TITLE: One-Step RT-qPCR for SARS-CoV-2 Wastewater Surveillance: N1, PMMoV, BCoV, SOC, CrAssphage, Bacteroides rRNA, 18S rRNA 
AUTHORS: Hannah Greenwald, Lauren Kennedy, Vinson Fan, Rose Kantor, Kara Nelson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The following protocol describes the reverse transcription quantitative polymerase chain reaction (RT-qPCR) methods that we have used at the University of California, Berkeley in the Nelson Lab and COVID-WEB (covid-web.org) for downstream analysis of extracted wastewater samples for the purpose of SARS-CoV-2 surveillance. We have been successfully using this protocol paired with the Sewage, Salt, Silica, and SARS-CoV-2 (4S) method. We are including multiple assays that we have used throughout this project, although not all of them may be in active use. This method may need to be adapted based on your lab's application, available equipment, and safety requirements. It is intended for use in a BSL2+ lab with extra precautions for processing environmental specimens for SARS-CoV-2 as described in the CDC Interim Laboratory Biosafety Guidelines.</div><div class = "text-block">Version 1 included the following assays: N1, PMMoV, BCoV, and SOC.</div><div class = "text-block">Version 2 was amended to include the following assays: crAssphage, Bacteroides HF183 rRNA, and human 18S rRNA.</div></div>

[STEPS]
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Clean and set up: Clean the work surface and equipment (including recently calibrated pipettes, vortex, tube spinner, and personal gloves) with 10% bleach followed by 70% ethanol followed by .
We worked with University of California, Berkeley EH&S to adapt our protocol for safety considerations. We conduct all of our experimental steps in a class II biosafety cabinet in a BSL2 lab.
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Gather supplies: Gather materials needed for the assay that you are completing (see Assay Details section), including samples, probe, forward primer, reverse primer, master mix, water, and standard. Place on ice. Unless otherwise stated, primers are ordered as LabReady DNA oligos from IDT and arrive at a concentration of .  Probes are ordered as LabReady PrimeTime 5' FAM/ZEN/3' IBFQ (Purification:HPLC Purification from IDT) and arrive at a concentration of
Unless otherwise stated, the forward primer ( ), reverse primer ( ), and probe (  ) are mixed together with PCR water ( ) to create a primer/probe mix for more efficient sample processing. The resulting concentrations in the mix are   for each primer and   probe.
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Keep highly concentrated standards separate from all other components.
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From this point forward, all reagents should be kept below 4C-- either in cooling racks or on ice. Ensure components do not freeze again once thawed before they are used
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Create the Reaction Mix. Assay-specific details are included in section 2.
The reaction mix (sometimes referred to as “Master mix”) contains all of the ingredients needed for RT-qPCR, except for the sample/template itself.
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Calculate the total volume of reaction mix needed. Include 15 μL per well in addition to 10-20% excess (to account for losses and pipetting error).
All assays are set up as 20 μL reactions with 5 μL of template.
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Combine ,  the primer/probe mix, and DNase/RNase free water in
When preparing the reaction mix, keep all reagents cold-- either in cooling racks or on ice. Add components starting with the largest volume.
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Create the standard dilutions. Options for standards for each assay as well as recommended dilution concentrations are described in the following section ("Assay Details").
The standard stock should be resuspended in low EDTA TE buffer with added carrier RNA at a concentration of 0.1-1 mg/L and then aliquotted into single-use Lobind tubes (to avoid freeze-thaw) stored at -80 C. Standard dilutions should be made fresh each day from a newly thawed standard stock.
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Aliquot water in the correct volumes into strip tubes.
Ensure aliquoted water is the correct volume. Pipette back up the water and check for a gap in the tip or water left in the tube.
We find that making dilutions in ~100 uL total volume works well for us. For example, in a 10-fold dilution, 10 uL of the previous standard dilution would be added to 90 uL of water.
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Pipette standard stock solution into the top dilution then proceed with the serial dilution.
Mix thoroughly between each dilution by capping and vortexing, then spin down.
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Change gloves and clean your work station after handling standards.
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Aliquot reaction mix and sample or standard into a 96-well plate
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Place a 96 well plate on ice or in a cool rack. The plates we use in our QuantStudio 3 qPCR machine are. We also use cool racks (IsoFreeze, see below) in place of ice.
Check your instrument for plate compatibility by determining if your plates should be unskirted, semi-skirted, or full-skirted in addition to  the well volume requirements. We recommend using clear plates over colored plates because it is easier to view all wells and corresponding labels.
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Add 15 μL of reaction mix to each well. Before adding to the plate, ensure the mixture is well mixed but avoid bubbles (pipette up and down).
Using a multichannel pipette can expedite this process. Visually inspect the plate afterwards to ensure every well contains reaction mix as intended.
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Add 5 μL of well-mixed sample RNA, no template control (DNase/RNase free water), or standard dilution to each well. Pipette up and down to mix.
No template control (NTC): a qPCR negative control with respect to the target where DNase/RNase free water is input to the reaction instead of sample.
After designing the plate in the qPCR instrument software, print the layout for reference in the lab. This "cheat sheet" can also include volumes of reaction mix components and volumes for standard dilutions.
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Seal the plate with .
Use a rubber plate sealer to press the adhesive film securely onto the plate. Then, using the edge, score around the wells of the plate, between every column, and between every row to prevent cross-contamination.
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Gently vortex the plate to mix and spin down in a plate spinner until all liquid is in the bottom of the wells and bubbles have popped or risen to the surface.
Be sure to inspect the plate to verify that all wells have the same volume in the intended wells.
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Place the plate in the qPCR machine, load the previously designed plate file with cycling conditions included, and start. The qPCR machine we use is a cloud-connected QuantStudio3 Real-Time PCR System: We use the following cycling conditions used for all assays listed in this protocol:  ABCD1StepTemperature (C)Time (min:sec)Number of Cycles2UNG Incubation252:0013Rerverse Transcription5015:0014Polymerase Activation952:0015Denaturation950:03456Annealing/Extension550:30
The instrument can be set to Fast cycling mode. Set data collection during the annealing/extension step
ABCD1StepTemperature (C)Time (min:sec)Number of Cycles2UNG Incubation252:0013Rerverse Transcription5015:0014Polymerase Activation952:0015Denaturation950:03456Annealing/Extension550:30
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Follow-up QA/QCYour full SARS-CoV-2 RT-qPCR protocol should include steps listed in the following subsections.
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Ensure MIQE guidelines are met using the following resource:
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Assess RT-PCR inhibitionInhibition testing could include:Spike and dilute methods Internal positive control addition Direct dilute methods For more information, check the following resources:
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Normalize SARS-CoV-2 signal using a fecal concentration controlThe fecal concentration control target could be:Pepper Mild Mottle virusHuman 18S rRNABacteroides HF183crAssphageFor more information, check the following resources:
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Assess concentration and/or extraction consistencyProcess control(s) could include:proxy virus (e.g., bovine coronavirus) free-RNA (e.g., synthetic oligomer construct)For more information, check the following resources:
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SARS-CoV-2 nucleocapsid N gene (N1) The SARS-CoV-2 nucleocapsid N gene (N1) is one of the CDC assays for SARS-CoV-2 and is our primary target for SARS-CoV-2.
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N1 Reaction Mix RecipeThearrive at a premixed concentration of 15 uM, and the following recipe can be used for the reaction mix:  ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA8.5 μL4Primer/probe mixF: 500 nM, R: 500 nM, P: 125 nMF: 500 nM, R: 500 nM, P: 125 nM1.5 μL5Total15 μL
ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA8.5 μL4Primer/probe mixF: 500 nM, R: 500 nM, P: 125 nMF: 500 nM, R: 500 nM, P: 125 nM1.5 μL5Total15 μL
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N1 Reaction Mix Recipe -- with optional Vetmax Xeno assayThe  arrive at a premixed concentration of 15 uM. For this assay,   and   can be added  in order to assess inhibition in samples (through internal positive control addition). The N1 probe uses a FAM reporter while the Xeno assay uses a VIC reporter, allowing them to be duplexed. The following recipe can be used for the reaction mix:  ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3Water--7.6 μL4Primer/probe mixF: 500 nM, R: 500 nM, P: 125 nMF: 500 nM, R: 500 nM, P: 125 nM1.5 μL5Xeno AssayProprietaryProprietary0.8 μL6Xeno RNA10000 copies/μL50 copies/μL0.1 μL7Total15 μL
ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3Water--7.6 μL4Primer/probe mixF: 500 nM, R: 500 nM, P: 125 nMF: 500 nM, R: 500 nM, P: 125 nM1.5 μL5Xeno AssayProprietaryProprietary0.8 μL6Xeno RNA10000 copies/μL50 copies/μL0.1 μL7Total15 μL
Note that the internal positive control assay and RNA are added to the reaction mix. Therefore, the no template controls (NTCs) will become positive for Vetmax Xeno and can serve as a baseline for inhibition testing.
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N1 primers/probe/amplicon sequencesThe N1 assay has an amplicon length of 72 nucleotides.   AB1forwardGACCCCAAAATCAGCGAAAT2reverseTCTGGTTACTGCCAGTTGAATCTG3probeFAM/ACCCCGCATTACGTTTGGTGGACC/IABkFQ4ampliconGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGA
AB1forwardGACCCCAAAATCAGCGAAAT2reverseTCTGGTTACTGCCAGTTGAATCTG3probeFAM/ACCCCGCATTACGTTTGGTGGACC/IABkFQ4ampliconGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGA
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N1 StandardThere are many options to use as N1 standard. We have used and had success with the following options: Twist Bioscience offers a complete genome synthetic RNA standard: DNA plasmids ordered and linearized for use as a DNA standard:
Our standard curves include the following concentrations: 5, 10, 20, 100, 1000, 10000, and 100000 gene copies/reaction.
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Pepper Mild Mottle virus coat protein gene (PMMoV)Pepper mild mottle virus can be used as a fecal concentration control to normalize N1 signal. The Pepper Mild Mottle virus coat protein gene (PMMoV) primers and probe sequences that we include here were used by Haramoto et. al, 2013.
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PMMoV Reaction Mix RecipeThe PMMoV primer/probe mix is made by adding  of forward primer (LabReady 100 uM),  of reverse primer (LabReady 100 uM), and  of probe (LabReady 100 uM) to  of DNAse/RNAse free water or low EDTA TE buffer. The following recipe can be used for the reaction mix:  ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA9 μL4Primer/probe mixF: 8 uM, R: 8 uM, P: 4 uMF: 400 nM, R: 400 nM, P: 200 nM1 μL5Total15 μL
20 µl
20 µl
10 µl
200 µl
ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA9 μL4Primer/probe mixF: 8 uM, R: 8 uM, P: 4 uMF: 400 nM, R: 400 nM, P: 200 nM1 μL5Total15 μL
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PMMoV primer/probe/amplicon sequences The PMMoV assay has an amplicon length of 68 nucleotides. Note that the original publication (Haramoto et. al, 2013) had a probe with an MGB quencher as a melt temperature modifier, but the ZEN/IBFQ quencher has been working for us.   AB1forwardGAGTGGTTTGACCTTAACGTTTGA2reverseTTGTCGGTTGCAATGCAAGT3probeFAM/CCTACCGAAGCAAATG/ZEN/IABkFQ4amplicon (Accession AB716964)GAGTGGTTTGACCTTAACGTTTGAGCGGCCTACCGAAGCAAATGTCGCACTTGCATTGCAACCGACAA
AB1forwardGAGTGGTTTGACCTTAACGTTTGA2reverseTTGTCGGTTGCAATGCAAGT3probeFAM/CCTACCGAAGCAAATG/ZEN/IABkFQ4amplicon (Accession AB716964)GAGTGGTTTGACCTTAACGTTTGAGCGGCCTACCGAAGCAAATGTCGCACTTGCATTGCAACCGACAA
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PMMoV StandardWe have successfully used the following PMMoV standards:RNA Ultramer from IDTDNA gBlock from IDTThe standard sequence is based on the amplicon plus additional 5 nucleotides from the NCBI sequence (Accession: AB71694) added to each end: AAUGAGAGUGGUUUGACCUUAACGUUUGAGCGGCCUACCGAAGCAAAUGUCGCACUUGCAUUGCAACCGACAAUUGCA
Our standard curves include the following concentrations: 102, 103, 104, 105,106, 107, and 108 gene copies/reaction.
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Bovine coronavirus transmembrane protein gene (BCoV)Bovine coronavirus can be used as a spike-in process control. The bovine coronavirus transmembrane protein gene (BCoV) primers and probe sequences that we include here were used by Decaro, et. al 2008.
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BCoV Reaction Mix RecipeThe BCoV primer/probe mix is made by adding  of forward primer (LabReady 100 uM),  of reverse primer (LabReady 100 uM), and  of probe (LabReady 100 uM) to  of DNAse/RNAse free water or low EDTA TE buffer.  ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA7.59 μL4Primer/probe mixF: 7.5 uM, R: 7.5 uM, P: 2.1 nMF: 900 nM, R: 900 nM, P: 250 nM2.41 μL5Total15 μL
18 µl
18 µl
5 µl
200 µl
ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA7.59 μL4Primer/probe mixF: 7.5 uM, R: 7.5 uM, P: 2.1 nMF: 900 nM, R: 900 nM, P: 250 nM2.41 μL5Total15 μL
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BCoV primers/probe/ampliconThe BCoV assay has an amplicon length of 85 nucleotides.   AB1forwardCTGGAAGTTGGTGGAGTT2reverseATTATCGGCCTAACATACATC3probeFAM/CCTTCATATCTATACACATCAAGTTGTT/IABkFQ4amplicon (Accession AF39154)CTGGAAGTTGGTGGAGTTTCAACCCAGAAACAAACAACTTGATGTGTATAGATATGAAGGGAAGGATGTATGTTAGGCCGATAAT
AB1forwardCTGGAAGTTGGTGGAGTT2reverseATTATCGGCCTAACATACATC3probeFAM/CCTTCATATCTATACACATCAAGTTGTT/IABkFQ4amplicon (Accession AF39154)CTGGAAGTTGGTGGAGTTTCAACCCAGAAACAAACAACTTGATGTGTATAGATATGAAGGGAAGGATGTATGTTAGGCCGATAAT
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BCoV StandardWe have successfully used the following types of standards for this assay:RNA Ultramer from IDTDNA gBlock from IDT
The standard curve range will depend on the amount spiked into the sample and the expected recovery. Our standard curves include the following concentrations: 103, 104, 105,106, 107, and 108   gene copies/reaction.
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Synthetic Oligomer Construct T33-21 free-RNA (SOC) We designed an assay that targets RNA that is not naturally present in wastewater to serve as a free-RNA recovery control that we could spike into our samples. To this end, we in vitro transcribed RNA from a synthetic oligomer construct, T33-21 (12-mer) from Hsia et. al 2016.
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SOC reaction mix recipe  ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA8.5 μL4Primer/probe mixF: 6.7 uM, R: 6.7 uM, P: 1.7 uMF: 500 nM, R: 500 nM, P: 125 nM1.5 μL5Total15 μL
ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA8.5 μL4Primer/probe mixF: 6.7 uM, R: 6.7 uM, P: 1.7 uMF: 500 nM, R: 500 nM, P: 125 nM1.5 μL5Total15 μL
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SOC primers/probe/amplicon sequencesThe SOC assay amplicon length is 89 nucleotides.    AB1forwardCCACCAAAGTGGGCGATAAA2reverseGGTGCCATTCGCCTCAATAA3probeFAM/TGGCGGTGAGGAAGTTTGGAAAGA/IABkFQ4ampliconCCACCAAAGTGGGCGATAAAGGCAGCACCCGTTTATTTGGCGGTGAGGAAGTTTGGAAAGATAGCCCGATTATTGAGGCGAATGGCACC
AB1forwardCCACCAAAGTGGGCGATAAA2reverseGGTGCCATTCGCCTCAATAA3probeFAM/TGGCGGTGAGGAAGTTTGGAAAGA/IABkFQ4ampliconCCACCAAAGTGGGCGATAAAGGCAGCACCCGTTTATTTGGCGGTGAGGAAGTTTGGAAAGATAGCCCGATTATTGAGGCGAATGGCACC
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SOC standard and spike-in SOC RNA standards were generated by in vitro transcription of an IDT gBlock corresponding to the amplicon with a T7 promoter added to the 5' end of the sequence. We used as per manufacturer's specifications to generate crude RNA and digested with DNase I to remove the gBlock template. We purified this crude RNA with  and quantified the standard by Nanodrop.
The standard curve range will depend on the amount spiked into the sample and the expected recovery. Our standard curves include the following concentrations:  104, 105,106, 107,108, and 109 gene copies/reaction.
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CrAssphage CPQ_056 (crAssphage)CrAssphage has been proposed as a fecal concentration control to normalize N1 signal. The primers and probe described in this protocol were designed by Stachler et. al, 2017.
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Bacteroides 16S ribosomal RNA HF183/BacR287 (Bacteroides) Bacteroides has been proposed as a fecal concentration control to normalize N1 signal, although we have not had success with it for SARS-CoV-2 WBE. The primers and probe for this assay were previously presented in Green et. al, 2014.
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Human 18S ribosomal subunit RNA (18S rRNA)The human 18S rRNA assay was designed by our group (first presented in Whitney et. al, 2020) for use as a fecal concentration control to normalize N1 signal. Although we have not had success with it for SARS-CoV-2 normalization, it may be a promising degradation control, indicating when samples may no longer reflect accurate target values.
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18S rRNA Reaction Mix Recipe  ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA8.5 μL4Primer/probe mixF: 6.7 uM, R: 6.7 uM, P: 1.7 uMF: 500 nM, R: 500 nM, P: 125 nM1.5 μL5Total15 μL
ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA8.5 μL4Primer/probe mixF: 6.7 uM, R: 6.7 uM, P: 1.7 uMF: 500 nM, R: 500 nM, P: 125 nM1.5 μL5Total15 μL
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CrAssphage Reaction Mix Recipe  ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA8.5 μL4Primer/probe mixF: 6.7 uM, R: 6.7 uM, P: 1.7 uMF: 500 nM, R: 500 nM, P: 125 nM1.5 μL5Total15 μL
ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA8.5 μL4Primer/probe mixF: 6.7 uM, R: 6.7 uM, P: 1.7 uMF: 500 nM, R: 500 nM, P: 125 nM1.5 μL5Total15 μL
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CrAssphage primers/probe/ampliconThe crAssphage CPQ_056 amplicon is 126 nucleotides long. forward 056F1CAGAAGTACAAACTCCTAAAAAA CGTAGAGreverse 056R1GATGACCAATAAACAAGCCATTA GCprobe 056P1FAM-AATAACGATTTACGTGATGTAACamplicon (Accession AF39154)CAGAAGTACAAACTCCTAAAAAACGTAGAGGTAGAGGTATTAATAACGATTTACGTGATGTAACTCGTAAAAAGTTTGATGAACATACTGATTGTAATAAAGCTAATGGCTTGTTTATTGGTCATC
forward 056F1CAGAAGTACAAACTCCTAAAAAA CGTAGAGreverse 056R1GATGACCAATAAACAAGCCATTA GCprobe 056P1FAM-AATAACGATTTACGTGATGTAACamplicon (Accession AF39154)CAGAAGTACAAACTCCTAAAAAACGTAGAGGTAGAGGTATTAATAACGATTTACGTGATGTAACTCGTAAAAAGTTTGATGAACATACTGATTGTAATAAAGCTAATGGCTTGTTTATTGGTCATC
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CrAssphage StandardFor the crAssphage standard, we ordered a custom geneBlocks DNA (Integrated DNA Technologies).
Our standard curve includes the following concentrations: 1e3, 1e4, 1e5, 1e6, 1e7, 1e8, 1e9 gene copies/reaction.
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Bacteroides Reaction Mix Recipe  ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA8.5 μL4Primer/probe mixF: 6.7 uM, R: 6.7 uM, P: 1.7 uMF: 500 nM, R: 500 nM, P: 125 nM1.5 μL5Total15 μL
ABCD1ReagentStock ConcentrationReaction ConcentrationVolume per reaction2Taqman Master Mix4x1x5 μL3WaterNANA8.5 μL4Primer/probe mixF: 6.7 uM, R: 6.7 uM, P: 1.7 uMF: 500 nM, R: 500 nM, P: 125 nM1.5 μL5Total15 μL
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Bacteroides primers/probe/ampliconThe Bacteroides amplicon is 126 nucleotides long.forwardATCATGAGTTCACATGTCCGreverseCTTCCTCTCAGAACCCCTATCCprobeFAM/AT CGT TGA C/ZEN/T AGG TGG GCC GTT AC/IBFQamplicon (Accession MT464394.1)ATCATGAGTTCACATGTCCGCATGATTAAAGGTATTTTCCGGTAGACGATGGGGATGCGTTCCATTAGATAGTAGGCGGGGTAACGGCCCACCTAGTCAACGATGGATAGGGGTTCTGAGAGGAAG
forwardATCATGAGTTCACATGTCCGreverseCTTCCTCTCAGAACCCCTATCCprobeFAM/AT CGT TGA C/ZEN/T AGG TGG GCC GTT AC/IBFQamplicon (Accession MT464394.1)ATCATGAGTTCACATGTCCGCATGATTAAAGGTATTTTCCGGTAGACGATGGGGATGCGTTCCATTAGATAGTAGGCGGGGTAACGGCCCACCTAGTCAACGATGGATAGGGGTTCTGAGAGGAAG
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Bacteroides StandardBacteroides RNA standards were generated by in vitro transcription of an IDT gBlock corresponding to the amplicon with a T7 promoter added to the 5' end of the sequence. We used as per manufacturer's specifications to generate crude RNA and digested with DNase I to remove the gBlock template. We purified this crude RNA with  and quantified the standard by Nanodrop.
Our standard curve includes the following concentrations: 2e2, 2e3, 2e4, 2e5, 2e6, 2e7, 2e8 gene copies/reaction.
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18S rRNA primers/probe/ampliconThe 18S rRNA amplicon is 138 nucleotides long.forwardGGTTCCTTTGGTCGCTCGCTreverseGGGCTGACCGGGTTGGTTTTprobeFAM/AG AGC TAA T/ZEN/A CAT GCC GAC GGG C/IBFQ/amplicon (Accession 6G18_2)GGTTCCTTTGGTCGCTCGCTCCTCTCCTACTTGGATAACTGTGGTAATTCTAGAGCTAATACATGCCGACGGGCGCTGACCCCCTTCGCGGGGGGGATGCGTGCATTTATCAGATCAAAACCAACCCGGTCAGCCC
forwardGGTTCCTTTGGTCGCTCGCTreverseGGGCTGACCGGGTTGGTTTTprobeFAM/AG AGC TAA T/ZEN/A CAT GCC GAC GGG C/IBFQ/amplicon (Accession 6G18_2)GGTTCCTTTGGTCGCTCGCTCCTCTCCTACTTGGATAACTGTGGTAATTCTAGAGCTAATACATGCCGACGGGCGCTGACCCCCTTCGCGGGGGGGATGCGTGCATTTATCAGATCAAAACCAACCCGGTCAGCCC
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18S rRNA Standard18S RNA standards were generated by in vitro transcription of an IDT gBlock corresponding to the amplicon with a T7 promoter added to the 5' end of the sequence. We used as per manufacturer's specifications to generate crude RNA and digested with DNase I to remove the gBlock template. We purified this crude RNA with  and quantified the standard by Nanodrop.
Our standard curve includes the following concentrations: 1e2, 1e3, 1e4, 1e5, 1e6, 1e7, 1e8 gene copies/reaction.