In silico PCR Verification and Simplex Real-Time PCR Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) from East Coast Malaysian Clinical Isolates
Keywords:In-silico PCR, real-time PCR SYBR Green I, methicillin-resistant Staphylococcus aureus (MRSA), staphylocoagulase (coa), nuclease (nuc) and methicillin-resistant (mecA) genes
AbstractThe aims of this study were to validate the primers developed for molecular-based detection and identification of Malaysian clinical isolates of methicilin-resistance Staphylococcus aureus (MRSA) using in-silico Polymerase Chain Reaction (PCR) and real-time PCR SYBR with Green I. Rapid molecular diagnostic and risk assessment of the MRSA are possible by real-time PCR SYBR Green I. However, validation of such primers for real-life samples is expensive and time consuming. Hence, development and verification of real-time PCR primers by in-silico PCR can be the first step in the selection of the most appropriate primers. Three species-specific markers were chosen targeting coa (staphylocoagulase), nuc (thermonuclease) and mecA (methicillin-resistance) and were specifically verified against 35 selected S. aureus strains by using in-silico PCR. For the actual laboratory verification, all of the 3 genes were detected with a single specific melting curve peak (Tm at 76.16 ± 0.8 °C, 78.50 ± 0.4 °C and 74.41 ± 0.6 °C for a coa, nuc and mecA respectively) in 32 bacterial strains including ATCC reference strains. Thus, there is no disagreement between both in-silico PCR and real-time PCR verification and validation of the primers designed for the detection and identification of MRSA in this study. The potential of using a bioinformatics approach (in-silico PCR) before selecting primer pairs for a given study may enable researchers to accept or reject the potential primer pairs for downstream experimental (in vitro) PCR without wasting any chemicals as well as related cost.
K Hiramatsu. Vancomycin-resistance Staphylococcus aureus: a new model of antibiotic resistance. Lancet Infect. Dis. 2001; 1, 147-55.
FJ Schmitz, AC Fluit, M Luckefah, B Engler, B Hofmann, J Verhoef, HP Heinz, U Hadding and ME Jone. The effect of reserpine, an inhibitor of multidrug efflux pumps, on the in-vitro activities of ciprofloxacin, 27 sparfloxacin and moxifloxacin against clinical isolates of Staphylococcus aureus. J. Antimicrob. Chemother. 1998; 42, 807-10.
TY Tan. Use of molecular techniques for the detection of antibiotic resistance in bacteria. Expet. Rev. Mol. Diagn. 2003; 3, 93-103.
CJ Papasian and B Garrison. Evaluation of a rapid slide agglutination test for identification of Staphylococcus aureus. Diagn. Microbiol. Infect. Dis. 1999; 33, 201-3.
MB Miller, H Meyer, E Rogers and PH Gilligan. Comparison of conventional susceptibility testing, penicillin-binding protein 2a latex agglutination testing, and mecA real-time PCR for detection of oxacillin resistance in Staphylococcus aureus and coagulase-negative Staphylococcus. J. Clin. Microbiol. 2005; 43, 3450-2.
V Perreten, L Vorlet-Fawer, P Slickers, R Ehricht, P Kuhnert and J Frey. Microarray-based detection of 90 antibiotic resistance genes of grampositive bacteria. J. Clin. Microbiol. 2005; 43, 2291-302.
M Kubista, JM Andrade, M Bengtsson, A Forootan, J Jonák, K Lind, R Sindelka, R Sjöback, B Sjögreen, L Strömbom, A Ståhlberg and N Zoric. The real-time polymerase chain reaction. Mol. Aspects Med. 2006; 27, 95-125.
AM Costa, I Kay and S Palladino. Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction. Diagn. Microbiol. Infect. Dis. 2005; 51, 13-7.
NS Sabet, GM Subramaniam, P Navartnam and SD Sekaran. Detection of methicillin and aminoglycoside-resistant genes and simultaneous identification of S. aureus using triplex real-time PCR TaqMan assay. J. Microbiol. Methods 2007; 68, 157-62.
KA Abd-Elsalam. Bioinformatics tools and guidelines for PCR primer design. African J. Biotechnol. 2003; 2, 91-5.
HM Nam, V Srinivasan, BE Gillespie, SE Murinda and SP Oliver. Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples. Int. J. Food Microbiol. 2005; 102, 161-71.
Z Suhaili, SA Johari, M Mohtar, ART Abdullah, A Ahmad and AM Ali. Detection of Malaysian methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates using simplex and duplex real-time PCR. World J. Microbiol. Biotech. 2009; 25, 253-8.
AJ Saiful, M Mastura, S Zarizal, MI Mazurah, M Shuhaimi and AM Ali. Detection of methicillin-resistant Staphylococcus aureus using mecA/nuc genes and antibiotic susceptibility profile of Malaysian clinical isolates. World J. Microbiol. Biotechnol. 2006; 22, 1289-94.
J Bikandi, RS Millán, A Rementeria and J Garaizar. In silico analysis of complete bacterial genomes: PCR, AFLP-PCR, and endonuclease restriction. Bioinformatics 2004; 20, 798-9.
Singh VK and Kumar A. PCR Primer Design. Mol. Biol. Today 2001; 2, 27-32.
S Rozen and H Skaletsky. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 2002; 132, 365-86.
JM Rouillard, M Zuker and E Gulari. OligoArray 2.0: design of oligonucleotide probes for DNA microarrays using a thermodynamic approach. Nucleic Acids Res. 2003; 31, 3057-62.
RJ Blick, AT Revel and EJ Hansen. FindGDPs: identification of primers for labeling microbial transcriptomes for DNA microarray analysis. Bioinformatics 2003; 19, 1718-9.
MG Mitchell and M Ziman. An In silico investigation into the discovery of novel Cis-acting elements within the intronic regions of human PAX7. Nat. Sci. 2006; 4, 69-85.
M Lexa, J Horak and B Brobohaty. Virtual PCR. Bioinformatics 2001; 17, 192-3.
AC Fluit, MR Visser and FJ Schmitz. Molecular detection of antimicrobial resistance. Clin. Microbiol. Rev. 2001; 14, 836-71.
B Strommenger, C Kettlitz, G Werner and W Witte. Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistances in Staphylococcus aureus. J Clin. Microbio. 2003; 41, 4089-94.
FC Tenover, MV Lancaster, BC Hill and K Hiramatsu. Characterization of staphylococci with reduced susceptibilities to vancomycin and other glycopeptides. J. Clin. Microbiol. 1998; 36, 1020-7.
AA Rushdy, MS Salama and AS Othman. Detection of Methicillin/Oxacillin resistant Staphylococcus aureus isolated from some clinical Hospitals in Cairo using mecA/nuc genes and antibiotic susceptibility profile. Int. J. Argi. Biol. 2007; 9, 800-6.
B Shopsin. Use of coagulase gene (CoA) repeat region nucleotide sequences for typing of methicillin-resistant Staphylococcus aureus strains. J. Clin. Microbiol. 2000; 38, 3453-6.
A Japoni, AV Alborzi, M Rasouli and B Pourabbas. Modified DNA extraction for rapid PCR detection of methicillin-resistant staphylococci. Iranian Biomed. J. 2004; 8, 161-5.
How to Cite
Copyright (c) 2013 Walailak University
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.