Development and validation of a molecular beacon probe-based real-time polymerase chain reaction assay for rapid detection of methicillin resistance in Staphylococcus aureus.
BACKGROUND
A rapid, real-time, duplex, fluorescent molecular beacon probe-based polymerase chain reaction (PCR) assay was recently developed for the detection of methicillin-resistant Staphylococcus aureus.


OBJECTIVE
To describe the development and validation of this unique assay.


METHODS
Prospective laboratory analysis.


METHODS
Urban health region/centralized diagnostic microbiology laboratory. BACTERIAL STRAINS: One hundred eighty-one previously characterized clinical and American Type Culture Collection isolates, including 50 strains each of methicillin-resistant and methicillin-sensitive S aureus, plus 50 strains of coagulase-negative staphylococci and 31 nonstaphylococcal isolates to ensure assay specificity.


METHODS
Assays were performed on purified genomic DNA extracted from growing bacterial colonies. Two sets of oligonucleotide primers were used to specifically amplify the mecA and nuc genes, followed by detection of amplicons using fluorophore-labeled molecular beacon probes. Assays were performed on the Mx4000 Multiplex Quantitative PCR System (Stratagene Inc, La Jolla, Calif).


METHODS
(1) Assay sensitivity and specificity, and (2) analytical sensitivity.


RESULTS
The assay demonstrated 100% sensitivity and 100% specificity, and accurately characterized isolates as methicillin-resistant S aureus, methicillin-sensitive S aureus, or methicillin-resistant coagulase-negative staphylococci, with test results available in 2.5 hours. The analytical sensitivity of the assay was determined to be between 6 and 60 genomic equivalents.


CONCLUSIONS
This assay is rapid, accurate, easy to perform, and is compatible with other real-time PCR instruments, making it a suitable alternative to conventional PCR methodologies.
Elsayed
Sameer
S
Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, Alberta, Canada. sameer.elsayed@cls.ab.ca
Chow
Barbara L
BL
Hamilton
Nina L
NL
Gregson
Daniel B
DB
Pitout
Johann D D
JD
Church
Deirdre L
DL
D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000235:genetics; D003030:Coagulase / Q000172:deficiency / Q000502:physiology; D004720:Endonucleases / Q000235:genetics; D004755:Enterobacteriaceae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D016983:Enterococcus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D006602:Hexosyltransferases; D008712:Methicillin / Q000378:metabolism* / Q000627:therapeutic use; D016106:Methicillin Resistance / Q000235:genetics*; D008836:Micrococcal Nuclease; D015335:Molecular Probes / Q000235:genetics*; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D011446:Prospective Studies; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000175:diagnosis / Q000188:drug therapy / Q000378:metabolism; D013211:Staphylococcus aureus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification*; D013291:Streptococcus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D013997:Time Factors; D019037:Urban Health Services
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003030:Coagulase; D005456:Fluorescent Dyes; D015335:Molecular Probes; D046915:Penicillin-Binding Proteins; C074002:nuc protein, staphylococcus; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D004720:Endonucleases; D008836:Micrococcal Nuclease; D002267:Muramoylpentapeptide Carboxypeptidase; D008712:Methicillin