Apr 04, 2019

Public workspaceDetection of bacterial pathogens and drug resistance markers using Rolling circle amplification

DOI
dx.doi.org/10.17504/protocols.io.zp8f5rw
  • Malte Kühnemund1,
  • Ivan Hernandez1,
  • Mohd Istiaq Sharif1,
  • Matteo Cornaglia2,
  • Martin A.M. Gijs2,
  • Mats Nilsson1
  • 1Stockholm University, Science for Life Laboratory;
  • 2EPFL - EPF Lausanne
  • Molecular Diagnostics - Mats Nilsson Group
Ivan Hernandez
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DOI: dx.doi.org/10.17504/protocols.io.zp8f5rw
External link: https://academic.oup.com/nar/article/45/8/e59/2888443
Protocol Citation: Malte Kühnemund, Ivan Hernandez, Mohd Istiaq Sharif, Matteo Cornaglia, Martin A.M. Gijs, Mats Nilsson 2019. Detection of bacterial pathogens and drug resistance markers using Rolling circle amplification. protocols.io https://dx.doi.org/10.17504/protocols.io.zp8f5rw
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 02, 2019
Last Modified: April 04, 2019
Protocol Integer ID: 21984
Keywords: Padlock probes, rolling circle amplification, bacteria, antibiotic resistance markers
Abstract
Here is described the protocol used to detect bacteria and antibiotic resistance markers using padlock probes and Rolling circle amplification. This method allows for single-molecule/digital quantification in a semi-quantitative way.
Materials
Materials

Ampligase buffer 10x (Epicenter)

200 mM Tris-HCl (pH 8.3), 250 mM KCl, 100 mM MgCl2, 5 mM NAD 0.1% Triton® X-100

Phi29 buffer 10x (ThermoScientific)

330 mM Tris-acetate pH-7.9, 100 mM Mg-acetate, 660 mM K-acetate, 1% (v/v) Tween 20, 10 mM DTT

Labeling buffer 2x

20 mM Tris-HCl pH-8.0, 20 mM EDTA, 0.1% Tween-20, 2 M NaCl

Padlock probes

5' phosphorylated padlock probes are obtained from IDT and resuspended in IDTE buffer (pH 8.0) to 100 μM for long storage at -20°C.

A working aliquot of 1 μM/each containing all the probes in ddH2O. Storage at -20°C is recommended.

Detection oligos

Fluorophore-tagged oligos are obtained from IDT and resuspended in IDTE buffer (pH 8.0) to 100 μM for long storage at -20°C.

A working aliquot of 1 μM/each containing all the detection probes in ddH2O. Ambar tubes and storage at -20°C is recommended.

dNTPs

Prepare a working aliquot of 2.5 mM/each in water.

BSA

Prepare a working aliquot of 2 μg/µl in water.

Dilute padlock probes to 10 nM. Is recommended to prepare a fresh dilution for each experiment.
Denature extracted DNA from culture isolate or clinical sample by heating atTemperature95 °C Duration00:05:00 .Put immediately on ice




Prepare ligation mix for the total number of reactions you require
Initial conc.Final conc.x1 reaction
Probe mix10 nM100 pM0.2
BSA2 µg/µl0.2 µg/µl2
Ampligase Buffer10 ×1 ×2
Ampligase 5 U/µl0.25 U/µl0.5
mQ H2O- 5.3
Final mix volume (µl)10
Add 10 µl of master mix to each assay tube and add 10 µl of the denatured DNA from Step 1.

Incubate the assay tubes at:

Temperature95 °C Duration00:00:30
Temperature55 °C Duration00:40:00
Prepare RCA mix for the total number of reactions you require
Initial conc.Final conc.x1 reaction
dNTP's2,5mM125 µM1.5
phi 29 buffer10x1 x3
BSA2 µg/µl0.2 µg/µl3
phi 29 polymerase10 U/uL400 mU/uL1.2
mQ H2O 1.3
Final mix volume (µl)10
Add 10 µL to each tube assay containing the ligation reaction

Incubate the assays tubes at:

Temperature37 °C Duration03:00:00
Temperature65 °C Duration00:02:00
Note
Note 1. In principle the longer the amplification time the longer amplification products and thus stronger signals one can get. Phi29 polymerase is reported to be stable for up to 12 h. For quantifying rolling circle products spread on microscope slides 1 h amplification time is enough to get robust signals. For microfluidic enrichment ≥ 2 h amplification is recommended.

Note
Note 2. Ampligase buffer can also be used in this step. Volumes need to be adjusted in that case

Prepare the labeling mix for the total number of reactions you require

Initial conc.Final conc.x1 reaction
Labelling buffer2x1x15
Detection oligo mix1 µM5nM0.3
mQ H2O14.4
Final mix volume (µl)30
Total volume (µl)60
Labeling master mix

Add 30 µL to each tube assay containing the Rreaction

Incubate at:

Temperature75 °C Duration00:02:00
Temperature55 °C Duration00:15:00

Note
Note 3. For optimal results using microfluidic enrichment, filtering this mix through a 0.1 µmfilter (Millipore). This will filter out fluorophore aggregates or cristals that may cause background.

If the initial concentration of samples is higher than 100 femtomolar, pipette a 5-10 µL drop on a Superfrost Microscope Slide (Thermofisher) and put a 24x24 mm cover slip (Menzel) on top to spread it.

Incubate the slide at room temperature for Duration00:10:00 and you can proceed to image using fluorescence microscopy. 10x or 20x objectives are recommended.

If the concentration is lower than 100 femtomolar, microfluidic enrichment is required.