A multiplex Taqman PCR assay for MRSA detection from whole blood.
Methicillin-resistant Staphylococcus aureus (MRSA) causes a wide range of hospital and community-acquired infections worldwide. MRSA is associated with worse clinical outcomes that can lead to multiple organ failure, septic shock, and death, making timely diagnosis of MRSA infections very crucial. I...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2023-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0294782&type=printable |
| _version_ | 1827587747380985856 |
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| author | Suhanya Duraiswamy Sushama Agarwalla Khoi Sheng Lok Yee Yung Tse Ruige Wu Zhiping Wang |
| author_facet | Suhanya Duraiswamy Sushama Agarwalla Khoi Sheng Lok Yee Yung Tse Ruige Wu Zhiping Wang |
| author_sort | Suhanya Duraiswamy |
| collection | DOAJ |
| description | Methicillin-resistant Staphylococcus aureus (MRSA) causes a wide range of hospital and community-acquired infections worldwide. MRSA is associated with worse clinical outcomes that can lead to multiple organ failure, septic shock, and death, making timely diagnosis of MRSA infections very crucial. In the present work, we develop a method that enables the positive enrichment of bacteria from spiked whole blood using protein coated magnetic beads, followed by their lysis, and detection by a real-time multiplex PCR directly. The assay targeted bacterial 16S rRNA, S. aureus (spa) and methicillin resistance (mecA). In addition, an internal control (lambda phage) was added to determine the assay's true negative. To validate this assay, staphylococcal and non-staphylococcal bacterial strains were used. The three-markers used in this study were detected as expected by monomicrobial and poly-microbial models of the S. aureus and coagulase-negative staphylococci (CoNS). The thermal cycling completed within 30 mins, delivering 100% specificity. The detection LoD of the pre-processing step was ∼ 1 CFU/mL from 2-5mL of whole blood and that of PCR was ∼ 1pg of NA. However, the combined protocol led to a lower detection limit of 100-1000 MRSA CFUs/mL. The main issue with the method developed is in the pre-processing of blood which will be the subject of our future study. |
| first_indexed | 2024-03-09T00:22:28Z |
| format | Article |
| id | doaj.art-86a3a216778c42558864f6ed1b488c4e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-03-09T00:22:28Z |
| publishDate | 2023-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-86a3a216778c42558864f6ed1b488c4e2023-12-12T05:33:25ZengPublic Library of Science (PLoS)PLoS ONE1932-62032023-01-011811e029478210.1371/journal.pone.0294782A multiplex Taqman PCR assay for MRSA detection from whole blood.Suhanya DuraiswamySushama AgarwallaKhoi Sheng LokYee Yung TseRuige WuZhiping WangMethicillin-resistant Staphylococcus aureus (MRSA) causes a wide range of hospital and community-acquired infections worldwide. MRSA is associated with worse clinical outcomes that can lead to multiple organ failure, septic shock, and death, making timely diagnosis of MRSA infections very crucial. In the present work, we develop a method that enables the positive enrichment of bacteria from spiked whole blood using protein coated magnetic beads, followed by their lysis, and detection by a real-time multiplex PCR directly. The assay targeted bacterial 16S rRNA, S. aureus (spa) and methicillin resistance (mecA). In addition, an internal control (lambda phage) was added to determine the assay's true negative. To validate this assay, staphylococcal and non-staphylococcal bacterial strains were used. The three-markers used in this study were detected as expected by monomicrobial and poly-microbial models of the S. aureus and coagulase-negative staphylococci (CoNS). The thermal cycling completed within 30 mins, delivering 100% specificity. The detection LoD of the pre-processing step was ∼ 1 CFU/mL from 2-5mL of whole blood and that of PCR was ∼ 1pg of NA. However, the combined protocol led to a lower detection limit of 100-1000 MRSA CFUs/mL. The main issue with the method developed is in the pre-processing of blood which will be the subject of our future study.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0294782&type=printable |
| spellingShingle | Suhanya Duraiswamy Sushama Agarwalla Khoi Sheng Lok Yee Yung Tse Ruige Wu Zhiping Wang A multiplex Taqman PCR assay for MRSA detection from whole blood. PLoS ONE |
| title | A multiplex Taqman PCR assay for MRSA detection from whole blood. |
| title_full | A multiplex Taqman PCR assay for MRSA detection from whole blood. |
| title_fullStr | A multiplex Taqman PCR assay for MRSA detection from whole blood. |
| title_full_unstemmed | A multiplex Taqman PCR assay for MRSA detection from whole blood. |
| title_short | A multiplex Taqman PCR assay for MRSA detection from whole blood. |
| title_sort | multiplex taqman pcr assay for mrsa detection from whole blood |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0294782&type=printable |
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