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Evaluation of a duplex real-time PCR assay to detect MRSA from broth culture, human sera seeded with MRSA and from patient's serum.

ABSTRACT: The need for rapid methods in order to precisely detect methicillin-resistant Staphylococcus aureus (MRSA) is extensively acknowledged. This study evaluated a quantitative real-time PCR assay targeting mecA (encoding high level resistance to methicillin) and femB (a specific genomic marker for S. aureus) genes to detect MRSA from broth culture, from serum seeded with MRSA and straight from the patient's serum. One hundred and thirty-five clinical isolates of MRSA strains and different species were utilised in this study. In addition, a pilot study with 9 patients' serum samples was performed. The sensitivity and specificity values for this assay were 99% and 100% respectively. The detection limit for this method was 1.23×10(2) CFU/ml from the serum seeded with MRSA cells and the limiting concentration of DNA for detection was 18 fg, which equates to 5.14 genomic DNA copies. In addition, this assay detected MRSA from patient's serum (7 out of 9) with sensitivity of 77.8%. Overall, the assay was rapid, efficient, sensitive and easy to perform.

SUBMITTER: Awad S

PROVIDER: S-EPMC3842574 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Evaluation of a duplex real-time PCR assay to detect MRSA from broth culture, human sera seeded with MRSA and from patient's serum.

Awad Sawsan S Alshami Issam I Alharbi Ahmed Eid AE

Bioinformation 20131111 18

The need for rapid methods in order to precisely detect methicillin-resistant Staphylococcus aureus (MRSA) is extensively acknowledged. This study evaluated a quantitative real-time PCR assay targeting mecA (encoding high level resistance to methicillin) and femB (a specific genomic marker for S. aureus) genes to detect MRSA from broth culture, from serum seeded with MRSA and straight from the patient's serum. One hundred and thirty-five clinical isolates of MRSA strains and different species we ...[more]

PMID: 24307766

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Project description:We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/ microl to 1 ng/ microl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/ microl to 1 ng/ microl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/ microl to 1 ng/ microl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/ microl to 1 ng/ microl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler and the LightCycler platforms. The assay may be useful for the early detection of smallpox virus infections should such infections occur as a result of a deliberate or an accidental recurrence.

Dataset Information

Evaluation of a duplex real-time PCR assay to detect MRSA from broth culture, human sera seeded with MRSA and from patient's serum.

Publications

Evaluation of a duplex real-time PCR assay to detect MRSA from broth culture, human sera seeded with MRSA and from patient's serum.

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