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, Environ. Sci. Technol, vol.46, pp.11566-11574

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, Annexe A : Métagénomique ciblée de la diversité

, Au cours de mes travaux de thèse, j'ai contribué à la rédaction d'un article méthodologique intitulé "Targeted metagenomics of microbial diversity in free-living amoebae and water samples" visant à être publié dans une nouvelle édition du livre Legionella : Methods and Protocols. Ma contribution à ce chapitre de livre, présenté ci-dessous, a consisté à la rédaction des sections expliquant la méthodologie à appliquer pour isoler l'ADN d'échantillons d'eau et pour la sélection du couple d'amorces utilisé

, Store after sterilization either by autoclaving at 121°C for 20 minutes, or by thorough flame sterilization of each funnel, Stainless-steel filtration funnel (Sartorius, reference 16828)

, Vacuum pump (Millipore EZ-Stream TM

, Sterile cellulose acetate membrane of 0, vol.22

, Sterile nitrocellulose membrane of 3 µm porosity

, Stainless steel forceps

, Page Amoeba Saline: 4 mM MgSO4, 2.5 mM Na2HPO4, 2.5 mM KH2PO4, 0.4 mM CaCl2, 4 mM Sodium citrate. Prepare extemporaneously stock solutions of 0, p.4

, Filter sterilize each solution through a 0.22 µm sterile filter. For preparing 1 L of Page Amoeba Saline (PAS) buffer, use the stock solutions previously prepared, and add 10 mL of MgSO4, Na2HPO4:2H2O 0.25 M and KH2PO4 0.25M, respectively, 8 mL of CaCl2:2H2O 0.05 M, as well as 1 g of sodium citrate. Complete to 1 L with distilled water, and autoclave at 121°C for 20 minutes

, Prepare one liter of PAS buffer as described above, and supplement it with 15 g/L of agar. Sterilize by autoclaving at 121°C for 20 minutes. Pour into Petri dishes in sterile condition

, Saccharomyces cerevisiae strain L40 (ATCC MYA 3332)

. Sabouraud, Adjust the pH at 6, and add water for up to 1 L. Sterilize by autoclaving at 121°C for 20 minutes

, Strike a suspension of S. cerevisiae on an agar plate, and incubate for 5 days at 30°C. Resuspend one isolated colony into 500 µL of PAS buffer, and spread evenly onto a Sabouraud agar plate. Collect and resuspend a whole fully-grown S. cerevisiae culture by adding 3 mL of PAS buffer onto one Petri dish, using a bacterial cell spreader, and collect the suspension into a clean 15 mL plastic tube. Determine the cell concentration of a 1:100 dilution in PAS of the suspension, by measuring the optical density at 600 nm (one unit of optical density correspond to approximately 1x10 7 yeasts / mL), Saccharomyces cerevisiae strain L40 (ATCC MYA 3332) is used as a nonbacterial food source for enriching samples in free-living amoebae

, Inverted microscope with 10x and 20x phase contrast magnification. DNA extractions and preparation 1. Lysis tubes: add 500 mg of Glass beads

, to non-skirted empty FastPrep® 2 mL tubes, reference G1277)

, Seal tubes using clear caps for FastPrep® tubes (MP Biomedicals; reference 115068), reference 115076)

, FastPrep® 24 Classic instrument (MP Biomedical

, Proteinase K 20 mg / mL, > 600 mAU / mL (QIAGEN; reference 19131)

, DNeasy PowerWater kit (QIAGEN; reference 14900-50-NF)

, Methods 1.1. Water sampling and filtration 1. Sample water at desired sites and transfer 1 L in a sterile bottle

, Mount the filtration unit by assembling the filtration funnels with the pump. Place the outlet tubing either in a sink

, Using sterile forceps, take one appropriate filter (3 µm porosity nitrocellulose filter when screening for amoebae, or 0.22 µm cellulose acetate filter when screening for total bacteria) and place it onto the frit filter support, Dislodge the funnel from the filtration ramp

, Switch on the vacuum pump and filter water samples by gently pouring the water into the filter-containing funnels

, 500 µL of S. cerevisiae suspension (prepared as described above) onto one NNA-PAS Petri dish, using a bacterial cell spreader

, Dislodge the funnel from the filtration ramp. Cut the filter in two halves using sterile razor blade or scalpel, directly onto the frit filter support

, Using sterile forceps, transfer the pieces of filter upside down onto NNA-PAS seeded with S. cerevisiae

, Close the Petri dish and seal it using Parafilm®. Incubate at 30°C for up to one month

, Every two to three days, check the cultures for the presence of amoebae around the filters, by phase contrast microscopy, using 10x and/or 20x magnification

, DNA isolation from amoeba cultures 1. Dispose Petri dishes under a laminar flow, prior to opening, in order to work in aseptic conditions

, Scrap the surface of the agar plate using a sterile inoculation loop, from cultures showing signs of amoebal development (i.e. formation of migration fronts

, Resuspend the collected microorganisms in 200 µL of PAS buffer

, Add proteinase K to the samples, to a final concentration of 2 mg / mL. Incubate the sample at 40°C for 30 minutes

, Transfer the suspension into a sterile lysis tubes, vol.20

, Transfer the suspension into a sterile microtube, and proceed to DNA extraction, using commercially available kits

, Store DNA extractions at -20°C until further use, or -80°C for long term storage. DNA isolation from water samples 1. Filter 1 L of water sample on a cellulose acetate filter (0.22 µm) as described above

, Using sterile forceps, transfer one filter into beads tube provided in the DNeasy PowerWater kit, designed for the isolation of genomic DNA from filtered water

, Proceed to the DNA extraction according to manufacturer recommendations, eluting DNA in a final volume of 100 µL

, Add 5µL of 3 M sodium acetate, and 200 µL of ice-cold molecular biology grade ethanol. Mix thoroughly and incubate for 2 hours to overnight at -20°C

, Centrifuge at 10000 g for 15 minutes at 4°C, and discard the supernatant. Let air dry, and resuspend the pellet in 20 µL of elution buffer provided in the DNeasy PowerWater kit

J. K. Goodrich, S. C. Di-rienzi, A. C. Poole, O. Koren, W. A. Walters et al., Store DNA extractions at -20°C until further use, or -80°C for long term storage. References 1, Cell, vol.158, pp.250-262, 2014.

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