Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic Staphylococcus aureus
Background/Purpose: Among DNA-based techniques, polymerase chain reaction (PCR) is the most widely accepted molecular tool for the detection of pathogens. However, the technique involves several reagents and multiple pipetting steps that often lead to error-prone results. Additionally, the reagents...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2018-04-01
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| Series: | Journal of Microbiology, Immunology and Infection |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1684118216300330 |
| _version_ | 1818892587152441344 |
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| author | Sowmya Nagaraj Shylaja Ramlal Joseph Kingston Harsh Vardhan Batra |
| author_facet | Sowmya Nagaraj Shylaja Ramlal Joseph Kingston Harsh Vardhan Batra |
| author_sort | Sowmya Nagaraj |
| collection | DOAJ |
| description | Background/Purpose: Among DNA-based techniques, polymerase chain reaction (PCR) is the most widely accepted molecular tool for the detection of pathogens. However, the technique involves several reagents and multiple pipetting steps that often lead to error-prone results. Additionally, the reagents entail a cold-chain facility to maintain their stability during storage and transportation. The main aim of the present study was to simplify the utility of a pre-optimized multiplex PCR format that was developed to detect toxigenic strains of Staphylococcus aureus by providing stable, pre-mixed, and ready-to-use master mix in a lyophilized formulation. Methods: Master mix containing all reagents except the template was lyophilized in the presence of an excipient lyoprotectant to achieve long-term stability without altering the sensitivity, specificity and PCR performance. Bromophenol blue was also included in the master mix to reduce the risk of external contamination during gel loading. The stability of lyophilized master mix was analyzed at different temperatures. The PCR performance was also examined after exposure of master mix to notable temperature fluctuations during transportation. Results: The shelf-life of lyophilized master mix was estimated to be 1.5 months at ambient temperature and 6 months at 4°C. Stability was unaffected by temperature fluctuations during transportation even in cold-chain-free conditions, thus reducing the cost required for cold storage. Conclusion: The sensitive, cost-effective, ready-to-use, and ambient temperature stable formulation could be implemented as a detection tool in food analysis and diagnostic laboratories and hospitals and for on-field application outside the laboratories, as well as for detection of toxigenic strains of S. aureus. Keywords: Bromophenol blue, Cold-chain-free, Lyoprotectant, Multiplex polymerase chain reaction, Stabilization, Staphylococcus aureus |
| first_indexed | 2024-12-19T17:59:04Z |
| format | Article |
| id | doaj.art-f0e52dde9e124000bea83dd7ce5a4fa7 |
| institution | Directory Open Access Journal |
| issn | 1684-1182 |
| language | English |
| last_indexed | 2024-12-19T17:59:04Z |
| publishDate | 2018-04-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Microbiology, Immunology and Infection |
| spelling | doaj.art-f0e52dde9e124000bea83dd7ce5a4fa72022-12-21T20:11:44ZengElsevierJournal of Microbiology, Immunology and Infection1684-11822018-04-01512191198Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic Staphylococcus aureusSowmya Nagaraj0Shylaja Ramlal1Joseph Kingston2Harsh Vardhan Batra3Microbiology Division, Defence Food Research Laboratory, Siddarthanagar, Mysore, Karnataka, IndiaCorresponding author. Microbiology Division, Defence Food Research Laboratory, Siddarthanagar, Mysore, Karnataka, 570011, India.; Microbiology Division, Defence Food Research Laboratory, Siddarthanagar, Mysore, Karnataka, IndiaMicrobiology Division, Defence Food Research Laboratory, Siddarthanagar, Mysore, Karnataka, IndiaMicrobiology Division, Defence Food Research Laboratory, Siddarthanagar, Mysore, Karnataka, IndiaBackground/Purpose: Among DNA-based techniques, polymerase chain reaction (PCR) is the most widely accepted molecular tool for the detection of pathogens. However, the technique involves several reagents and multiple pipetting steps that often lead to error-prone results. Additionally, the reagents entail a cold-chain facility to maintain their stability during storage and transportation. The main aim of the present study was to simplify the utility of a pre-optimized multiplex PCR format that was developed to detect toxigenic strains of Staphylococcus aureus by providing stable, pre-mixed, and ready-to-use master mix in a lyophilized formulation. Methods: Master mix containing all reagents except the template was lyophilized in the presence of an excipient lyoprotectant to achieve long-term stability without altering the sensitivity, specificity and PCR performance. Bromophenol blue was also included in the master mix to reduce the risk of external contamination during gel loading. The stability of lyophilized master mix was analyzed at different temperatures. The PCR performance was also examined after exposure of master mix to notable temperature fluctuations during transportation. Results: The shelf-life of lyophilized master mix was estimated to be 1.5 months at ambient temperature and 6 months at 4°C. Stability was unaffected by temperature fluctuations during transportation even in cold-chain-free conditions, thus reducing the cost required for cold storage. Conclusion: The sensitive, cost-effective, ready-to-use, and ambient temperature stable formulation could be implemented as a detection tool in food analysis and diagnostic laboratories and hospitals and for on-field application outside the laboratories, as well as for detection of toxigenic strains of S. aureus. Keywords: Bromophenol blue, Cold-chain-free, Lyoprotectant, Multiplex polymerase chain reaction, Stabilization, Staphylococcus aureushttp://www.sciencedirect.com/science/article/pii/S1684118216300330 |
| spellingShingle | Sowmya Nagaraj Shylaja Ramlal Joseph Kingston Harsh Vardhan Batra Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic Staphylococcus aureus Journal of Microbiology, Immunology and Infection |
| title | Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic Staphylococcus aureus |
| title_full | Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic Staphylococcus aureus |
| title_fullStr | Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic Staphylococcus aureus |
| title_full_unstemmed | Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic Staphylococcus aureus |
| title_short | Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic Staphylococcus aureus |
| title_sort | thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic staphylococcus aureus |
| url | http://www.sciencedirect.com/science/article/pii/S1684118216300330 |
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