TITLE: AMR Detection by dPCR
AUTHORS: Carlos Goller

[DESCRIPTION]
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: 
Read Digital PCR for beginners
Read Fundamentals of Digital PCR
Watch Principles of dPCR Explained

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. 

[GUIDELINES]
Additional Resources:
QIAcuity Probe PCR Kit Protocol

[STEPS]
SECTION: Metagenomic DNA Extraction
1. 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.
SECTION: Metagenomic DNA Extraction
4. Weigh 250 mg into QIAGEN PowerSoil Pro bead beating tubes.

SECTION: Metagenomic DNA Extraction
5. Extract DNA following the QIAGEN PowerSoil Pro protocol in triplicate.
SECTION: Metagenomic DNA Extraction
6. Elute in 50 µL 5 of CD6 Elution buffer.

SECTION: Metagenomic DNA Extraction
7. Quantify DNA by NanoDrop and use the Qubit BR DNA kit.
SECTION: Metagenomic DNA Extraction
8. Run 1 µL  of sample on Agilent TapeStation using the Genomic DNA tape and reagents.

 


SECTION: Digital PCR Setup
9. Obtain 0.8 micromolar (µM)  Forward and 0.8 micromolar (µM)  Reverse Primer for the blaCTX-M-1 antimicrobial resistance gene. 

SECTION: Digital PCR Setup
10. Obtain probe 0.4 micromolar (µM)   and dilute it with 625 µL  of TE Buffer. 

SECTION: Digital PCR Setup
11. Transfer 100 µL 1 of diluted probe into tubes via pipette in a biosafety hood.

SECTION: Digital PCR Setup
12. Transfer 400 µL of buffer solution with 40 µL of both the reverse and forward primers, and 20 µL  of the probe to make the primer probe mix

SECTION: Digital PCR Setup
13. The primer probe mix, 3 µL of the master mix, and 0.15 µL  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 (1 µL  per reaction)

SECTION: Digital PCR Setup
14. Transfer mixtures from the PCR plate to the QIAGEN Nanoplate (96 wells, 8.5k partitions) using the epMotion 5075. 


SECTION: Digital PCR Setup
15. Seal the Nanoplate properly with the provided seal and use roller to ensure proper seal.
SECTION: Digital PCR Setup
16. 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. 
SECTION: Reagent Concentrations and Calculations
17. Table 1. Concentrations of stocks and working solutions for primer mix and probes.

 

SECTION: Reagent Concentrations and Calculations
18. Table 2. List of reagents used for digital PCR on the QIAGEN QIAcuity instrument.

 

SECTION: Reagent Concentrations and Calculations
19. Table 3. Thermocycling Conditions.

 

SECTION: Reagent Concentrations and Calculations
20. 
 

SECTION: Reagent Concentrations and Calculations
21. 
 

SECTION: Metagenomic DNA Extraction
2.  
SECTION: Metagenomic DNA Extraction
3.