Development Of A Thermostabilized Lviijl Tip Lex Pcr Assay For The Rapid Detection Of Lviethicillin-Resistant Sl'aphylococcus Aureus
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Date
2010-03
Authors
Al-Talib, Hassanain I
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
The 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.
Description
Keywords
Multiplex PCR assay for the , rapid detection of MRSA.