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Updated: Jul 11, 2025
Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus
Published on: September 5, 2013
Identification of pneumococcal serotypes with individual recognition of vaccine types by a highly multiplexed
Shujuan Zhou1, Jie Che2, Xuran Wang1
1Engineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, China.
Background:
Pneumococcus serotyping is important for monitoring serotype epidemiology, vaccine-induced serotypes replacement and emerging pathogenic serotypes. However, the lack of high-resolution serotyping tools has hindered its widespread implementation.
Methods:
We devised a single-step, multiplex real-time polymerase chain reaction (PCR)-based MeltArray approach termed PneumoSero that can identify 92 serotypes with individual recognition of 54 serotypes, including all 24 currently available vaccine types. The limit of detection (LOD) and the ability to coexisting serotypes were studied, followed by analytical evaluation using 92 reference pneumococcal strains and 125 non-pneumococcal strains, and clinical evaluation using 471 pneumococcus isolates and 46 pneumococcus-positive clinical samples.
Results:
The LODs varied with serotypes from 50 to 100 copies per reaction and 10 % of the minor serotypes were detectable in samples containing two mixed serotypes. Analytical evaluation presented 100 % accuracy in both 92 reference pneumococcal strains and 125 non-pneumococcal strains. Clinical evaluation of 471 pneumococcus isolates displayed full concordance with Sanger sequencing results. The 46 clinical specimens yielded 45 typeable results and one untypeable result. Of the 45 typeable samples, 41 were of a single serotype and four were of mixed serotypes, all of which were confirmed by Sanger sequencing or separate PCR assays.
Conclusion:
We conclude that the PneumoSero assay can be implemented as a routine tool for pneumococcal serotyping in standard microbiology laboratories and even in clinical settings.
Insights
A new multiplex real-time PCR assay, PneumoSero, accurately identifies 92 pneumococcus serotypes, including all 24 vaccine types. This high-resolution tool is suitable for routine use in clinical microbiology labs.
Area of Science:
- Microbiology
- Molecular Biology
- Epidemiology
Background:
- Pneumococcus serotyping is crucial for tracking disease epidemiology and vaccine effectiveness.
- Current serotyping methods lack the high resolution needed for widespread implementation.
Purpose of the Study:
- To develop a high-resolution, multiplex real-time PCR assay for comprehensive pneumococcus serotyping.
- To evaluate the accuracy and clinical utility of the novel PneumoSero assay.
Main Methods:
- Developed PneumoSero, a single-step, multiplex real-time PCR-based MeltArray assay.
- Assessed limit of detection and ability to detect coexisting serotypes.
- Validated the assay using reference strains, non-pneumococcal strains, and clinical isolates.
Main Results:
- PneumoSero identifies 92 serotypes, with individual recognition of 54 types, including all 24 vaccine types.
- Achieved 100% accuracy for both pneumococcal and non-pneumococcal strains in analytical evaluations.
- Clinical evaluation showed high concordance with Sanger sequencing, with 45 out of 46 clinical samples yielding typeable results.
Conclusions:
- The PneumoSero assay provides accurate and high-resolution pneumococcus serotyping.
- It is suitable for implementation as a routine tool in standard microbiology and clinical settings.
