In silico PCR verification and simplex real-time PCR detection of methicillin-resistant Staphylococcus aureus (MRSA) from east coast Malaysian clinical isolates
The 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 dia...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Institute of Research and Development, Walailak University
2013
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| Online Access: | http://psasir.upm.edu.my/id/eprint/28043/1/28043.pdf |
| _version_ | 1796971250418974720 |
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| author | Suhaili, Zarizal Johari, Saiful Azmi Sajili, Mohammad Hailmi Yahya, Azifah Zakaria, Zainul Amiruddin Mohd Desa, Mohd Nasir Ali, Abdul Manaf |
| author_facet | Suhaili, Zarizal Johari, Saiful Azmi Sajili, Mohammad Hailmi Yahya, Azifah Zakaria, Zainul Amiruddin Mohd Desa, Mohd Nasir Ali, Abdul Manaf |
| author_sort | Suhaili, Zarizal |
| collection | UPM |
| description | The 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. |
| first_indexed | 2024-03-06T08:10:11Z |
| format | Article |
| id | upm.eprints-28043 |
| institution | Universiti Putra Malaysia |
| language | English |
| last_indexed | 2024-03-06T08:10:11Z |
| publishDate | 2013 |
| publisher | Institute of Research and Development, Walailak University |
| record_format | dspace |
| spelling | upm.eprints-280432016-04-25T07:11:27Z http://psasir.upm.edu.my/id/eprint/28043/ In silico PCR verification and simplex real-time PCR detection of methicillin-resistant Staphylococcus aureus (MRSA) from east coast Malaysian clinical isolates Suhaili, Zarizal Johari, Saiful Azmi Sajili, Mohammad Hailmi Yahya, Azifah Zakaria, Zainul Amiruddin Mohd Desa, Mohd Nasir Ali, Abdul Manaf The 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. Institute of Research and Development, Walailak University 2013 Article PeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/28043/1/28043.pdf Suhaili, Zarizal and Johari, Saiful Azmi and Sajili, Mohammad Hailmi and Yahya, Azifah and Zakaria, Zainul Amiruddin and Mohd Desa, Mohd Nasir and Ali, Abdul Manaf (2013) In silico PCR verification and simplex real-time PCR detection of methicillin-resistant Staphylococcus aureus (MRSA) from east coast Malaysian clinical isolates. Walailak Journal of Science and Technology, 10 (3). pp. 237-246. ISSN 1686-3933; ESSN: 2228-835X http://wjst.wu.ac.th/index.php/wjst/article/view/262 |
| spellingShingle | Suhaili, Zarizal Johari, Saiful Azmi Sajili, Mohammad Hailmi Yahya, Azifah Zakaria, Zainul Amiruddin Mohd Desa, Mohd Nasir Ali, Abdul Manaf In silico PCR verification and simplex real-time PCR detection of methicillin-resistant Staphylococcus aureus (MRSA) from east coast Malaysian clinical isolates |
| title | In silico PCR verification and simplex real-time PCR detection of methicillin-resistant Staphylococcus aureus (MRSA) from east coast Malaysian clinical isolates |
| title_full | In silico PCR verification and simplex real-time PCR detection of methicillin-resistant Staphylococcus aureus (MRSA) from east coast Malaysian clinical isolates |
| title_fullStr | In silico PCR verification and simplex real-time PCR detection of methicillin-resistant Staphylococcus aureus (MRSA) from east coast Malaysian clinical isolates |
| title_full_unstemmed | In silico PCR verification and simplex real-time PCR detection of methicillin-resistant Staphylococcus aureus (MRSA) from east coast Malaysian clinical isolates |
| title_short | In silico PCR verification and simplex real-time PCR detection of methicillin-resistant Staphylococcus aureus (MRSA) from east coast Malaysian clinical isolates |
| title_sort | in silico pcr verification and simplex real time pcr detection of methicillin resistant staphylococcus aureus mrsa from east coast malaysian clinical isolates |
| url | http://psasir.upm.edu.my/id/eprint/28043/1/28043.pdf |
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