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Published on: July 9, 2012
Real-Time PCR to Identify Staphylococci and Assay for Virulence from Blood
1Department of Biological Sciences, Landmark University, Km 4 Ipetu Omu Aran Road, Omu-Aran, PMB 1001, Kwara State, Nigeria. okolie.charles@lmu.edu.ng.
Abstract:
The genus Staphylococcus includes pathogenic and non-pathogenic facultative anaerobes. Due to the plethora of virulence factors encoded in its genome, the species Staphylococcus aureus is known to be the most pathogenic. S. aureus strains harboring genes encoding virulence and antibiotic resistance are of public health importance. In clinical samples, however, pathogenic S. aureus is often mixed with putatively less pathogenic coagulase-negative staphylococci (CoNS), both of which can harbor mecA, the genetic driver for staphylococcal methicillin-resistance. In this chapter, the detailed practical procedure for operating a real-time pentaplex PCR assay in blood cultures is described. The pentaplex real-time PCR assay simultaneously detects markers for the presence of bacteria (16S rRNA), coagulase-negative staphylococcus (cns), S. aureus (spa), Panton-Valentine leukocidin (pvl), and methicillin resistance (mecA).
Insights
This study details a pentaplex real-time PCR assay for rapid detection of bacteria, including Staphylococcus aureus and methicillin resistance (mecA) in blood cultures. This method aids in identifying critical pathogens and antibiotic resistance markers simultaneously.
Area of Science:
- Clinical Microbiology
- Molecular Diagnostics
- Bacterial Pathogenesis
Background:
- Staphylococcus species encompass both pathogenic and non-pathogenic strains, with Staphylococcus aureus being highly virulent due to numerous virulence factors.
- Coagulase-negative staphylococci (CoNS) often co-exist with S. aureus in clinical samples, complicating diagnosis.
- The mecA gene confers methicillin resistance in staphylococci, posing a significant public health challenge.
Purpose of the Study:
- To describe a practical procedure for a pentaplex real-time PCR assay.
- To enable simultaneous detection of bacterial presence, S. aureus, and methicillin resistance in blood cultures.
Main Methods:
- Development and implementation of a pentaplex real-time PCR assay.
- The assay targets specific genetic markers: 16S rRNA (bacteria), cns (CoNS), spa (S. aureus), pvl (Panton-Valentine leukocidin), and mecA (methicillin resistance).
Main Results:
- The described pentaplex real-time PCR assay allows for the simultaneous detection of multiple targets in a single reaction.
- This method provides rapid identification of bacterial pathogens and key resistance genes directly from blood cultures.
Conclusions:
- The pentaplex real-time PCR assay is a valuable tool for the rapid and simultaneous detection of bacteria, S. aureus, and methicillin resistance in clinical settings.
- This diagnostic approach can significantly aid in timely clinical decision-making for patients with suspected staphylococcal infections.
