Five microliter (µl) of extract with known RNA content (ng/µl) was combined with 5 µl DNAse/RNAse free water (Sigma, The Netherlands). SARS-CoV-2 rRT-PCR was performed on the E-gen and N-gen as described [16 (link)], with minor modifications. The PCR reaction is a multiplex for SARS-CoV-2 and Phocine Distemper Virus (PDV) and is performed in a total reaction volume of 25 µl using 10 µl input and 15 µl PCR mix, containing 1xTaqMan® Fast Virus 1-Step Master Mix (Applied Biosystems, Foster City, CA, USA), DNAse/RNAse free water (Sigma, The Netherlands), 400 nM SARS-CoV-2 forward and reverse primer, 200 nM SARS-CoV-2 probe, 300 nM PDV forward primer (5’-cgggtgccttttacaagaac), 300 nM PDV reverse primer (5’-ttctttcctcaacctcgtcc) and100nM PDV probe (NED-aag ggc caa ttc t-MGBNFQ). The ABI PRISM 7500 (Life technologies, USA) was used for the amplification and detection using the profile of 2 min 50 °C, 20 s 95 °C, followed by 45 cycles of 3 s 95 °C and 32 s 60 °C. Since the RNA from kidney and lung tissues were isolated without the addition of PDV as described above, the PDV reaction was in this case redundant.
1xtaqman fast virus 1 step master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 1xTaqMan™ Fast Virus 1-Step Master Mix is a ready-to-use solution for the detection and quantification of viral RNA. It combines a reverse transcriptase and a DNA polymerase in a single reaction mix, enabling one-step RT-qPCR analysis.
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Lab products found in correlation
4 protocols using 1xtaqman fast virus 1 step master mix
1
SARS-CoV-2 Multiplex rRT-PCR Assay
2
Viral Respiratory Pathogen Identification
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PV/PMV infection was defined as a positive polymerase chain reaction (PCR) test of a respiratory sample for RSV, hMPV, or PIV 1‐4. Viral RNA detection was performed in nasopharyngeal swabs, nasal washes, sputum, or bronchoalveolar lavage specimens. RNA was extracted using the NucliSense EasyMag (bioMérieux). From 2008 until 2014, all respiratory samples were tested by a laboratory‐developed real‐time PCR‐test (LDT), using 1xTaqMan Fast Virus 1‐Step Master Mix (Applied Biosystems), as described previously.28 From then onward the FilmArray respiratory panel (BioFire Diagnostics) was implemented in the laboratory alongside the LDT and used for priority testing of respiratory viruses, including RSV, hMPV, and PIV 1‐4. Results were available within 24 hours after sample collection for the LDT and within 3 hours for the FilmArray panel.
3
USCDC N2 Assay for SARS-CoV-2 Detection
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The USCDC N2 assay for SARS-CoV-2 detection was purchased from Integrated DNA Technologies. Serial dilutions of RNA targets were mixed with 1x USCDC N2 assay, 1x
TaqMan™ Fast Virus 1-Step Master Mix (Applied Biosystems) and loaded on Roche Light Cycler. The reactions were incubated at 55°C for 5 min followed by 95°C for 20 s (one cycle), 95°C 10 s and 60°C 30 s (45 cycles). Fluorescent signals were monitored at the 60°C step, and Cq values (also called Ct values) were calculated automatically using Roche Light Cycler software.
TaqMan™ Fast Virus 1-Step Master Mix (Applied Biosystems) and loaded on Roche Light Cycler. The reactions were incubated at 55°C for 5 min followed by 95°C for 20 s (one cycle), 95°C 10 s and 60°C 30 s (45 cycles). Fluorescent signals were monitored at the 60°C step, and Cq values (also called Ct values) were calculated automatically using Roche Light Cycler software.
4
USCDC N2 Assay for SARS-CoV-2 Detection
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The USCDC N2 assay for SARS-CoV-2 detection was purchased from Integrated DNA Technologies. Serial dilutions of RNA targets were mixed with 1x USCDC N2 assay, 1x
TaqMan™ Fast Virus 1-Step Master Mix (Applied Biosystems) and loaded on Roche Light Cycler. The reactions were incubated at 55°C for 5 min followed by 95°C for 20 s (one cycle), 95°C 10 s and 60°C 30 s (45 cycles). Fluorescent signals were monitored at the 60°C step, and Cq values (also called Ct values) were calculated automatically using Roche Light Cycler software.
TaqMan™ Fast Virus 1-Step Master Mix (Applied Biosystems) and loaded on Roche Light Cycler. The reactions were incubated at 55°C for 5 min followed by 95°C for 20 s (one cycle), 95°C 10 s and 60°C 30 s (45 cycles). Fluorescent signals were monitored at the 60°C step, and Cq values (also called Ct values) were calculated automatically using Roche Light Cycler software.
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