during the COVID-19 Pandemic: An Initiative Using the Japan Sports–Cyber Physical System (JS–CPS) of the Sports Research Innovation Project (SRIP). International Journal of Environmental Research and Public Health, 18(18), 9865.

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SARS-CoV-2 Neutralization Assay Using VeroE6/TMPRSS2 Cells

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SARS-CoV-2 neutralization assay was performed using VeroE6/TMPRSS2 cells (34 (link)) that were obtained from the JCRB Cell Bank, Ibaraki, Japan. The cells (5 × 10³ cells per well, 50 μl) were seeded in 96-well culture plates and incubated at 37°C for 18 hours before infection. Monobodies were fourfold serially diluted (from 400 nM to 24.4 pM), and 20 μl of the diluted monobodies were added into each culture well. Cells were infected with 10 μl of SARS-CoV-2 (10⁵ TCID₅₀/ml) for 1 hour at 37°C. The supernatant was removed, and 80 μl of Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) was supplemented with 10% fetal bovine serum and penicillin (100 U/ml) and streptomycin (100 μg/ml) (Thermo Fisher Scientific). After incubation at 37°C supplied with 5% CO₂ for 36 hours, the culture supernatants were harvested. The SARS-CoV-2 RNA amounts in the supernatants were measured by RT-qPCR using the SARS-CoV-2 Direct Detection RT-qPCR Kit (Takara Bio). The IC₅₀ values were determined using the GraphPad Prism version 6.0.

Kondo T., Iwatani Y., Matsuoka K., Fujino T., Umemoto S., Yokomaku Y., Ishizaki K., Kito S., Sezaki T., Hayashi G, & Murakami H. (2020). Antibody-like proteins that capture and neutralize SARS-CoV-2. Science Advances, 6(42), eabd3916.

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Quantifying Viral RNA and SARS-CoV-2 in Saliva

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Viral RNA of HCoV-229E was purified using TRIzol LS Reagent (Thermo Fisher Scientific). Viral RNA copy numbers were quantified using QuantiTect Probe RT-PCR Kit (Qiagen GmbH, Hilden, Germany) and PCR thermal cycler Dice (Takara). Primers and probes (Eurofins Genomics, Ebersberg, Germany) specific to the viral RNA-dependent RNA polymerase (RdRp) are listed in Supplementary Table S7. For standard of genome copy number, the partial nucleotides of RdRp (position 998-1999 of HCoV-229E, GenBank Accession number MT438700) was amplified using forward and reverse primers listed in Supplementary Table S7 and cloned into pCAGGS plasmid using Gibson assembly kit (New England Biolabs, Germany).
Saliva samples were examined by SARS-CoV-2 Direct Detection RT-qPCR Kit (Takara Bio, Otsu, Shiga, Japan) using Roche Light Cycler 96 for the detection of SARS-CoV-2 according to the manufacturer instructions. Primers and probes recommended by the CDC for N1, N2, and RNase P targets in a multiplex reaction are listed in Supplementary Table S7. In SARS-CoV-2 positive samples, viral copy numbers were calculated based on the C_t value of RT-qPCR.

Taniguchi M., Minami S., Ono C., Hamajima R., Morimura A., Hamaguchi S., Akeda Y., Kanai Y., Kobayashi T., Kamitani W., Terada Y., Suzuki K., Hatori N., Yamagishi Y., Washizu N., Takei H., Sakamoto O., Naono N., Tatematsu K., Washio T., Matsuura Y, & Tomono K. (2021). Combining machine learning and nanopore construction creates an artificial intelligence nanopore for coronavirus detection. Nature Communications, 12, 3726.

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Real-time RT-PCR for SARS-CoV-2 Detection

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Real-time RT-PCR was performed as previously described^{31 (link),32 (link)}. In brief, 5 μl of culture supernatant was mixed with 5 μl of 2× RNA lysis buffer (2% Triton X-100, 50 mM KCl, 100 mM Tris HCl (pH 7.4), 40% glycerol, 0.8 U μl⁻¹ recombinant RNase inhibitor (Takara, catalogue no. 2313B)) and incubated at room temperature for 10 min. RNase-free water (90 μl) was added, and the diluted sample (2.5 μl) was used as the template for real-time RT-PCR performed according to the manufacturer’s protocol using the One Step TB Green PrimeScript PLUS RT-PCR kit (Takara, catalogue no. RR096A) and the following primers: forward N, 5′-AGCCTCTTCTCGTTCCTCATC AC-3′; and reverse N, 5′-CCGCCATTGCCAGCCATT C-3′. The copy number of viral RNA was standardized with a SARS-CoV-2 direct detection RT-qPCR kit (Takara, catalogue no. RC300A). The fluorescent signal was acquired using a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific), a CFX Connect Real-Time PCR Detection System (Bio-Rad) or a 7500 Real Time PCR System (Applied Biosystems).

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