Development Of A Thermostabilized Lviijl Tip Lex Pcr Assay For The Rapid Detection Of Lviethicillin-Resistant Sl'aphylococcus Aureus

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dc.contributor.authorAl-Talib, Hassanain I
dc.date.accessioned2017-02-16T07:40:17Z
dc.date.available2017-02-16T07:40:17Z
dc.date.issued2010-03
dc.description.abstractThe emergence of methicillin-resistant Staphylococcus aureus (MRSA) is responsible for nosocomial and community-acquired infections. The conventional culture test takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a multiplex PCR assay for the rapid detection of MRSA. The assay simultaneously detected five genes, namely 16S rRNA of the Staphylococcus genus,femA of S. aureus, mecA that encodes methicillin resistance, lukS that encodes production of Panton- Valentine leukocidin (PVL), a necrotizing cytotoxin and one internal control. Unique and specific primer pairs were designed to amplify the 5 genes with the PCR products ranging from 151 to 759 bp. The specificity of the primers was confirmed by DNA sequencing of the multiplex PCR products and BLAST analysis. The sensitivity and specificity of multiplex PCR assay was evaluated against the conventional culture method. The multiplex PCR was thermostabilized and an accelerated stability test was evaluated at room temperature, 37°C and 10°C. The analytical sensitivity of the assay was found to be I 0 ng at the DNA level while the analytical specificity was evaluated with 34 reference staphylococci and non-staphylococcal strains and was found to be 100%. The thermostabilized multiplex PCR mix stored at 1 0°C was stable up to two and a half years by the accelerated stability test. The diagnostic accuracy was determined using a total of 231 nasal swabs clinical isolates. Among these 231 swabs, only 207 showed positive growth for staphylococci. Of these 207, 59 \vere found to be S. aureus while 148 were coagulase-negative staphylococci. Out of the 59 S. aureus isolates, 14 were found to be resistant to methicillin and expressed mecA gene. The presence of an internal control in the multiplex PCR assay helped to rule out false negative cases. The multiplex PCR assay is robust and can give information about the 5 genes that are essential for the identification of the Staphylococcus species and their methicillin resistance pattern. The PCR assay developed in this study can be used as an effective tool for screening and diagnosis ofMRSA nasal carrier in hospitals and community.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/3742
dc.language.isoenen_US
dc.publisherUniversiti Sains Malaysiaen_US
dc.subjectMultiplex PCR assay for theen_US
dc.subjectrapid detection of MRSA.en_US
dc.titleDevelopment Of A Thermostabilized Lviijl Tip Lex Pcr Assay For The Rapid Detection Of Lviethicillin-Resistant Sl'aphylococcus Aureusen_US
dc.typeThesisen_US
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