The Streptococcus uberis MLST scheme uses fragments of the following seven house-keeping genes:
Glucose kinase (gki)
D-ala-D-ala ligase (ddl)
Thymidine kinase (tdk)
Carbamate kinase (arcC)
Triosephosphate isomerase (tpi)
Acetyl CoA acetyl-transferase (yqiL)
Please note that all protocols here refer to fast-ramping thermal cyclers (such as ABI GeneAmp PCR 2700 or DNA engine) using 48/96 microtitre plates. Any other plates/thermal cycler combinations will require you to optimize your conditions.
Purification of chromosomal DNA from S. uberis
- Grow S. uberis strains at 37°C for 16-20h in "Universals" containing 10 ml of liquid media (Todd Hewitt Broth or Brain Heart Infusion).
- Prepare chromosomal DNA by harvesting bacterial cells from multiples of 1.5 ml of culture by centrifugation (13000 x g for 5 min at room temperature). Typically 3ml of bacterial culture yields 50-100µg DNA.
- Resuspend cell pellet by vortexing with 0.5 ml of TE buffer1 [10mM Tris, 5 mM EDTA (pH 7.8)]. Re-pellet cells (13000 x g for 5 min at room temperature), remove supernatant and re-suspend in 375µl of fresh cell wall disruption buffer2.
- Incubate at 37°C for 30 min.
- Lyse cells by addition of 20µl Lysis buffer3 and 3µl of Proteinase K4 - shake briefly to mix (the sample should clear). Incubate at 37°C for 1h.
- Add 200µl saturated NaCl (approx. 6.0M) to precipitate protein cell wall material and agitate (not whirli-mix!) for 15s. Centrifuge (13,000 x g) for 10 min to obtain a firm pellet.
- Remove 400-450µl of supernatant carefully to a fresh, labeled tube. Re-pellet if strings of precipitated material are lifted, and discard tube containing pellet. Add an equal volume of Tris-equilibrated phenol:chloroform:isoamyl alcohol (25:24:1 molecular biology grade, Sigma).
- Agitate to mix (shake or a brief whirli-mix) and centrifuge (13,000 x g at RT) for 3 min to separate phases.
- Retain 300-450µl of the upper aqueous phase avoiding the interface (leave some if in doubt). Precipitate DNA by adding 2 volumes of cold ethanol and hold at 4°C for up to 2h.
- Precipitate DNA by centrifugation at 13,000 x g for 5 min. Wash with cold 70% ethanol (centrifuge again at 13,000 x g for 5 min, and discard ethanol). Thoroughly air-dry the pellet.
- Allow to re-suspend slowly (~1/2h) at 4°C in 30-50µl of TE buffer (10mM Tris; 1mM EDTA pH 8.0) containing RNAase A (made DNAase-free by boiling for 5 min) at 20µg/ml. Do not shear by pipetting or vortexing. Incubate for 15-30 min at 37°C.
- Determine DNA concentration by assaying the absorbance of a dilution (typically 1/50 in water) of the preparation at 260nm. 1.0 OD260 corresponds to 50µg/ml of chromosomal DNA.
- Store DNA at -20°C (but try to avoid repeated freeze thaw).
- 1 TE buffer:for 10 ml, mix 0.1ml of 1M Tris-HCl pH 7.5 with 0.1ml of 0.5M EDTA pH 8.0 and 9.8ml of sterile molecular biology grade water.
- 2 cell wall disruption buffer:[30u/ml mutanolysin; 10mg/ml lysozyme in TE buffer]. For 10 ml, add 100mg dessicated lysozyme and 60µl of stock mutanolysin reagent (stock prepared in sterile water at 5000 units per ml) to 10ml TE buffer (prepared as above). Mix, store on ice until used.
- 3 Lysis buffer:SDS (20% w/v) in 50 mM Tris, 20 mM EDTA pH 7.8. For 100ml, dissolve 20g SDS in 80ml molecular biology grade water, add 5ml 1M Tris-HCl (pH 7.5), 4ml 0.5M EDTA (pH 8.0), make up to 100ml with water, mix & store buffer at ambient temperature.
- 4 Proteinase K:[stock 20mg/ml]. For 5ml, dissolve 0.1g in 5ml of sterile molecular biology grade water, aliquot and store frozen at < -20°C
Prepare a mastermix on ice by combining the reagents shown below:
Reagent per reaction for 48 reactions for 96 reactions 2 x Qiagen Taq PCR Mastermix* 15µ 780µl 1560µ Sterile dH2O 6µl 312µl 624µl 10 pmol/µl forward primer 3µl 156µl 312µl 10 pmol/µl reverse primer 3µl 156µl 312µl
*2 x Qiagen mastermix (catalogue no. 201445) contains 3 mM MgCl2, 400 µM each dNTP and 0.05 units/µl Taq DNA polymerase.
Remember to set up a negative PCR control, which consists of reactions components and NO added template DNA.
The primers used in MLST analysis of S. uberis are listed below.
Gene Ta PCR fragment Length (bp) Primer Sequence MLST Sequence length (bp) gki 55 564 5'-GACCGGACCCAAAACACAGTCACAGGTGCTTTT
455 recP 60 531 5'-AATTCAGGTCACCCTGGCTTACCAATGGGTGCAGCC
372 ddl 60 503 5'-GTCTATATTGAAGGTAATGACTTGGAAGACTGT
357 tdk 55 793 5'-TATTTTCATTTCATAATAAGTTAGTGGATTTAGTAA
500 arcC 55 518 5'-GTTTGTGACGCAAAATCTTTATCGATAACA
419 tpi 60 471 5'-GTTATTGGTCATTCAGAACGTCGTGATTACTTC
373 yqiL 55 574 5'-TTTCTTCTTTGAAACGATTATTTTTAAGTGCTTCAG
- Aliquot 27 µl of mastermix into each well of a microtitre plate, and add 3 µl 50 ng/µl purified Streptococcal chromosomal DNA as required (DNA is quantified using a GeneQuant). Seal plate with adhesive PCR film (ABgene catalogue no. AB-0558).
Perform large scale PCR amplification on an ABI GeneAmp PCR 2700 block, using the following cycling parameters:
Step 1) 94°C 5 minutes Step 2) 40 cycles of: 94°C 30 seconds Annealing temp. (Ta) 30 seconds 72°C 45 seconds Step 3) 72°C 7 minutes Step 4) 4°C hold
For annealing temperatures, see primer table.
- Purify DNA using the MinElute 96 well purification kit from Qiagen (catalogue no. 28053), as directed (omitting the optional wash), and elute DNA in 25 µl of sterile distilled water. Seal plates with adhesive film and store at -20°C until used.
Sequence analysis is performed using the BigDyeTM Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Use of other sequencing reagents will require optimization.
- Assay the DNA concentration of the purified PCR products, using the GeneQuant, and dilute in sterile dH2O to approximately 10 ng/µl.
Prepare a mastermix using a single MLST primer (see primer table) as shown below, setting up both a forward primer and reverse primer reaction for each PCR product:
Reagent Per reaction For 48 reactions BigDye ready reaction mix 1µl 50µl BigDye 5X sequencing buffer 3.5µl 175µl Primer (3.2 pmol/µl) 1µl 50µl Sterile distilled water 13.5µl 675µl
Aliquot 19 µl of the BigDye mastermix into each well of a 96 well plate, and add 1 µl of 10 ng/µl PCR products as required. Seal the plate with adhesive PCR film.
If using gel-based high throughput sequencing technology (such as the ABI 377), as opposed to capillary based (ABI 3700), we would recommend combining forward and reverse sequencing reactions (ideally alternating these across the microtitre plate) on one 96 well plate. This facilitates accurate tracking of the sequence data following electrophoresis on the 96 well sequencing gel.
Place the 96-well plate into the GeneAmp 2700 thermal cycler. The cycling program required is as follows:
Step 1) 96 °C for 1 minute Step 2) 25 cycles of: 96 °C for 20 seconds. 50 °C for 10 seconds. 60 °C for 4 minutes. Step 3) Hold at 4 C.
Plates can be stored at -20°C prior to clean-up (maximum of 2 days), but we would recommend storage after the following clean-up protocol.
Clean-up of sequencing reactions
- Spin plates for 1 minute at 1000 rpm and 4°C in a refrigerated centrifuge with a micro-titre plate rotor.
To each of the wells on the 96-well plate containing sequencing reactions add the following in order:
2 µl of 125 mM EDTA
2 µl of 3M sodium acetate (pH 5.2)
50 µl of 100% ethanol
Please note the quality of the ethanol is crucial for obtaining 'clean' sequencing reactions. We recommend using molecular biology grade ethanol were possible, and avoiding the use of 'old' bottles of ethanol which are likely to be < 100% due to the absorption of atmospheric water. Buying small bottles of ethanol, ensuring rapid turnover of stock, is therefore ideal.
- Seal the plate (ABgene plate seals - catalogue no. AB-0580) and mix by inverting four times. Leave the plate at room temperature for 15 minutes to precipitate the extension products.
- Place the plate(s), plus balance if required, into a centrifuge with micro-titre plate rotor and spin for 40 minutes at 2500 x g and 4 °C.
Immediately after centrifugation, carefully remove the adhesive cover from the plate whilst holding it firmly, and discard the ethanol wash by inverting the plate onto a paper towel folded to the size of the plate.
If this is not done immediately, spin the plates for an additional 2 minutes before discarding the ethanol wash.
- Place the inverted plate with a fresh paper towel into the centrifuge and spin at 185 x g for 1 minute at 4 °C, removing all residual ethanol.
- Add 70 µl of 70% ethanol to each well, seal the plate, and invert a few times to mix.
- Spin the plate for 15 minutes at 1650 x g and 4 °C.
- Repeat steps 5 and 6. Allow plate to air dry (it is very important that plates are 100% dry).
- Seal the dry plate and store at 4 °C until analysis (Plates are normally used within 10 days after preparation).