Simultaneous species identification and detection of methicillin resistance in staphylococci using triplex real-time PCR assay

Sabet, Negar Shafiei; Subramaniam, G.; Parasakthi, N; Sekaran, Shamala Devi

doi:10.1016/j.diagmicrobio.2006.02.013

Cited by 24 publications

(18 citation statements)

References 21 publications

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“…Some of the newer assays, although sensitive and specific, require complicated procedures for processing or detection (14) or are not commercially available (37). Those that are available commercially do not discriminate between MSSA and MRSA (16,22) or require a subcultured isolate (18,33), which is the aforementioned time constraint with culture-based methods.…”

Section: Discussionmentioning

confidence: 99%

“…Nucleic acid amplification methods can provide same-day results once the blood cultures are positive for microbial growth; however, the majority of presently described assays are not designed to differentiate between MSSA and MRSA. Additionally, those nucleic acid amplification procedures that can differentiate between MSSA and MRSA often require labor-intensive protocols, technologically advanced equipment, and a colony isolated from subculture, or such assays are not readily available (14,17,28,30,33,37,42). The detection of mecA or MRSA in positive blood culture bottles using molecular methods has previously been very successful (4,36), but there are few reports on the rapid differentiation between MSSA and MRSA among other gram-positive organisms causing bacteremia (29,34,37).…”

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confidence: 99%

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Clinical Validation of the Molecular BD GeneOhm StaphSR Assay for Direct Detection of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus in Positive Blood Cultures

et al. 2007

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The rapid detection of Staphylococcus aureus bacteremia and a swift determination of methicillin susceptibility has serious clinical implications affecting patient mortality. This study evaluated the StaphSR assay (BD GeneOhm, San Diego, CA), a real-time PCR assay, for the identification and differentiation of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) from 300 positive blood cultures. The BD GeneOhm StaphSR assay was performed and interpreted according to the manufacturer's recommendations. Positive blood cultures (containing predominantly gram-positive cocci in clusters) were subcultured on 5% sheep blood agar plates. After 18 to 24 h of incubation, isolates morphologically consistent with S. aureus were presumptively identified by latex agglutination (Staphaurex Plus; Remel, Lenexa, KS). Susceptibility testing was initially performed with the Phoenix automated microbiology system (BD Diagnostics, Sparks, MD). Additional susceptibility testing of samples with discrepant results was done using BBL oxacillin screen agar (BD Diagnostics, Sparks, MD), oxacillin and cefoxitin Etests (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar, an immunoassay for penicillin binding protein 2 (Denka Seiken Co., Tokyo, Japan), and mecA PCR. The sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm StaphSR assay for MSSA detection were 98.9, 96.7, 93.6, and 99.5%, respectively. For the detection of MRSA, the BD GeneOhm StaphSR assay was 100% sensitive and 98.4% specific; positive and negative predictive values for MRSA detection were 92.6 and 100%, respectively. Inhibition was seen with only one sample, and the issue was resolved upon retesting. The BD GeneOhm StaphSR assay appears to be a valuable diagnostic tool for quickly differentiating bacteremia caused by MSSA and MRSA from that caused by other gram-positive cocci.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Clinical Validation of the Molecular BD GeneOhm StaphSR Assay for Direct Detection of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus in Positive Blood Cultures

et al. 2007

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“…The predominant target was nuc gene used in either conventional PCR (Ercolini et al 2004;Pinto et al 2005;Ikeda et al 2005;Cremonesi et al 2005) or quantitative real-time PCR (Hein et al 2005;Alarcon et al 2006). Other popular targets were species-specific regions of the DNA coding for 16S or 23S rRNA or coa gene (Cremonesi et al 2005;Baron et al 2004;Sabet et al 2006) and putative transcriptional regulator genes (Liu et al 2005;Goto et al 2007). These S.-aureus-specific genes were successfully amplified along with se (ent) genes coding for enterotoxins in multiplex conventional PCR (Ercolini et al 2004;Pinto et al 2005;Ikeda et al 2005;Tamarapu et al 2001;Cremonesi et al 2007) or at simultaneous species identification and detection of methicillin resistance using multiplex real-time PCR assay (Baron et al 2004).…”

Section: Introductionmentioning

confidence: 99%

Rapid and Sensitive Detection of Staphylococcus aureus in Food Using Selective Enrichment and Real-Time PCR Targeting a New Gene Marker

et al. 2008

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Staphylococcus aureus is a bacterial pathogen considered a principal etiological agent of food poisoning. The aim of this study was to develop and evaluate a rapid and sensitive method for the detection of S. aureus in food by using selective enrichment and a new species-specific real-time polymerase chain reaction (PCR). Specific primers and a TaqMan probe targeted to specific S. aureus gene encoding for acriflavine resistance protein were designed. The real-time PCR was highly specific for S. aureus with 100% inclusivity and 100% exclusivity determined using 83 S. aureus strains and 64 non-S.-aureus strains. PCR detection limit of 6.8×10 1 and 3.4×10 1 CFU ml −1 were obtained with 100% and 70% detection probability, respectively. The single selective enrichment based on the study of different enrichment conditions was selected and a lysis by boiling was used to obtain bacterial DNA. Out of 112 food samples analyzed, 61 were positive by the PCR-based method and 53 by the standard method. Out of ten food matrices artificially contaminated at a level of 10°CFU g −1 , ten and six were positive by the respective methods. Moreover, 10°CFU 10 g −1 was detected in all ten artificially contaminated samples after a large-scale enrichment using PCR-based detection, in contrast to seven false negative by standard detection. The developed method facilitated the detection of S. aureus on the next day after the sample reception. This method can be used for S. aureus detection as a faster, highly specific, and more sensitive alternative to microbiological method with the potential for providing of improved food-processing hygiene control.

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“…However, this type of PCR assay still quite laborious and time-consuming since it requires the preparation and PCR product characterization using gel electrophoresis (Costa et al 2005;Kearns et al 1999). Recently, a number of real-time PCR studies based on fluorescent probes have been carried out to detect MRSA from various clinical sources using TaqMan probes (Sabet et al 2006(Sabet et al , 2007Costa et al 2005). However, these assays require the availability of primers and probes that must be selected according to very rigid conditions, which cannot always be easily applied (Nam et al 2005).…”

Section: Introductionmentioning

confidence: 99%

Detection of Malaysian methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates using simplex and duplex real-time PCR

Suhaili

Johari

Mohtar

et al. 2008

World J Microbiol Biotechnol

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The aim of this study was to develop a methicillin-resistant Staphylococcus aureus (MRSA) detection method based on the melting temperature analysis profiling of S. aureus clinical isolates from three different hospitals in Malaysia. Simplex and duplex real-time PCR assay was used for the simultaneous detection of nuc (species-specific) and mecA (methicillin-resistance) genes in a single SYBR Green I real-time PCR tube assay. Evaluations were based on the melting temperature (T m ) analysis of the amplicons using 23 S. aureus clinical isolates including three ATCC S. aureus standard strains. Real-time PCR amplification products with melting peaks at 78.39 ± 0.4°C and 74.41 ± 0.6°C were detected for nuc and mecA genes, respectively. Each real-time PCR assay was completed within two hours. This rapid genotypic method is useful for the detection of resistant determinant (mecA) and identification of S. aureus (nuc) clinical isolates, thus benefiting patient therapy in hospitals.

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Simultaneous species identification and detection of methicillin resistance in staphylococci using triplex real-time PCR assay

Cited by 24 publications

References 21 publications

Clinical Validation of the Molecular BD GeneOhm StaphSR Assay for Direct Detection of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus in Positive Blood Cultures

Clinical Validation of the Molecular BD GeneOhm StaphSR Assay for Direct Detection of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus in Positive Blood Cultures

Rapid and Sensitive Detection of Staphylococcus aureus in Food Using Selective Enrichment and Real-Time PCR Targeting a New Gene Marker

Detection of Malaysian methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates using simplex and duplex real-time PCR

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