A simple and inexpensive RT-qPCR panel for human respiratory viruses

I have set up a very small lab for identification of human respiratory viruses for educational and research purposes. After some trial-and-error, the following is what I've found to be the simplest and most cost effective. It should go without saying that biosafety protocols and appropriate regulations must be followed. Feel free to contact me with any questions.

Materials

• qPCR machine:
  • Biomeme Franklin ($10,000 USD): https://shop.biomeme.com/collections/devices/products/franklin-real-time-pcr-thermocycler
    • Requires custom empty tubes: https://shop.biomeme.com/collections/accessories/products/biomeme-go-strips-100-qty
  • I’ve also had success with the miniPCR mini8 ($650) using visual quantification, but it’s labor intensive to get even approximately useful Ct values.
    • https://www.minipcr.com/product/minipcr-mini8-thermal-cycler-color/
    • https://www.minipcr.com/product/covid-qpcr/
• RNA extraction and purification:
  • Biomeme M1 RNA ($1,440 for 96 samples)
  • Or Biobasic EZ-10 Spin Column Viral Total RNA Extraction Kit ($158 for 50 samples)
• RT-PCR master Mix
  • NEB Luna Universal Probe qPCR Master Mix
• Custom primers and probes
  • IDT Custom ssDNA oligos and PrimeTime qPCR probes
    • Ordering primers and probes separately instead of using the PrimeTime assay affords more flexibility and cost savings
    • Probes can be rather expensive so I tried with primers only and dye chemistry but found off-target amplification was inevitable and so it was unreliable to the point of impracticality
• Microcentrifuge - eg. miniPCR gyro plus
• Optional: Vortexer - eg. Vornado
• Nasal swabs and viral transport tubes, micropipettes (Amazon), tips, tubes and other basic lab supplies (Biobasic)

Method

• Order primers and probes for 15 common respiratory viruses along with one human internal control - B2M.
  • See panel design and sequences and their corresponding citations: https://docs.google.com/spreadsheets/d/1Unzecg72gMkiTvilp634tVyCdSa6qWtwTcD0QKhtI0g/edit#gid=1750282749
• Make up working stocks of the 6 multiplex assays (primers+probes) according to concentrations in the panel design
• Extract and purify sample(s) following Biomeme M1 / EZ-10 instructions
  • Note that the Biomeme M1 RNA extraction kit seems to reliably extract DNA as well but there could be some loss of sensitivity for the DNA targets.
  • If desired, can pool multiple samples together, re-running individual samples for the positive assay if any.
• Mix 60µl Luna MM with 6µl Luna RT in tube labeled “RTMM”
• Prepare wells as follows
  • 1: RTMM 11µl, RVP1 2µl, sample 7µl
  • 2: RTMM 11µl, PIVP 2µl, sample 7µl
  • 3: Luna MM 10µl, DVP 2µl, sample 8µl
  • 4: RTMM 11µl, INF 2µl, sample 7µl
  • 5: RTMM 11µl, COV1 3µl, sample 6µl
  • 6: RTMM 11µl, COV2 3µl, sample 6µl
• Run PCR according to Luna instructions, eg.:
  • Reverse Transcription 50°C, 600 sec
  • Initial denaturation 95°C, 60 sec
  • Denaturation 95°C, 10 sec
  • Anneal / Extension 60°C, 30 sec
  • Number of cycles 45
• In Biomeme cloud analysis software, view curves and adjust thresholds to ensure only curves with exponential growth are captured
• A good sample will result in the B2M control (COV2 grn channel) having a Cq of less than 25