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Open Access Methodology article

Effectiveness of the standard and an alternative set of Streptococcus pneumoniae multi locus sequence typing primers

Paul Adamiak1, Otto G Vanderkooi134, James D Kellner123, Anthony B Schryvers1, Julie A Bettinger5 and Joenel Alcantara1*

Author Affiliations

1 Department of Microbiology, Immunology and Infectious Diseases, 3330 Hospital Dr. NW, Calgary, AB T2N 4 N1, Canada

2 Alberta Children’s Hospital Research Institute for Child and Maternal Health, 3330 Hospital Dr. NW, T2N 4N1 Calgary, AB, Canada

3 Department of Pediatrics University of Calgary, Alberta Children’s Hospital, 2888 Shaganappi Trail NW, T3B 6A8 Calgary, AB, Canada

4 Department of Pathology and Laboratory Medicine Calgary Laboratory Services, 3535 Research Rd NW, T2L 2 K8 Calgary, AB, Canada

5 Vaccine Evaluation Center BC Children’s Hospital, University of British Columbia, 4480 Oak St, V6H 3 V4 Vancouver, British Colombia, Canada

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BMC Microbiology 2014, 14:143  doi:10.1186/1471-2180-14-143

Published: 3 June 2014

Abstract

Background

Multi-locus sequence typing (MLST) is a portable, broadly applicable method for classifying bacterial isolates at an intra-species level. This methodology provides clinical and scientific investigators with a standardized means of monitoring evolution within bacterial populations. MLST uses the DNA sequences from a set of genes such that each unique combination of sequences defines an isolate’s sequence type. In order to reliably determine the sequence of a typing gene, matching sequence reads for both strands of the gene must be obtained. This study assesses the ability of both the standard, and an alternative set of, Streptococcus pneumoniae MLST primers to completely sequence, in both directions, the required typing alleles.

Results

The results demonstrated that for five (aroE, recP, spi, xpt, ddl) of the seven S. pneumoniae typing alleles, the standard primers were unable to obtain the complete forward and reverse sequences. This is due to the standard primers annealing too closely to the target regions, and current sequencing technology failing to sequence the bases that are too close to the primer. The alternative primer set described here, which includes a combination of primers proposed by the CDC and several designed as part of this study, addresses this limitation by annealing to highly conserved segments further from the target region. This primer set was subsequently employed to sequence type 105 S. pneumoniae isolates collected by the Canadian Immunization Monitoring Program ACTive (IMPACT) over a period of 18 years.

Conclusions

The inability of several of the standard S. pneumoniae MLST primers to fully sequence the required region was consistently observed and is the result of a shift in sequencing technology occurring after the original primers were designed. The results presented here introduce clear documentation describing this phenomenon into the literature, and provide additional guidance, through the introduction of a widely validated set of alternative primers, to research groups seeking to undertake S. pneumoniae MLST based studies.

Keywords:
Multi-locus sequence typing; MLST; Invasive pneumococcal disease; Molecular epidemiology; Streptococcus pneumoniae; Bacterial typing