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Multiple-locus variable number tandem repeat analysis for Streptococcus pneumoniae: comparison with PFGE and MLST.

Elberse KE, Nunes S, Sá-Leão R, van der Heide HG, Schouls LM - PLoS ONE (2011)

Bottom Line: Furthermore, the Wallace of MLVA to clonal complex of MLST was even higher: 99.5%.For some isolates belonging to a single MLST clonal complex although displaying different serotypes, MLVA was more discriminatory, generating groups according to serotype or serogroup.In the companion paper published simultaneously in this issue we applied the MLVA to assess the pneumococcal population structure of isolates causing invasive disease in The Netherlands before the introduction of the 7-valent conjugate vaccine.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Infectious Diseases and Perinatal Screening, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands. Karin.Elberse@rivm.nl

ABSTRACT
In the era of pneumococcal conjugate vaccines, surveillance of pneumococcal disease and carriage remains of utmost importance as important changes may occur in the population. To monitor these alterations reliable genotyping methods are required for large-scale applications. We introduced a high throughput multiple-locus variable number tandem repeat analysis (MLVA) and compared this method with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The MLVA described here is based on 8 BOX loci that are amplified in two multiplex PCRs. The labeled PCR products are sized on an automated DNA sequencer to accurately determine the number of tandem repeats. The composite of the number of repeats of the BOX loci makes up a numerical profile that is used for identification and clustering. In this study, MLVA was performed on 263 carriage isolates that were previously characterized by MLST and PFGE. MLVA, MLST and PFGE (cut-off of 80%) yielded 164, 120, and 87 types, respectively. The three typing methods had Simpson's diversity indices of 98.5% or higher. Congruence between MLST and MLVA was high. The Wallace of MLVA to MLST was 0.874, meaning that if two strains had the same MLVA type they had an 88% chance of having the same MLST type. Furthermore, the Wallace of MLVA to clonal complex of MLST was even higher: 99.5%. For some isolates belonging to a single MLST clonal complex although displaying different serotypes, MLVA was more discriminatory, generating groups according to serotype or serogroup. Overall, MLVA is a promising genotyping method that is easy to perform and a relatively cheap alternative to PFGE and MLST. In the companion paper published simultaneously in this issue we applied the MLVA to assess the pneumococcal population structure of isolates causing invasive disease in The Netherlands before the introduction of the 7-valent conjugate vaccine.

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Schematic representation of the pneumococcus R6 genome indicating the location of the 8 BOX loci that are used in the MLVA scheme.
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pone-0019668-g002: Schematic representation of the pneumococcus R6 genome indicating the location of the 8 BOX loci that are used in the MLVA scheme.

Mentions: In the genome sequence of isolate R6 (GenBank number NC_003098), one of the publicly available genomes, 127 BOX tandem repeats were found. Thirteen randomly chosen BOX loci scattered throughout the R6 genome to be used in the MLVA scheme were tested on a panel of 84 isolates. Eight BOX loci were chosen and primers were designed targeting the flanking regions of the BoxA and BoxC elements (Figure 2, Table 1). One primer of each primer set was fluorescently labeled with FAM, NED, VIC or PET. For BOX loci 4 and 6 respectively 3 and 4 primers were used to enhance amplification within all isolates. The 8 loci were amplified in 2 separate multiplex PCRs, creating mixtures each with 4 different fluorescent labels enabling analysis of 4 BOX loci in a single fragment analysis reaction (Table 1). Amplification of the loci was done in Applied Biosystems 9700 PCR machines. The 25 µl PCR reaction mixtures consisted of Qiagen multiplex PCR mix, 10 µM of each primer and 2 µl of 1∶10 lysates diluted in sterile water. Lysates were prepared by suspending a loop full of pneumococci in 500 µl TE (10 mM Tris.HCl and 1 mM EDTA, pH 8) followed by heating for 10 minutes by 95°C. Amplification was performed using the following PCR program: 15 min 95°C, 25 cycles of 30 sec 95°C, 1 min 54°C and 1 min 72°C followed by a 30 min incubating at 68°C to ensure complete addition of the extra 3′adenosin by the terminal transferase activity of the Taq polymerase. Of the PCR product mixtures, 2 µl aliquots, diluted 1∶200 in water, were mixed with 10 µl of 1∶200 diluted Genescan 1200 LIZ-marker (Applied Biosystems, Foster City, U.S.A.). The product was heated for 5 minutes at 95°C for denaturation and sized on the AB 3730 DNA sequencer using the Fragment Analysis module.


Multiple-locus variable number tandem repeat analysis for Streptococcus pneumoniae: comparison with PFGE and MLST.

Elberse KE, Nunes S, Sá-Leão R, van der Heide HG, Schouls LM - PLoS ONE (2011)

Schematic representation of the pneumococcus R6 genome indicating the location of the 8 BOX loci that are used in the MLVA scheme.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102655&req=5

pone-0019668-g002: Schematic representation of the pneumococcus R6 genome indicating the location of the 8 BOX loci that are used in the MLVA scheme.
Mentions: In the genome sequence of isolate R6 (GenBank number NC_003098), one of the publicly available genomes, 127 BOX tandem repeats were found. Thirteen randomly chosen BOX loci scattered throughout the R6 genome to be used in the MLVA scheme were tested on a panel of 84 isolates. Eight BOX loci were chosen and primers were designed targeting the flanking regions of the BoxA and BoxC elements (Figure 2, Table 1). One primer of each primer set was fluorescently labeled with FAM, NED, VIC or PET. For BOX loci 4 and 6 respectively 3 and 4 primers were used to enhance amplification within all isolates. The 8 loci were amplified in 2 separate multiplex PCRs, creating mixtures each with 4 different fluorescent labels enabling analysis of 4 BOX loci in a single fragment analysis reaction (Table 1). Amplification of the loci was done in Applied Biosystems 9700 PCR machines. The 25 µl PCR reaction mixtures consisted of Qiagen multiplex PCR mix, 10 µM of each primer and 2 µl of 1∶10 lysates diluted in sterile water. Lysates were prepared by suspending a loop full of pneumococci in 500 µl TE (10 mM Tris.HCl and 1 mM EDTA, pH 8) followed by heating for 10 minutes by 95°C. Amplification was performed using the following PCR program: 15 min 95°C, 25 cycles of 30 sec 95°C, 1 min 54°C and 1 min 72°C followed by a 30 min incubating at 68°C to ensure complete addition of the extra 3′adenosin by the terminal transferase activity of the Taq polymerase. Of the PCR product mixtures, 2 µl aliquots, diluted 1∶200 in water, were mixed with 10 µl of 1∶200 diluted Genescan 1200 LIZ-marker (Applied Biosystems, Foster City, U.S.A.). The product was heated for 5 minutes at 95°C for denaturation and sized on the AB 3730 DNA sequencer using the Fragment Analysis module.

Bottom Line: Furthermore, the Wallace of MLVA to clonal complex of MLST was even higher: 99.5%.For some isolates belonging to a single MLST clonal complex although displaying different serotypes, MLVA was more discriminatory, generating groups according to serotype or serogroup.In the companion paper published simultaneously in this issue we applied the MLVA to assess the pneumococcal population structure of isolates causing invasive disease in The Netherlands before the introduction of the 7-valent conjugate vaccine.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Infectious Diseases and Perinatal Screening, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands. Karin.Elberse@rivm.nl

ABSTRACT
In the era of pneumococcal conjugate vaccines, surveillance of pneumococcal disease and carriage remains of utmost importance as important changes may occur in the population. To monitor these alterations reliable genotyping methods are required for large-scale applications. We introduced a high throughput multiple-locus variable number tandem repeat analysis (MLVA) and compared this method with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The MLVA described here is based on 8 BOX loci that are amplified in two multiplex PCRs. The labeled PCR products are sized on an automated DNA sequencer to accurately determine the number of tandem repeats. The composite of the number of repeats of the BOX loci makes up a numerical profile that is used for identification and clustering. In this study, MLVA was performed on 263 carriage isolates that were previously characterized by MLST and PFGE. MLVA, MLST and PFGE (cut-off of 80%) yielded 164, 120, and 87 types, respectively. The three typing methods had Simpson's diversity indices of 98.5% or higher. Congruence between MLST and MLVA was high. The Wallace of MLVA to MLST was 0.874, meaning that if two strains had the same MLVA type they had an 88% chance of having the same MLST type. Furthermore, the Wallace of MLVA to clonal complex of MLST was even higher: 99.5%. For some isolates belonging to a single MLST clonal complex although displaying different serotypes, MLVA was more discriminatory, generating groups according to serotype or serogroup. Overall, MLVA is a promising genotyping method that is easy to perform and a relatively cheap alternative to PFGE and MLST. In the companion paper published simultaneously in this issue we applied the MLVA to assess the pneumococcal population structure of isolates causing invasive disease in The Netherlands before the introduction of the 7-valent conjugate vaccine.

Show MeSH