Real‐time multiplex PCR for direct detection of methicillin‐resistant Staphylococcus aureus (MRSA) in clinical samples enriched by broth culture

Söderquist, Bo; Neander, Marita; Dienus, Olaf; Zimmermann, Johanna; Berglund, Carolina; Matussek, Andreas; Mölling, Paula

doi:10.1111/j.1600-0463.2011.02849.x

Cited by 6 publications

(3 citation statements)

References 19 publications

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“…The assay provides a valuable tool for the rapid and accurate characterization of staphylococci, as automatic DNA extraction directly from sample, amplification cycles and post-PCR analyses are completed in less than 3 hours. Comparing to the assay developed by Söderquist et al [17], our assay doesn't need to pre-treat clinical samples by broth enriched method. Our assay was validate starting directly from heavy contaminated clinical samples (rectal swab and pharingeal swab) during multidrug resistant screening program and show a very high sensititity and specificity.…”

Section: Resultsmentioning

confidence: 97%

Real-time PCR assay for detection of Staphylococcus aureus, Panton-Valentine Leucocidin and Methicillin Resistance directly from clinical samples

Galia¹,

Ligozzi²,

Bertoncelli³

et al. 2019

AIMS Microbiology

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Rapid detection of Methicillin Resistant Staphylococcus aureus (MRSA) is an important concern for both treatment and implementation of infection control policies. The present study provides an ‘in house’ real-time PCR assay to detect directly nuc , pvl , and mecA genes. The assay is able to perform identification of MRSA, Methicillin-Sensitive S. aureus , Methicillin-Resistant Coagulase Negative Staphylococci and the Panton-Valentine leukocidin virulence gene from rectal and pharyngeal swab samples in a screening context. We found an analytical sensitivity of this current Triplex PCR assay of 514 CFU/mL. Analytical specificity was tested with different Gram-positive and Gram-negative species and yielded no false-positive PCR signal. The sensitivity and specificity of the Triplex Real Time PCR were both 100% for these targets when compared with the culture and conventional methods. This assay is readily adaptable for routine use in a microbiology laboratory, as it will enable the implementation of timely and properly guided therapy and infection control strategies.

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Section: Resultsmentioning

confidence: 97%

Real-time PCR assay for detection of Staphylococcus aureus, Panton-Valentine Leucocidin and Methicillin Resistance directly from clinical samples

Galia¹,

Ligozzi²,

Bertoncelli³

et al. 2019

AIMS Microbiology

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“…Real-time PCR technology has been used as an alternative to culture methods for the rapid detection of S. aureus and MRSA. Detection using real-time PCR may decrease the time of analysis to 18 h after consecutive broth enrichment in clinical samples [20] ; or <2 h in positive blood cultures [21] , [22] . However, most studies have used real-time PCR to detect MRSA in clinical samples and isolates and a few studies have evaluated the application of this method for the detection of MRSA in animals [23] , [24] and meat [15] , [25] , [26] .…”

Section: Introductionmentioning

confidence: 99%

Multiplex Real-Time PCR for Detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) Genes from Selective Enrichments from Animals and Retail Meat

et al. 2014

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The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68–0.88 (from substantial to almost perfect agreement) and 0.29–0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0–0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using the standard culture method.

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“…The sensitivity of this approach is high, and it could detect MRSA from a sample with 25 CFU of bacteria. Moreover, it provides a powerful approach to differentiate MRSA from clinical samples with different staphylococci [ 44 , 45 ]. The specificity could be improved to reduce false positive results by including 16S rRNA gene in the multiplex RT-PCR [ 46 ].…”

Section: Identification Of S Aureus Including Mmentioning

confidence: 99%

PCR-based Approaches for the Detection of Clinical Methicillin-resistant Staphylococcus aureus

Liu¹,

Zhang²,

Ji³

2016

TOMICROJ

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Staphylococcus aureus is an important pathogen that can cause a variety of infections, including superficial and systematic infections, in humans and animals. The persistent emergence of multidrug resistant S. aureus, particularly methicillin-resistant S. aureus, has caused dramatically economic burden and concerns in the public health due to limited options of treatment of MRSA infections. In order to make a correct choice of treatment for physicians and understand the prevalence of MRSA, it is extremely critical to precisely and timely diagnose the pathogen that induces a specific infection of patients and to reveal the antibiotic resistant profile of the pathogen. In this review, we outlined different PCR-based approaches that have been successfully utilized for the rapid detection of S. aureus, including MRSA and MSSA, directly from various clinical specimens. The sensitivity and specificity of detections were pointed out. Both advantages and disadvantages of listed approaches were discussed. Importantly, an alternative approach is necessary to further confirm the detection results from the molecular diagnostic assays.

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Real‐time multiplex PCR for direct detection of methicillin‐resistant Staphylococcus aureus (MRSA) in clinical samples enriched by broth culture

Cited by 6 publications

References 19 publications

Real-time PCR assay for detection of <em>Staphylococcus aureus</em>, Panton-Valentine Leucocidin and Methicillin Resistance directly from clinical samples

Real-time PCR assay for detection of <em>Staphylococcus aureus</em>, Panton-Valentine Leucocidin and Methicillin Resistance directly from clinical samples

Multiplex Real-Time PCR for Detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) Genes from Selective Enrichments from Animals and Retail Meat

PCR-based Approaches for the Detection of Clinical Methicillin-resistant Staphylococcus aureus

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