Automated Pneumococcal MLST
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Automated Pneumococcal MLST Using Liquid-Handling Robotics and a Capillary DNA Sequencer Johanna Jefferies,1 Stuart C. Clarke,1,2,* Mathew A. Diggle,2 Andrew Smith,3 Chris Dowson,4 and Tim Mitchell1 Abstract Multilocus sequence typing (MLST) is used by the Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) as a routine method for the characterization of certain bacterial pathogens. The SMPRL recently started performing MLST on strains of Streptococcus pneumoniae, and here we describe a fully automated method for MLST using a 96-well-format liquid-handling robot and a 96-capillary automated DNA sequencer. Index Entries: Multilocus sequence typing; DNA sequencing; polymerase chain reaction; automation.
1. Introduction Multilocus sequence typing (MLST) is a nucleotide-based typing method that uses data from housekeeping genes in order to provide a sequence type (ST). MLST provides molecular typing data that are highly discriminatory and electronically portable between laboratories, and is therefore suitable for investigating the genetic relatedness of bacteria. The method has been validated with some important pathogens including Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae (1,2). The Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) provides a national service in Scotland for the laboratory confirmation and typing of strains from meningococcal, pneumococcal and H. influenzae disease. The development of a semiautomated MLST method for the characterization of N. meningitidis has been reported previously (3). Here we describe an improved protocol for the analysis of large numbers of S. pneumoniae isolates using liquid handling robotics and a capillary DNA sequencer.
2. Materials 1. Columbia-agar plates (Oxoid, Basingstoke, UK). 2. 1× Reddymix polymerase chain reaction (PCR) mastermix. 3. Oligonucleotide primers. 4. Ninety-six-well Thermosprint microtitre plates. 5. Serum tubes. 6. Energy transfer (ET)-terminator sequencing kit. 7. Millipore Multiscreen™ 96- or 384-PCR plates. 8. Millipore Multiscreen™ 96- or 384-SEQ plates. 9. Sterile distilled water (18 MΩ purity). 10. Vacuum manifold system. 11. Liquid-handling robot (RoboAmp 4200, MWG, Milton Keynes, UK). 12. Automated 96-well capillary sequencer (Amersham Biosciences, Little Chalfont, UK). 13. Sequence analysis software.
3. Methods 3.1. Phenotypic Characterization of Pneumococcal Strains All strains were isolated on Columbia-agar plates at 37°C under anaerobic conditions by use
*Author to whom all correspondence and reprint requests should be addressed: 1Scottish Meningococcus and Pneumococcus Reference Laboratory, North Glasgow University Hospital NHS Trust, Department of Microbiology, Stobhill Hospital, Balornock Rd., Glasgow, G21 3UW, UK. E-mail:
[email protected]. 2Institute of Biomedical and Life Sciences, University of Glasgow. 3Glasgow Dental School and Hospital, Glasgow. 4Department of Biological Sciences, Warwick University. Molecular Biotechnology 2003 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2003/24:3/303–307/$20.00
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of an anaerobic pack (Oxoid). Serogrouping was performed by coagglutination (4,5) (see Note 1).
3.2. Genotypic Characterization of Pneumococcal Strains Characterization of strains by this MLST method utilized a liquid-handling robot and an automated capillary electrophoresis system. This allowed automation of most of the procedures, thus facilitating high-throughput processing (see Note 2).
3.2.1. PCR Sample Preparation Clinical isolates of S. pneumoniae were plated on Columbia-agar plates and incubated overnight under anaerobic conditions at 37°C (see Note 1). 1. A sweep was taken of 8–10 fresh colonies and was added to 200 µL of 18-MΩ distilled water in a 200 (L thin-walled PCR tube (ABgene, Epsom, UK). 2. This was quickly heated to 80°C using a Primus-96 thermocycler (MWG) at the maximum ramp rate (3°C). This temperature was maintained for 4 min. 3. The tubes are removed from the machine and the suspension centrifuged at 15,000 rpm for 3 min, after which the supernatant is removed to a fresh tube. The supernatant can then be used as a template in PCR reactions to amplify pneumococcal genes.
3.2.2. PCR Setup 1. Program the RoboAmp system according to the manufacturer’s instructions. 2. Keep all PCR components chilled at 4°C on the platform of the RoboAmp. 3. Master mixes for each of the 7 MLST loci should be prepared manually and placed in the chilled reagent rack of the platform. Each master mix is made up from 20 mL 1.1× Reddymix PCR master mix (ABgene, Epsom, UK), (0.75 U Taq polymerase, 75 mM; Tris-HCl, pH 8.8 at 25°C; 20 mM (NH4)2; 1.5 mM MgCl2, 0.01% (vol/vol) Tween-20; dATP, dCTP, dGTP and dTTP each at 0.2 mM; and red dye, plus 1 µL each of the forward and reverse PCR primers at a concentration of 50 pmol/µL using the fourdisposable-tip unit on the liquid-handling robot.
4. Transfer 22 µL of master mix per well into a 96-well Thermosprint plate (Web Scientific, Crewe, UK). 5. Program the robot to add 3 µL of template DNA automatically to each of the wells. 6. Store the plate used for the PCR setup at 4°C on the RoboAmp platform throughout. 7. Once setup is completed automatically, seal the plate and place it in one of the two integral MWG thermocyclers, using the RoboAmp to do this. Using the RoboAmp in this way makes it possible to set up between 1 and 24 strains of S. pneumoniae for MLST at one time.
3.2.3. PCR Conditions The conditions used to amplify the 7 MLST loci of S. pneumoniae have been altered from those originally described (1,2) to a step down cycle developed for amplification of MLST loci N. meningitidis (3). Once the cycle is complete, the plate should be automatically removed from the thermocycler and placed at 4°C on one of the 4 chilled racks of the RoboAmp platform. 3.2.4. PCR Cleanup Cleanup of the PCR reactions is done to remove excess primer and unused dNTPs. Use commercially available filtration plates (Multiscreen 384PCR) (6) and the integrated vacuum manifold of the RoboAmp platform (see Note 3). 1. Using the RoboAmp, transfer the full volume of each PCR reaction, 25 µL, to a separate well of the Multiscreen plate, which should be previously placed on the vacuum manifold. 2. Once all reaction mixtures have been transferred, apply a vacuum of 450 mbar for 20 min until the wells are dry. 3. Elute the amplified DNA from the membrane by adding of 20 µL of 18-MΩ distilled H2O, and follow this by repeat pipetting by the robot for 100 repetitions at 50% pipetting speed. 4. Automatically transfer 2 × 9 µL of each cleaned PCR product to two chilled 96-well plates that are ready for the sequence reaction.
3.2.5. Sequence Setup Sequencing is done with dideoxy dye-labeled terminators. At the start of the sequence setup
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Automated Pneumococcal MLST procedure manually set up the sequence master mixes for forward and reverse nucleotide sequence readings of each of the 7 MSLT genes of S. pneumoniae. 1. Place 14 serum tubes (Sarstedt, Leicester, UK) in the chilled reagent rack of the RoboAmp platform. 2. Each sequence master mix should be made up from 10 µL of sequence mix (DYEnamic™ ET Terminator sequencing premix, Amersham Biosciences, Little Chalfont, UK) plus 1 µL of forward or reverse primer (5 pmol/µL) per 20 µL of reaction mixture. 3. Automatically pipet 11 µL of master mix into the wells of the two 96-well plates containing the clean PCR products. 4. Once this transfer is complete, automatically transfer the plates into the two thermocyclers of the RoboAmp platform.
3.2.6. Sequence Reaction Conditions 1. Program the thermocycler with the following cycle: 95°C for 2 min, 30 cycles each at 95°C for 15 s, 50°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min (3). 2. Once the cycle has finished, automatically remove the plates from the thermocyclers and place them onto racks of the RoboAmp that have been chilled to 4°C.
3.2.7. Sequence Cleanup 1. Perform sequence cleanup in a manner similar to that for the PCR cleanup described above, using a vacuum manifold and Multiscreen SEQ plates. In this case use a 384-SEQ plate (6) (see Note 3). 2. Transfer the full 20-µL volume of each sequence reaction to the wells of the plate and apply the vacuum to a pressure of 850 mbar. Continue this for 40 min, by which time the wells should be dry. 3. Wash the sequence products deposited on the membrane by adding 10 µL of 18-MΩ distilled H2O (dH2O), and then reapply the vaccum for 20 min until the wells are dry. 4. Elute sequence products in 50 µL 18-MΩ dH2O, repipetted 50 times at 50% pipetting speed.
305 Transfer 20 µL of each reaction product into a skirted 96-well plate (ABgene). 5. Store plates at 4°C on the RoboAmp platform, or remove and freeze at –20°C until loading directly into the automated capillary sequencer.
3.2.8. DNA Sequencing Sequencing is performed with the automated MegaBACE 1000 96-capillary sequencer (see Note 4). 1. Using the skirted 96-well plate direct from the RoboAmp load samples into the sequencer and inject them into the capillaries at a voltage of 3 kV for 40 s. 2. Apply a voltage of 9 kV over 100 min to separate the dye-labeled DNA fragments generated in the sequencing reaction.
3.2.9. Sequence Interpretation Sequences are automatically read from the sequencer with the integrated Cimaron v1.53 Slim Phredify base-calling software. 1. View raw data for each sequence as an electropherogram and convert to FASTA (text) format; this again can be done using the software integral to the MegaBACE 1000. 2. Download the text files into a local database of pneumococcal MLST alleles (7) in which each sequence will be automatically compared against all other similar alleles to produce an allele number. This step can also be accomplished through the MLST website (www.mlst.net) or NCBI Blast server (www.ncbi.nih.gov/blast). 3. Once all 7 gene loci for a given isolate have been assigned allele numbers, input the resulting 7-digit number into the MLST web site (www.mlst.net/new). The sequence type (ST) can then be determined from the particular combination of alleles. 4. When a new allele number or ST is detected, send the trace file data via the spreadsheet on the MLST web page to the MLST database curator for verification before its addition to the S. pneumoniae MLST database. A new allele number will be given to the appropriate locus and a new ST type will be assigned to that combination of alleles.
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4. Results This semiautomated protocol for pneumococcal MLST was validated on more than 200 pneumococcal isolates. Through use of the experience gained at the SMPRL for the analysis of meningococci by MLST, the protocol was improved to incorporate better DNA cleanup steps, higher throughput, and better nucleotide sequence analysis. Most of the pneumococci processed for MLST according to this protocol were clinical strains isolated in Scotland and typed for clinical or academic reasons. Like the original semiautomated protocol, the current protocol has proven highly reproducible and alleviates most of the manual tasks involved in producing MLST data. The SMPRL will continue utilizing this protocol for clinical and academic needs, enabling thousands of strains of bacteria to be characterized every year. Although we have described this method for pneumococcal MLST, it can be altered for MLST of other bacteria.
When seven alleles are used for MLST, as is the case for pneumococcal MLST, the specificity is such that when the technique was validated with 295 strains of pneumoccci, the mean number of alleles per locus was 25.1, potentially distinguishing > 6 × 109 genotypes (2). Thus, the resolving power of MLST as an analytical tool is high. MLST can be used to gain information on all pneumococci, whether or not they express a capsule. The information can then be used in conjunction with serologic data. In order for a system involving the amplification and sequencing of multiple loci to be employed in a reference-laboratory environment, where high numbers of isolates are analyzed, it is essential to implement some form of automation. This can reduce cross-contamination, enable continuity of performance, and reduce the cost per sample, all key goals in settings in which throughput is high. The semiautomated protocol described here fulfills these requirements.
5. Discussion Around 600 cases of invasive pneumococcal disease are reported in Scotland each year. Isolates from reported cases of disease are sent to the SMPRL where serotyping and MLST is performed. Although most strains of S. pneumoniae are serotyped successfully, strains that, for any reason, cannot be serotyped still require characterization. The most commonly occurring serotypes in Scotland are 4, 6, 9, 14, 19, and 23. Knowledge of serotype is very useful in determining epidemiologic data, since the distribution of serotypes associated with disease varies according to the age of the patient and also geographically (8–10). In addition, certain clones of pneumococci are antibiotic-resistant, and serotyping helps identify such clones. However, serotyping alone is often not sufficient to fully identify such clones, making methods such as MLST important. MLST can identify antibiotic-resistant clones as well as strains causing case clusters of pneumococcal disease. Moreover, new conjugate pneumococcal vaccines are being developed, and good epidemiological data is required for this. Importantly, such data must be comparable for different countries.
1. This method was used to analyze a collection of 150 pneumococci. All strains used in this study were clinical isolates of S. pneumoniae sent to SMPRL in Glasgow from routine diagnostic bacteriology laboratories in Scotland. 2. The RoboAmp platform used in the study is a fully automated robotic platform, that comprises four 96-well racks (two of which can be chilled to 4°C); a reagent rack for 2-mL serum tubes or 1.5-mL centrifuge tubes, which is also temperature controlled; two programmable thermocylers with motorized lids; a robotic arm that can grip and move 96- or 384-well plates around the platform; a liquid-handling robotic pipetting system that can be fitted with 4 washable or disposable tips, each handling up to 200 µL liquid at a time; and eight temperature-controlled storage racks for 96- or 384-well plates or Millipore plates. The RoboAmp permits automation of most of the procedures involved in DNA amplification and sequence labeling of the amplified products. 3. The Millipore Multiscreen 384-PCR and 384SEQ plates facilitate the removal of unincorporated primers and dNTPs from the amplification reactions, and the removal of unincorporated
6. Notes
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Automated Pneumococcal MLST dye-terminators and primers from the sequenceing reactions. If not removed, these products can interfere with upstream processes such as dye labeling after amplification and DNA sequencing after dye labeling. 4. The capillary DNA sequencer used in this protocol was the MegaBACE. It is an automated 96capillary electrophoresis system that requires little user intervention; removes the need to cast and load gels, as with traditional slab-gel-based electrophoresis systems; and identifies 96 DNA sequences of about 500 nucleotides each in the span of approx 1 h and 40 min.
Acknowledgments The authors appreciate additional technical support provided by Chris Sullivan at the SMPRL. Funding for the robot liquid handling systems and DNA sequencers was provided by the Meningitis Association (Scotland) and the National Services Division of the Scottish Executive. The project in which the present work was done is funded by the Chief Scientist Office of the Department of Health at the Scottish Executive. References 1. Maiden, M.C., Bygraves, J. A., Feil, E., et al. (1998) Multilocus sequence typing: A portable approach to the identification of clones within populations of
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pathogenic microorganisms. Proc. Natl. Acad. Sci. USA 95, 3140–3145. Enright, M.C. and B.G. Spratt (1998) A multilocus sequence typing scheme for Streptococcus pneumoniae: Identification of clones associated with serious invasive disease. Microbiology 144(Pt 11), 3049–3060. Clarke, S.C., M.A. Diggle, and G.F. Edwards (2001) Semiautomation of multilocus sequence typing for the characterisation of clinical isolates of Neisseria meningitidis. J. Clin. Microbiol. 39, 3066–3071. Smart, L.E. (1986) Serotyping of Streptococcus pneumoniae strains by coagglutination. J. Clin. Pathol. 39, 328–331. Smart, L.E. and J. Henrichsen (1986) An alternative approach to typing of Streptococcus pneumoniae strains by coagglutination. Acta. Pathol. Microbiol. Immunol. Scand. [B]. 94, 409–413. Clarke, S.C. and M.A. Diggle (2002) Automated PCR/sequence template purification. Mol. Biotechnol. 21, 221–224. Diggle, M.A. and S.C. Clarke (2002) Rapid assignment of nucleotide sequence data to allele types for multi-locus sequence analysis (MLSA) of bacteria using an adapted database and modified alignment program. J. Mol. Microbiol. Biotechnol. 4, 515–517. Clarke, S.C., Denham, B.C., Reid, J.A., et al. (2002) Pneumococcal disease in Scotland 1999. SCIEH Weekly Rep. 36, 62–63. Kalin, M. (1998) Pneumococcal serotypes and their clinical relevance. Thorax 53, 159–162. Scott, J.A., et al. (1996) Serogroup-specific epidemiology of Streptococcus pneumoniae: Associations with age, sex, and geography in 7,000 episodes of invasive disease. Clin. Infect. Dis. 22, 973–981.
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