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A limit of detection of 200 CFU/mL of Salmonella typhi spiked in various sample matrices were achieved in 30 min. The sample matrices were raw/unprocessed milk, commercially available milk, juice from packed bottles, fresh juice from carts, potable water, turbid water and calf serum. The complete protocol comprised of three steps: (a) cell lysis (b) nucleic acid amplification and (c) an in situ optical detection. The cell lysis was carried out using a simple heating based protocol, while the loop-mediated isothermal amplification of DNA was carried out by an in-house designed and fabricated system. The developed system consists of an aluminum block fitted with two cartridge heaters along with a thermocouple. The system was coupled to a light source and spectrometer for a simultaneous in situ detection. Primers specific for STY2879 gene were used to amplify the nucleic acid sequence, isolated from S. typhi cells. The protocol involves 15 min of cell lysis and DNA isolation followed by 15 min for isothermal amplification and simultaneous detection. No cross-reactivity of the primers were observed at 10(6) CFU/mL of Escherichia coli, Vibrio cholerae, Salmonella typhimurium, Salmonella paratyphi A, Pseudomonas aeruginosa, Bacillus cereus, Lysteria monocytogenes, Clostridium botulinum, Staphylococcus aureus and Salmonella havana. In addition, the system was able to detect S. typhi of 200 CFU/mL in a concoction of 10(6) CFU/mL of E. coli, 10(6) CFU/mL of V. cholerae, and 10(6) CFU/mL of hepatocyte-derived cellular carcinoma HUH7 cells. The proposed rapid diagnostic system shows a promising future in the field of food and medical diagnostics. |
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