Development of a new pentaplex real-time PCR assay for the identification of poly-microbial specimens containing Staphylococcus aureus and other staphylococci, with simultaneous detection of staphylococcal virulence and methicillin resistance markers

Okolie, Charles (2015) Development of a new pentaplex real-time PCR assay for the identification of poly-microbial specimens containing Staphylococcus aureus and other staphylococci, with simultaneous detection of staphylococcal virulence and methicillin resistance markers. Molecular and Cellular Probes, 29. pp. 144-150.

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Abstract

Staphylococcus aureus strains harbouring genes encoding virulence and antibiotic resistance are of public health importance. In clinical samples, pathogenic S. aureus is often mixed with putatively less pathogenic coagulase-negative staphylococci (CoNS), both of which can harbour mecA, the gene encoding staphylococcal methicillin-resistance. There have been previous attempts at distinguishing MRSA from MRCoNS, most of which were based on the detection of one of the pathognomonic markers of S. aureus, such as coa, nuc or spa. That approach might suffice for discrete colonies and mono-microbial samples; it is inadequate for identification of clinical specimens containing mixtures of S. aureus and CoNS. In the present study, a real-time pentaplex PCR assay has been developed which simultaneously detects markers for bacteria (16S rRNA), coagulase-negative staphylococcus (cns), S. aureus (spa), Panton- Valentine leukocidin (pvl) and methicillin resistance (mecA). Staphylococcal and non-staphylococcal bacterial strains (n ¼ 283) were used to validate the new assay. The applicability of this test to clinical samples was evaluated using spiked blood cultures (n ¼ 43) containing S. aureus and CoNS in monomicrobial and poly-microbial models, which showed that the 5 markers were all detected as expected. Cycling completes within 1 h, delivering 100% specificity, NPV and PPV with a detection limit of 1.0 � 101 to 3.0 � 101 colony forming units (CFU)/ml, suggesting direct applicability in routine diagnostic microbiology. This is the most multiplexed real-time PCR-based PVL-MRSA assay and the first detection of a unique marker for CoNS without recourse to the conventional elimination approach. There was no evidence that this new assay produced invalid/indeterminate test results.

Item Type: Article
Uncontrolled Keywords: Poly-microbial infections Staphylococci Differential diagnosis PVL
Subjects: Q Science > Q Science (General)
Q Science > QR Microbiology
R Medicine > R Medicine (General)
R Medicine > RB Pathology
R Medicine > RC Internal medicine
R Medicine > RL Dermatology
R Medicine > RZ Other systems of medicine
T Technology > T Technology (General)
Divisions: Faculty of Medicine, Health and Life Sciences > School of Biological Sciences
Depositing User: CHARLES OKOLIE
Date Deposited: 29 Feb 2016 12:32
Last Modified: 29 Feb 2016 12:32
URI: http://eprints.lmu.edu.ng/id/eprint/400

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