TITLE: Binary synthetic template oligonucleotide positive control for in-house diagnostic real-time RT-PCR
AUTHORS: Ian Mackay, Judy Northill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details how to make and use a pair of synthetic template oligonucleotides (STOs) for use as real-time reverse transcription polymerase chain reaction (RT-rPCR) controls. The method can also be used for PCR controls. This binary positive control approach is easy to replicate once mastered, quick to produce, results in enough RNA stock for years of PCR runs, and provides several benefits over the use of a wild-type positive control such as the nucleic acids extracted from virus culture or from a known previously-positive clinical specimen.</div><div class = "text-block">The method is an adaptation of a previsouly published approach [Ref 4; see Steps section] and has recently been described in some publications.[Ref 5]</div><div class = "text-block">Our approach to a positive control requires that each PCR run includes two additional reactions. </div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"><span>one controls for primer function (</span><span style = "font-weight:bold;">PRIMER </span><span>control). This STO includes hybridisation sites for the specific target assay's primers bouding a generic probe hybridisation target site</span></li><li style = "counter-reset:ol0;"><span>one controls for probe function (</span><span style = "font-weight:bold;">PROBE </span><span>control). This STO includes a hybridisation site for the specific target assay's probe(s), bounded by a pair of generic primer hybridisation target sites </span></li></ol></div></div>

[STEPS]
?. [Overview]
This protocol describes the design and use of a binary positive control system for use in clinical biospecimen testing. An earlier version of this method was previously published, as was this more recent version [Ref.1, Ref.2] The end products avoid the need to handle infectious wild-type virus or rely on previously positive clinical specimens for a PCR positive control source. Ours is a public health virology laboratory, so the example below uses a specific virus, but it could easily be adapted for non-viral targets. I have chosen an RNA virus (enterovirus, EV-D68) and a pre-existing real-time reverse transcription polymerase chain reaction (RT-rPCR) test. I've included comments on how to adapt this protocol for rPCR of a DNA virus. We will create two synthetic template oligonucleotides (STOs) for inclusion alongside a non-template (water) control in every RT-rPCR run we perform. Each template challenges either the virus test's primers (PRIMER control) or the virus test's probe (PROBE control), is run in a separate reaction mix and will generate distinct fluorescent signals when amplified in the presence of an appropriate reaction mix.This work is conducted in a physically distinct room that is separate from biospecimen extraction, template addition and post-PCR activities. After working in the HiCCO (high concentration clean oligonucleotide) room, the scientist may no longer enter extraction or template addition rooms until the following day after a shower and clothing change.The benefits of this approach include:reducing the risk of contamination of PCR reactions with wild-type virus nucleic acid control materialeliminating the risk of infection from handling new, emerging or exotic infectious materialonly need a target region's nucleotide sequence, removing difficulties and delays due to procuring hard to obtain and/or dangerous materials from overseas or in the early stages of an outbreakcreate an easy-to-renew, long-life source of test control materialpermitting independent verification of primer or probe performance in each test runverifying the RT and PCR reactions are performing within specificationscreating quantified reagents for internal quality assurance programs
?. [Purchase]
Once designed, the STOs can be ordered from your manufacturer of choice.One example product is IDT's "Ultramer" at 4nmole scale. Each comes dry and suitably purified to be used in the next in vitro transcription step after resuspension in molecular grade water. We recommend ordering STO DNA from a facility that is different to the one from which you purchase the diagnostics primers and probes. If that isn't possible, at least order the two set of reagents a week apart, not in a single batch.
?. [In vitro transcription]
Reconstitute each lyophilised STO DNA to a final concentration of 200µM (equivalent to 200 picomoles per microliter; pmol/µl), in their original tube, using nuclease-free water.Affix a printed label to the tube stating the date of reconstitution and final concentration. Add another label to the STO datasheet as well as reconstitution details, and file. Freeze unused reconstituted STO at -20ºC.In a 200μl (0.2ml) PCR tube, assemble the following reaction mix on the bench at room temperature (only thaw frozen reagents as needed, keep them in a cold brick, then immediately return them to the freezer when finished with them):  AB1  <b style="mso-bidi-font-weight:
  normal">Reagent
    <span style="mso-bidi-font-size:11.0pt;
  mso-bidi-font-family:Arial">Volume (µl)
  2Transcription Optimized 5X Buffer1  20
  3100mM  DTT  10
  4rRNasin (40U/µl)  2.5
  5rNTPs (A, G, C, U) at 2.5mM each2  20
  62,000-5,000ng of FWD strand2  STO DNA  3
  730-40U T7 RNA polymerase (kit has 500U or  ~20rxn at 1µl/@10-20U/rxn  2
  8  <span style="mso-bidi-font-size:
  11.0pt;mso-bidi-font-family:Arial">H2O
    42.5
  9  Total volume
    100
  Incubate the tube in a 96-well conventional thermocycler using with the following conditions:37ºC    60min4ºC      hold
AB1  <b style="mso-bidi-font-weight:
  normal">Reagent
    <span style="mso-bidi-font-size:11.0pt;
  mso-bidi-font-family:Arial">Volume (µl)
  2Transcription Optimized 5X Buffer1  20
  3100mM  DTT  10
  4rRNasin (40U/µl)  2.5
  5rNTPs (A, G, C, U) at 2.5mM each2  20
  62,000-5,000ng of FWD strand2  STO DNA  3
  730-40U T7 RNA polymerase (kit has 500U or  ~20rxn at 1µl/@10-20U/rxn  2
  8  <span style="mso-bidi-font-size:
  11.0pt;mso-bidi-font-family:Arial">H2O
    42.5
  9  Total volume
    100
?. [DNA template removal from in vitro transcribed RNA]
Stop the thermocycler and remove the tube.To this tube add the following reaction mix on the bench at room temperature (only thaw frozen reagents as needed, keep them in a cold brick, then immediately return them to the freezer when finished with them):  AB1  <b style="mso-bidi-font-weight:
  normal">Reagent
    <span style="mso-bidi-font-size:11.0pt;
  mso-bidi-font-family:Arial">Volume (µl)
  2ivtRNA preparation from 5  100
  3  <span style="font-family:&quot;Arial&quot;,sans-serif;
  color:black">10X TURBO DNase Buffer1 (0.1X total volume)
    12
  4TURBO DNase (4U; removes up to 20µg of genomic DNA)  2
  5  <span style="font-family:
  &quot;Arial&quot;,sans-serif">H2O
    6
  6  Total volume
    120
  Split by transferring 60µl into a new 200μl tube.Incubate both tubes in a 96-well conventional thermocycler using with the following conditions: 37ºC  30min4ºC  hold Remove each tube, add 1µl of TURBO DNase, mix by pipetting, return the tubes to the thermocycler and incubate them using the following cycling conditions: 37ºC  30min4ºC  holdRemove the tubes, combine contents into a fresh 1.5ml tube and add 14µl of thawed DNase Inactivation Reagent (10X) to each 0.2ml tube. Incubate at room temperature for 5min, flicking 2-3 times during incubationCentrifuge at 10,000xg for 1.5minUse a micropipette to carefully transfer the supernatant into a new nuclease-free 1.5ml tubeRemove each tube, add 1µl of TURBO DNase, mix by pipetting, return the tubes to the thermocycler and incubate them using the following cycling conditions:37ºC  30min4ºC  holdRemove the tubes, combine contents into a fresh 1.5ml tube and add 14µl of thawed DNase Inactivation Reagent (10X) to each 0.2ml tube. Incubate at room temperature for 5min, flicking 2-3 times during incubationCentrifuge at 10,000xg for 1.5minUse a micropipette to carefully transfer the supernatant into a new nuclease-free 1.5ml tubeRemove the tubes, combine contents into a fresh 1.5ml tube and add 14µl of thawed DNase Inactivation Reagent (10X) to each 0.2ml tube. Incubate at room temperature for 5min, flicking 2-3 times during incubationCentrifuge at 10,000xg for 1.5minUse a micropipette to carefully transfer the supernatant into a new nuclease-free 1.5ml tube
AB1  <b style="mso-bidi-font-weight:
  normal">Reagent
    <span style="mso-bidi-font-size:11.0pt;
  mso-bidi-font-family:Arial">Volume (µl)
  2ivtRNA preparation from 5  100
  3  <span style="font-family:&quot;Arial&quot;,sans-serif;
  color:black">10X TURBO DNase Buffer1 (0.1X total volume)
    12
  4TURBO DNase (4U; removes up to 20µg of genomic DNA)  2
  5  <span style="font-family:
  &quot;Arial&quot;,sans-serif">H2O
    6
  6  Total volume
    120
?. [Storage of in vitro transcribed RNA]
Divide the undiluted in vitro STO RNA preparation into two labelled tubes. Obtain an optical density on one aliquot of the undiluted material. Store in separate locations as a safeguard for freezer failure or access limitations during laboratory maintenance etc.. Store at -80°C where they will remain amplifiable for years. Location details should be recorded in full.
?. [Titration, testing and storing of dilutions]
PCR and RT-PCR are used to identify how much amplifiable STO DNA template remains in the RNA preparation and to crudely estimate the amplifiable RNA content of the preparation, respectively. The procedure is as follows: Thaw one vial, label as having been thawed Pre-label 10X 2ml screw-capped tubes Add 495µl RNase-free water to the first tube and 450µl to the other nine tubes Prepare a 10-fold serial dilution from 10-2 to 10-11 in the prepared tubes keeping tubes on ice between transfer and vortexing stepsIn a purpose-specific BSCII, transfer 5µl of undiluted stock in vitro transcribed STO RNA into the tube preloaded with 495µl water – this is the 10-2 dilution. We start at this dilution to save stock and avoid too much RNA being brought into the template addition area. Using a vortex in BSCII, subject the re-capped tube to three pulses of 2-5 seconds each. Return the stock to the freezer.Transfer 50µl to the next tube, this one preloaded with 450µl RNase-free water and repeat this process, with vortexing as described after each transfer until complete, discarding the final 50µlThe synPri or synPrb RNA STO control is added as if it was the biospecimen extract sample. The only difference is that  mixes must also incorporate the required UBE2D2 primers or probe to "complete" the missing pieces needed to amplify the hybrid templates.Sufficient PCR mix is made to test the entire dilution series in duplicate + NTC + a small excess. See an example in the Table below, and scale up as required. Add STO RNA/DNA to rPCR and RT-rPCR mixes in duplicate and aliquot into tubes, plate or ring appropriate to the machine being used and space required. We initially test the dilution series using both RT-rPCR and rPCR (specific PCR mixes, not an RT-rtPCR without the RT step) to identify the amount of amplifiable DNA remaining in the RNA preparation. There will always be some, but it's important to gauge how much. When using an RNA STO, if the rtPCR comes up positive it is still possible to create a stock of useful RNA STO so long as the last RT-PCR positive dilution is ≥1 log10 dilution greater (more dilute) than the last PCR positive dilution. This is an arbitrary value.If the final dilution to amplify by rPCR is The synPri or synPrb RNA STO control is added as if it was the biospecimen extract sample. The only difference is that  mixes must also incorporate the required UBE2D2 primers or probe to "complete" the missing pieces needed to amplify the hybrid templates.Sufficient PCR mix is made to test the entire dilution series in duplicate + NTC + a small excess. See an example in the Table below, and scale up as required. Add STO RNA/DNA to rPCR and RT-rPCR mixes in duplicate and aliquot into tubes, plate or ring appropriate to the machine being used and space required.We initially test the dilution series using both RT-rPCR and rPCR (specific PCR mixes, not an RT-rtPCR without the RT step) to identify the amount of amplifiable DNA remaining in the RNA preparation. There will always be some, but it's important to gauge how much. When using an RNA STO, if the rtPCR comes up positive it is still possible to create a stock of useful RNA STO so long as the last RT-PCR positive dilution is ≥1 log10 dilution greater (more dilute) than the last PCR positive dilution. This is an arbitrary value.  ABC1<span style="mso-bidi-font-size:11.0pt;
  line-height:115%;mso-bidi-font-family:Arial">Volumes (µl)PRIMER control (µl)PROBE control (µl)2rPCR/RT-rPCR Mastermix (incl water & virus-specific primers & probe)14.014.03UBE2D2 Fwd & Rev primer working stock (6µM & 18µM)-1.04UBE2D2 probe working stock (5µM)1.0-5synPrb STO at chosen dilution-5.06synPri STO at chosen dilution5.0-7Total volume  20
    20
   An example result for the EV-D68 is shown below. The RNA tested was the follwing dilution series of synPri and synPrb STO RNA: 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8, 10-9, 10-10, 10-11 The highest conentraiton generates a fluorescnet signal first (lowest CT≡highest template load), followed by the next dilution, then the next, and so on. Sigmoidal curves are ideally separated by ∼3.3 cycles.rPCR TESTING FOR RESIDUAL DNA CONTENTRT-rPCR TESTING FOR RESIDUAL RNA CONTENTFor routine use, I'd recommend 10-6 as the usual dilution for routine RT-rPCR control use, in this example. This dilution produced a good sigmoidal shaped curve of suitable height that fell between a CT of 20-25 in this test in our hands.The repeatability and reproducibility of this can be used in the subsequent assay validation process and for ongoing monitoring of the test's and control's behaviour over time.
ABC1<span style="mso-bidi-font-size:11.0pt;
  line-height:115%;mso-bidi-font-family:Arial">Volumes (µl)PRIMER control (µl)PROBE control (µl)2rPCR/RT-rPCR Mastermix (incl water & virus-specific primers & probe)14.014.03UBE2D2 Fwd & Rev primer working stock (6µM & 18µM)-1.04UBE2D2 probe working stock (5µM)1.0-5synPrb STO at chosen dilution-5.06synPri STO at chosen dilution5.0-7Total volume  20
    20
?. [Regular use]
Once testing is complete, the NEAT stocks are returned to long-term storage.Working dilutions that are of use (10-5 to 10-9 in the example above) can also be stored until needed.For regular use, stocks of synPri+UBE2D2 probe+water (PRIMER controls) and synPrb+UBE2D2 primers+water (PROBE controls) can be made and frozen. Batches of multi-use tubes and single-use tubes can be made depending on your laboratory's throughput. Each diagnostic rPCR or RT-rPCR run muct include one of each control mix tube.  ABC1Premade PRIMER Control mix21X100X3Hb5c2 Prb (100µM)0.05µl5µl4H2O4.95µl495µl5TOTAL5.0µl500µl  ABC1Premade PROBE Control mix21X100X3UBE2D2_01.2 (200µM)0.03µl3µl4UBE2D2_02.2 (200µM)0.09µl9µl5H2O4.88µl488µl6TOTAL5.0µl500µlA further variation can be adopted for eh high-throughput PCR laboratory. The mixes above are adjusted to 2.5ul final volumes. Separate virus oligonucleotide mixes (primers, probes and water) are prepared to a final 2.5ul volume. Bulk numbers of mastermix aliquots are made and stored. Then mastermix can be thawed and a range of different virus-specific RT-rPCR/rPCR can be run on the same cycler by efficiently combining appropriate oligonucleotide and control mixes with the generic RT-rPCR or rPCR mastermix, as required.
ABC1Premade PRIMER Control mix21X100X3Hb5c2 Prb (100µM)0.05µl5µl4H2O4.95µl495µl5TOTAL5.0µl500µl
ABC1Premade PROBE Control mix21X100X3UBE2D2_01.2 (200µM)0.03µl3µl4UBE2D2_02.2 (200µM)0.09µl9µl5H2O4.88µl488µl6TOTAL5.0µl500µl
?. [STO design]
We next design the two single-stranded DNA STOs, PRIMER control and PROBE control. These are designed to be rPCR-amplifiable and detectable templates. This means they each incorporate forward primer, reverse primer and probe hybridisation sequences in appropriate orientation. We will design these for an RT-PCR so they will include a 5' T7 (could also use SP6) transcription initiation site [Ref.3] preceding the primer-probe-primer sequence. If you are designing a DNA virus test, you can leave off the T7 primer sequence and skip over the in vitro transcription step as you won't need to make RNA.PRIMER CONTROL DESIGNa single amplifiable STO target sequence (this one is 94nt long) is designed (using Word, Excel or a sequence analysis software package like Geneious) by laying the oligonucleotides next to each other, separated by spacer sequences (bold).I find it helps to visualise the STO and use colour to identify the various hybridisation targets the single-strand is only as long as the primers+probe hybridisation sites, plus the T7 transcription initiation site (pink).the PRIMER control tests the virus assay primers, not the probe, so it contains virus primer hybridisation sites (blue) plus that for the UBE2D2 probe (orange). 
PROBE CONTROL DESIGNa single amplifiable STO target sequence (this one is 106nt long) is designed (using Word, Excel or a sequence analysis software package like Geneious) by laying the oligonucleotides next to each other, separated by spacer sequences (bold).the single-strand is only as long as the primers+probe hybridisation sites, plus the T7 transcription initiation site (pink).the PROBE control only tests the virus assay probe, not the primers, so it contains a virus probe hybridisation site (green) plus those for the UBE2D2 primers (orange).PROBE CONTROL DESIGNa single amplifiable STO target sequence (this one is 106nt long) is designed (using Word, Excel or a sequence analysis software package like Geneious) by laying the oligonucleotides next to each other, separated by spacer sequences (bold).the single-strand is only as long as the primers+probe hybridisation sites, plus the T7 transcription initiation site (pink).the PROBE control only tests the virus assay probe, not the primers, so it contains a virus probe hybridisation site (green) plus those for the UBE2D2 primers (orange).
?. [Nucleotide sequences and how they are used]
As an example we will use enterovirus species D genotype 68 (EV-D68) for the test target. We have previously described this assay here. This assay uses 2 forward primers to account for virus variation, but they hybridise to the same site so they are treated as a single oligo in the design of our PRIMER control STO. The PRIMER control STO design incorporates the EV-D68 primer hybridisation site sequences and a non-viral probe hybridisation site sequenceThe PROBE control STO design incorporates the EV-D68 probe hybridisation site sequence and a pair of non-viral primer hybridisation site sequencesWe use human gene sequences from the human E2 ubiquitin conjugating enzyme E2 D2 (UBE2D2; [Ref.4] GenBank accession U39317) for our non-viral primer and probe sequences.    ABC1Oligonucleotide nameRoleSequence (5' - 3')2UBE2D2_01.2UBE2D21 forward primerTGAAGAGAATCCACAAGGAATTGA3UBE2D2_02.2UBE2D2 reverse primerCAACAGGACCTGCTGAACACTG4UbcH5B_TMUBE2D2 probeCAL FLUOR ORG 5602 - TGATCTGGCACGGGACCCTCCA - BHQ15EV-D68-For1EV-D68 forward primerTGTTYCCACGGTTGAAAAYAA6EV-D68-For2EV-D68 forward primerTTCCCACGGTTGAAARNYRAC7EV-D68-RevEV-D68 reverse primerCAAGCTACACACGGGTTAGT8EV-D68-FAM-TM2018EV-D68 probeFAM2 - CCGTTAWCCGCTATAGTACTTCGAGAAACC - BHQ1
ABC1Oligonucleotide nameRoleSequence (5' - 3')2UBE2D2_01.2UBE2D21 forward primerTGAAGAGAATCCACAAGGAATTGA3UBE2D2_02.2UBE2D2 reverse primerCAACAGGACCTGCTGAACACTG4UbcH5B_TMUBE2D2 probeCAL FLUOR ORG 5602 - TGATCTGGCACGGGACCCTCCA - BHQ15EV-D68-For1EV-D68 forward primerTGTTYCCACGGTTGAAAAYAA6EV-D68-For2EV-D68 forward primerTTCCCACGGTTGAAARNYRAC7EV-D68-RevEV-D68 reverse primerCAAGCTACACACGGGTTAGT8EV-D68-FAM-TM2018EV-D68 probeFAM2 - CCGTTAWCCGCTATAGTACTTCGAGAAACC - BHQ1
?. [References]
Smith G, Smith I, Harrower B, Warrilow D, Bletchly C. A simple method for preparing synthetic controls for conventional and real-time PCR for the identification of endemic and exotic disease agents. J Virol Methods. 2006 Aug;135(2):229-34. https://www.ncbi.nlm.nih.gov/pubmed/16677717Hall-Mendelin S, Pyke AT, Moore PR, Mackay IM, McMahon JL, Ritchie SA, Taylor CT, Moore FA, van den Hurk AF. Assessment of Local Mosquito Species Incriminates Aedes aegypti as the Potential Vector of Zika Virus in Australia. PLoS Negl Trop Dis. 2016 Sep 19;10(9):e0004959. https://www.ncbi.nlm.nih.gov/pubmed/27643685Transcription of RNA templates by T7 RNA polymerase. Nucl. Acids Res. (1998) 26 (15): 3550-3554. Arnaud-Barbe et al.UBE2D2. https://en.wikipedia.org/wiki/UBE2D2Hamalainen HK, et al. Identification and validation of endogenous reference genes for expression profiling of T helper cell differentiation by quantitative real-time RT-PCR. Anal Biochem 2001; 299: 63–70. https://www.ncbi.nlm.nih.gov/pubmed/11726185
?. [Purification of in vitro transcribed RNA]
To purify RNA, we have summarized an RNeasy kit method below performed on the bench at room temperature:Add 250µl 100% ethanol to supernatant from 6, mix by pipetting up and down and transfer total to an RNeasy spin columnSpin at 10,000rpm for 15secTransfer RNeasy column to a fresh collection tube, discard old collection tubeAdd 500µL Buffer RPESpin at 10,000rpm for 15secTransfer RNeasy column to a fresh collection tube, discard old collection tubeAdd 500µL Buffer RPESpin at 10,000rpm for 2minTransfer RNeasy column to a nuclease-free 1.5ml tube (included), discard old collection tubeAdd 50µl RNase-free waterSpin at 10,000rpm for 1min to elute STO RNATransfer eluate to a labelled screw cap tube