AMR Detection by dPCR
Carlos Carlos Goller
Abstract
Overview
Microbes resistant to antimicrobial agents are a major worldwide health challenge. Rapid detection of antimicrobial resistant pathogens and surveillance efforts are critical. Information about antimicrobial resistance (AMR) genes is available in databases, and quantitative PCR (qPCR) has been proven suitable for detecting AMR genes (Abram et al. 2019; Galhano et al. 2021; Wu et al. 2022). In this research session, we will use the epMotion 5075 and a custom 3D-printed adapter to leverage the power of automation and the new QIAGEN QIAcuity Digital PCR system to detect and quantify a target antimicrobial resistance gene in metagenomic DNA samples from soils and compost. Take a virtual tour of the QIAcuity that we will be using for our research!
Digital PCR (dPCR) allows for absolute quantification of template DNA or RNA molecules based on Poisson statistics. But, what does that mean and how does dPCR even work? Read about the fundamentals of dPCR in these articles and watch this short video about dPCR:
During our second research session, we will set up the epMotion 5075 to dilute metagenomic DNA samples. We will set up dPCR by combining a commercial PCR master mix (includes: dNTPs, polymerase, buffers, Mg2+,water) with our research specific primers, probes, and diluted DNA template to carefully pipette into a QIAGEN Nanoplate with partitions for use in the QIAcuity. We will run dPCR on AMR genes from a mixed population of soil microbes to investigate whether soils used as our samples contain microbes with AMR genes.
Steps
Metagenomic DNA Extraction
Obtain soil and compost samples from different environments. We have access to soil and compost samples from Christopher Hopkins from efforts to learn about the composting process of “swine lagoon” sludge and have dried and “tar-like” samples. Your instructors (Horton and Phillips) also prepared solid metagenomic DNA samples from around the campus for comparison.
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Weigh 250mg into QIAGEN PowerSoil Pro bead beating tubes.
Extract DNA following the QIAGEN PowerSoil Pro protocol in triplicate.
Elute in 50µL 5 of CD6 Elution buffer.
Quantify DNA by NanoDrop and use the Qubit BR DNA kit.
Run 1µL of sample on Agilent TapeStation using the Genomic DNA tape and reagents.
Digital PCR Setup
Obtain 0.8micromolar (µM) Forward and 0.8micromolar (µM) Reverse Primer for the blaCTX-M-1 antimicrobial resistance gene.
Obtain probe 0.4micromolar (µM) and dilute it with 625µL of TE Buffer.
Transfer 100µL 1 of diluted probe into tubes via pipette in a biosafety hood.
Transfer 400µL of buffer solution with 40µL of both the reverse and forward primers, and 20µL of the probe to make the primer probe mix
The primer probe mix, 3µL of the master mix, and 0.15µL of the restriction EcoRI enzyme per reaction were dispensed into the PCR plate. Then, the extracted compost DNA was added to each well that contained the overall reaction mix (1µL per reaction)
Transfer mixtures from the PCR plate to the QIAGEN Nanoplate (96 wells, 8.5k partitions) using the epMotion 5075.
Seal the Nanoplate properly with the provided seal and use roller to ensure proper seal.
Transfer the plate to the QIAcuity instrument, remove the white plate tray, and load into the digital PCR using a template for a full plate with a FAM-based probe.
Reagent Concentrations and Calculations
Table 1. Concentrations of stocks and working solutions for primer mix and probes.
| A | B | C | D |
|---|---|---|---|
| Component | Initial Concentration | Final Concentration | Volume for 500 µl (in TE) |
| Forward Primer | 100 µM | 8 µM | 40 µl |
| Reverse Primer | 100 µM | 8 µM | 40 µl |
| Probe | 100 µM | 4 µM | 20 µl |
| TE Buffer | - | - | 400 µl |
Table 2. List of reagents used for digital PCR on the QIAGEN QIAcuity instrument.
| A | B | C | D |
|---|---|---|---|
| Component and Concentration | Volume per Reaction | Final Concentration in µM | Volume for 100 reactions in 96-well plate |
| 4X QIAcuity Probe PCR Master Mix | 3 µl | 1X | 300 µl Master Mix |
| 10X Primer Probe Mix (FAM channel) | 1.2 µl | 0.8 µM forward | 120 µl Primer Probe Mix |
| Restriction EcoRI Enzyme 0.25 U/µl EcoRI-HF®, NEB® | 0.15µl | 0.8 µM reverse | 15 µl NEB EcoRI-HF |
| RNase-free Water | 6.65µl | primer | 665 µl water |
| Template DNA | 1.0 µl | 0.4 µM probe | 100 µl of DNA |
| Total Reaction Volume | 12.0 µl | 0.25 U/µl | 1,200 µl |
Table 3. Thermocycling Conditions.
| A | B | C | D |
|---|---|---|---|
| Step | Time | Temperature (°C) | Number of Cycles |
| PCR initial heat activation | 2 min | 95°C | 1 cycle (to activate the enzyme) |
| Denaturation | 15 seconds | 95°C | 40 cycles |
| Combined annealing/ extension | 30 seconds | 60°C | 40 cycles |
Source: QIAGEN QIAcuity Probe Mix
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