TITLE: Detection of bacterial pathogens and drug resistance markers using Rolling circle amplification
AUTHORS: Malte Kühnemund, Ivan Hernandez, Mohd Istiaq Sharif, Matteo Cornaglia, Martin A.M. Gijs, Mats Nilsson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. 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 at.Put immediately on ice
?. Prepare ligation mix for the total  number of reactions you require  ABCD1 Initial conc.Final conc.x1 reaction2Probe mix10 nM100 pM0.23BSA2 µg/µl0.2 µg/µl24Ampligase Buffer10 ×1 ×25Ampligase&nbsp;5 U/µl0.25 U/µl0.56mQ H2O- 5.37  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:
ABCD1 Initial conc.Final conc.x1 reaction2Probe mix10 nM100 pM0.23BSA2 µg/µl0.2 µg/µl24Ampligase Buffer10 ×1 ×25Ampligase&nbsp;5 U/µl0.25 U/µl0.56mQ H2O- 5.37  Final mix volume (µl)10
?. Prepare RCA mix for the total  number of reactions you require  ABCD1 Initial conc.Final conc.x1 reaction2dNTP's2,5mM125 µM1.53phi 29 buffer10x1 x34BSA2 µg/µl0.2 µg/µl35phi 29 polymerase10 U/uL400 mU/uL1.26mQ H2O  1.37Final mix volume (µl)10 Add 10 µL to each tube assay containing the ligation reactionIncubate the assays tubes at:
ABCD1 Initial conc.Final conc.x1 reaction2dNTP's2,5mM125 µM1.53phi 29 buffer10x1 x34BSA2 µg/µl0.2 µg/µl35phi 29 polymerase10 U/uL400 mU/uL1.26mQ H2O  1.37Final mix volume (µl)10
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 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  ABCD1 Initial conc.Final conc.x1 reaction2Labelling buffer2x1x153Detection oligo mix1 µM5nM0.34mQ H2O14.45  Final mix volume (µl)306Total volume (µl)60Add 30 µL to each tube assay containing the RreactionIncubate at:
ABCD1 Initial conc.Final conc.x1 reaction2Labelling buffer2x1x153Detection oligo mix1 µM5nM0.34mQ H2O14.45  Final mix volume (µl)306Total volume (µl)60
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 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.