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Serotyping of Streptococcus pneumoniae by PCR – expansion of the method to identify the Hungarian serotypes

Abstract number: 1733_925

Dobay O., Nagy K., Amyes S., Rozgonyi F.

Objectives:Streptococcus pneumoniae has 90 different serotypes, and as certain serotypes are linked with antibiotic resistance, virulence or different types of diseases, it is essential for the epidemiological studies to determine the serotype of the isolates, in addition to genotyping. The conventional method is to use antisera, but it is time consuming, expensive and difficult to evaluate. Some workers have started determining the serotype by PCR and we have expanded this methodology to characterise the strains normally found in Hungary.

Methods: Conventional serotyping was performed with MAST antisera on glass slides. For the PCR, the grouping primers and primers for several individual serotypes described by Brito et al. (1, 3, 4, 6, 14, 18C, 19F, 19A, 23F) were used, but we designed a set of new primers to other serotypes that are also relatively common in Hungary (9V, 6A, 6B, 7F, 11A, 15A, 15B). The PCR was done in two stages. The first multiplex PCR reaction divided the strains into 6 different groups, based on the gel pattern. Then a second multiplex PCR reaction, which included the primers for the individual serotypes of each group (3 or 4), was performed.

Results: Isolates of known serotypes were tested first with the individual primers in single PCR reactions. Then the grouping reactions were tried with these strains. The multiplex PCR worked best with Tth polymerase, but in Taq buffer. We had to alter the annealing temperature and other parameters as well. After improving the method, we designed the new primers. These had to be suited for the existing reaction, i.e. had to work under the same cycling parameters that had originally been used; however these primers also had to be unique for the new serotype within the group and provide PCR products of different sizes for each serotype. When invasive isolates of unknown serotypes were tested by this method and the results compared with the conventional method, the same serotypes were identified.

Conclusions: The new 7-valent conjugate vaccine was introduced in Hungary very recently, therefore the examination of invasive strains is of great importance. We have extended the method of Brito et al. to identify Hungarian isolates, by designing primers for new serotypes. Serotyping of large numbers of isolates by the conventional method requires expensive resources, while the capability to identify serotypes by PCR is much simpler, is as sensitive and is more cost effective than conventional serotyping.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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