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Published on: January 17, 2014
Real-time pneumolysin polymerase chain reaction with melting curve analysis differentiates pneumococcus from other
Tarja Kaijalainen1, Annika Saukkoriipi, Aini Bloigu
1KTL, National Public Health Institute, P.O. Box 310, FIN-90101 Oulu, Finland. tarja. kaijalainen@ktl.fi
Abstract:
The majority of pneumococcal isolates can be identified by the conventional methods based on phenotypic characteristics. Occasionally, however, the differentiation of alpha-hemolytic streptococci from pneumococci, especially those isolated from nasopharynx, is problematic due to the discrepant results obtained by the conventional identification methods. Several gene technological methods based on the amplification of genes encoding pneumococcal virulence factors, such as pneumolysin (ply) and autolysin, have been used as additional identification methods. Recent studies have shown that especially the ply gene is frequently also present in nonpneumococcal alpha-hemolytic streptococci. In this study, we compared the commonly used phenotypic identification methods with nucleic acid-based methods, commercial AccuProbetrade mark, conventional pneumolysin polymerase chain reaction (Ply-PCR), and real-time Ply-PCR in the identification of alpha-hemolytic streptococcal strains isolated from 100 consecutive nasopharyngeal specimens. We also studied if melting curve analysis and sequencing of the amplification products of a ply gene fragment could be helpful in the identification. Our results suggest that the ply gene present in alpha-hemolytic streptococci differs from that present in pneumococcus, and that melting curve analysis would prove useful in the differentiation of these bacteria.
Insights
Differentiating pneumococci from other alpha-hemolytic streptococci can be challenging. This study found that analyzing the pneumolysin (ply) gene using melting curve analysis can effectively distinguish between these bacteria, improving diagnostic accuracy.
Area of Science:
- Microbiology
- Molecular Biology
- Clinical Diagnostics
Background:
- Conventional phenotypic methods sometimes struggle to differentiate Streptococcus pneumoniae from other alpha-hemolytic streptococci, particularly in nasopharyngeal isolates.
- Gene-based methods, like pneumolysin (ply) gene amplification, are increasingly used but the ply gene's presence in non-pneumococcal strains complicates identification.
- Accurate differentiation is crucial for appropriate clinical management and epidemiological studies.
Purpose of the Study:
- To compare phenotypic identification methods with nucleic acid-based techniques for identifying alpha-hemolytic streptococci from nasopharyngeal specimens.
- To evaluate the utility of conventional pneumolysin polymerase chain reaction (Ply-PCR) and real-time Ply-PCR in differentiating pneumococci.
- To assess the role of melting curve analysis and gene sequencing of ply amplification products in bacterial identification.
Main Methods:
- Phenotypic identification of alpha-hemolytic streptococci.
- Nucleic acid-based methods: commercial AccuProbe, conventional Ply-PCR, and real-time Ply-PCR.
- Analysis of 100 consecutive nasopharyngeal isolates.
- Melting curve analysis and sequencing of ply gene amplification products.
Main Results:
- The ply gene sequences in alpha-hemolytic streptococci were found to differ from those in true pneumococci.
- Melting curve analysis of ply gene fragments showed promise in differentiating between pneumococcal and non-pneumococcal alpha-hemolytic streptococci.
- Real-time Ply-PCR combined with melting curve analysis offers a potentially valuable diagnostic approach.
Conclusions:
- The genetic variations in the ply gene can be exploited for accurate differentiation.
- Melting curve analysis is a useful adjunct to PCR for distinguishing Streptococcus pneumoniae from other alpha-hemolytic streptococci.
- Nucleic acid-based methods, particularly when incorporating melting curve analysis, enhance the identification of clinically relevant streptococci.
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