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Updated: Mar 20, 2026
Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus
Published on: September 5, 2013
Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic
Sowmya Nagaraj1, Shylaja Ramlal1, Joseph Kingston1
1Microbiology Division, Defence Food Research Laboratory, Siddarthanagar, Mysore, Karnataka, India.
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.
Insights
A novel lyophilized master mix simplifies pathogen detection using multiplex polymerase chain reaction (PCR). This stable, ready-to-use formulation enhances accuracy and reduces costs for detecting toxigenic Staphylococcus aureus strains.
Area of Science:
- Molecular biology
- Biotechnology
Background:
- Polymerase chain reaction (PCR) is a key DNA-based method for pathogen detection.
- Traditional PCR requires multiple steps and reagents, increasing error risk and cold-chain dependency.
- Reagent stability and handling challenges limit PCR's field application.
Purpose of the Study:
- To develop a stable, ready-to-use lyophilized master mix for multiplex PCR.
- To simplify the detection of toxigenic Staphylococcus aureus strains.
- To overcome the limitations of traditional PCR reagent handling and storage.
Main Methods:
- Lyophilization of a pre-optimized multiplex PCR master mix with a lyoprotectant.
- Inclusion of bromophenol blue to prevent contamination during gel loading.
- Stability testing at various temperatures and assessment of PCR performance after temperature fluctuations.
Main Results:
- The lyophilized master mix demonstrated a shelf-life of 1.5 months at ambient temperature and 6 months at 4°C.
- Stability was maintained despite temperature fluctuations during cold-chain-free transportation.
- PCR sensitivity, specificity, and performance remained unaltered.
Conclusions:
- The developed formulation is sensitive, cost-effective, and stable at ambient temperatures.
- This ready-to-use master mix is suitable for food analysis, diagnostic labs, and on-field applications.
- It offers a simplified approach for detecting toxigenic S. aureus strains.
