The capsular polysaccharide, a well-known virulence factor and the serogroup/type determinant of pneumococci, prevents opsonization and phagosytosis of S.pneumoniae
. Pneumococcal capsular vaccines (PCV7 and PCV13) protect against disease and nasopharyngeal carriage due to the serotypes included in the vaccine formulations 
. Nevertheless, surveillance of serotype prevalence patterns is very important since the serotypes responsible for invasive disease can change over time 
. The recent availability of sequence information of all serotypes has led to an interest in molecular typing methods. The serogroup/type-specific primers yielding distinct amplification products have been designed and different multiplex PCR schemes, capable of amplifying most disease-associated serotypes by sequential reactions, have been reported since 2003 
. The results indicated that the multiplex PCR method accurately detects the serotypes and serogroups of pneumococcal isolates.
In our study, we attempted to devise a multiplex PCR assay to detect all PCV13-serogroups/types in one amplification reaction. For this purpose, a single multiplex PCR master mix containing 12 primers targeting PCV13-serogroups/types was constituted and the amplification conditions were optimized. The results showed that our one-step multiplex PCR assay was successfully designed and correctly identified PCV13-serogroups/types of invasive and nasopharyngeal isolates. Unlike the available multiplex PCR methods requiring performance of several sequential PCR assays to detect serogroups/types present in PCV13 
, our multiplex PCR assay determined the serogroup/type of an isolate by running only one PCR reaction. As previously described, there is however some cross-reactivity and the antiserum method would still be required in order to fully type the isolates of serogroup 6, 7, 9 and 18.
To our knowledge, this is the first report on identification of all PCV13-serogroups/types by performing a one-step multiplex PCR. Results of our experiments demonstrated the capacity of our multiplex PCR assay for typing all PCV13-serogroups/types. Additionally, non-vaccine types and α-hemolytic streptococci were also reliably identified by this assay based on the presence and absence of internal control cpsA product. Thus, as a specific and sensitive serotyping tool, our one-step multiplex PCR assay can be applied in most research and public health laboratories for screening large numbers of isolates in a single day.
Misinterpretation of the multiplex PCR gels has been reported to be a minor limitation of PCR-based serotyping 
. Taking this technical difficulty into consideration, we prepared M1 and M2 size standards. In our setting, using the serogroup/type-specific markers and extending the gel run assured precise discrimination of all amplicons, including serogroup 3, 5 and serotype 23F with size differences of as low as 9–13 bp.
While the results of many studies previously demonstrated that the multiplex PCR was highly accurate, fast and cost-effective method 
, there is yet no manufactured multiplex PCR system commercially available for determination of the most invasive serogroups/types targeted by PCV13. In its present form, our multiplex PCR assay could be manufactured as a commercial typing kit for rapid screening of pneumococcal isolates.
As a conclusion, the information gathered by our one-step multiplex PCR assay successfully guides the experimenter and significantly reduces the number of isolates that have to be serotyped by the traditional antiserum method. Therefore, we currently are working on additional multiplex PCR combinations covering predominant serotypes other than those in PCV13.