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MLST of Streptococcus uberis

Genes

The Streptococcus uberis MLST scheme uses fragments of the following seven house-keeping genes:

Glucose kinase (gki)
Transketolase (recP)
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

  1. 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).
  2. 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.
  3. 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.
  4. Incubate at 37°C for 30 min.
  5. 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.
  6. 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.
  7. 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).
  8. Agitate to mix (shake or a brief whirli-mix) and centrifuge (13,000 x g at RT) for 3 min to separate phases.
  9. 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.
  10. 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.
  11. 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.
  12. 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.
  13. Store DNA at -20°C (but try to avoid repeated freeze thaw).

PCR amplification

  1. Prepare a mastermix on ice by combining the reagents shown below:

    Reagentper reactionfor 48 reactionsfor 96 reactions
    2 x Qiagen Taq PCR Mastermix*15µ780µl1560µ
    Sterile dH2O6µl312µl624µl
    10 pmol/µl forward primer3µl156µl312µl
    10 pmol/µl reverse primer3µl156µl312µl

    *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.

    GeneTaPCR fragment Length (bp)Primer SequenceMLST Sequence length (bp)
    gki555645'-GACCGGACCCAAAACACAGTCACAGGTGCTTTT
    5'-AAGAGAATCTGGATTTAGGATATTTGAAATATT
    455
    recP605315'-AATTCAGGTCACCCTGGCTTACCAATGGGTGCAGCC
    5'-TGTGAAAGCCATTGATGTTGGACCATCAAGTGAAAT
    372
    ddl605035'-GTCTATATTGAAGGTAATGACTTGGAAGACTGT
    5'-TACATGGACCACTGAGTGAATCCAGGCATAGTATTC
    357
    tdk557935'-TATTTTCATTTCATAATAAGTTAGTGGATTTAGTAA
    5'-TTGATCATATATATTCATGTTATGAATCGTTCTCCT
    500
    arcC555185'-GTTTGTGACGCAAAATCTTTATCGATAACA
    5'-ACTCATGGTAACGGACCACAAGTTGGTAAC
    419
    tpi604715'-GTTATTGGTCATTCAGAACGTCGTGATTACTTC
    5'-GTCAAGTAATGCTAAGAAGCTATCTGCTTCAAGTGA
    373
    yqiL555745'-TTTCTTCTTTGAAACGATTATTTTTAAGTGCTTCAG
    5'-CAAGCTCTAAGAACACCAATTGGTGCATTCGGAGGA
    439

  2. 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).
  3. 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.

  4. 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.

Sequencing

Sequence analysis is performed using the BigDyeTM Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Use of other sequencing reagents will require optimization.

  1. Assay the DNA concentration of the purified PCR products, using the GeneQuant, and dilute in sterile dH2O to approximately 10 ng/µl.
  2. 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:

    ReagentPer reactionFor 48 reactions
    BigDye ready reaction mix1µl50µl
    BigDye 5X sequencing buffer3.5µl175µl
    Primer (3.2 pmol/µl)1µl50µl
    Sterile distilled water13.5µl675µl

  3. 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.

  4. 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

  1. Spin plates for 1 minute at 1000 rpm and 4°C in a refrigerated centrifuge with a micro-titre plate rotor.
  2. 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.

  3. 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.
  4. 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.
  5. 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.

  6. 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.
  7. Add 70 µl of 70% ethanol to each well, seal the plate, and invert a few times to mix.
  8. Spin the plate for 15 minutes at 1650 x g and 4 °C.
  9. Repeat steps 5 and 6. Allow plate to air dry (it is very important that plates are 100% dry).
  10. Seal the dry plate and store at 4 °C until analysis (Plates are normally used within 10 days after preparation).

Citing the database

The preferred format for citing this website in publications is:

This publication made use of the Streptococcus uberis MLST website (https://pubmlst.org/ suberis/) sited at the University of Oxford (Jolley et al. Wellcome Open Res 2018, 3:124 [version 1; referees: 2 approved]). The development of this site has been funded by the Wellcome Trust.

Status

Sequence database
Sequences: 459
Profiles:
MLST: 1,059
vMLST: 0

Isolate database
Isolates: 1,179
Last updated: 2018-10-29