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

A novel real-time PCR assay panel for detection of common respiratory pathogens in a convenient, strip-tube array format

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

Method

  • Order primers and probes for 15 common respiratory viruses along with one human internal control - B2M.
  • 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

Article TitleA novel real-time PCR assay panel for detection of common respiratory pathogens in a convenient, strip-tube array format


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Rick Byers3 months ago

Shoot, it looks like all my links got stripped out. Here's the key one for the panel design: https://docs.google.com/spreadsheets/d/1Unzecg72gMkiTvilp634tVyCdSa6qWtwTcD0QKhtI0g/edit?usp=drivesdk. I'll try to fix it up later.

Guy Rohkin3 months ago

Links seem accessible to me on my end from here~!

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