Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic Staphylococcus aureus.
OBJECTIVE
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.


CONCLUSIONS
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.
Microbiology Division, Defence Food Research Laboratory, Siddarthanagar, Mysore, Karnataka, India.
Sowmya
S
Nagaraj
Microbiology Division, Defence Food Research Laboratory, Siddarthanagar, Mysore, Karnataka, India. Electronic address: shylajaramlal@gmail.com.
Shylaja
S
Ramlal
Microbiology Division, Defence Food Research Laboratory, Siddarthanagar, Mysore, Karnataka, India.
Joseph
J
Kingston
Microbiology Division, Defence Food Research Laboratory, Siddarthanagar, Mysore, Karnataka, India.
Harsh Vardhan
HV
Batra
D004768:Enterotoxins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics*; D005504:Food Analysis / Q000191:economics / Q000379:methods; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics* / Q000379:methods*; D013203:Staphylococcal Infections / Q000175:diagnosis* / Q000382:microbiology; D013211:Staphylococcus aureus / Q000235:genetics* / Q000302:isolation & purification
D004768:Enterotoxins
Bromophenol blue; Cold-chain-free; Lyoprotectant; Multiplex polymerase chain reaction; Stabilization; Staphylococcus aureus