Immunomagnetic nanoparticle based quantitative PCR for rapid detection of Salmonella
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We have developed a rapid and sensitive method for immunomagnetic separation (IMS) of Salmonella along with their real time detection via PCR. Silica-coated magnetic nanoparticles were functionalized with carboxy groups to which anti-Salmonella antibody raised against heat-inactivated whole cells of Salmonella were covalently attached. The immuno-captured target cells were detected in beverages like milk and lemon juice by multiplex PCR and real time PCR with a detection limit of 104 cfu.mL−1 and 103 cfu.mL−1, respectively. We demonstrate that IMS can be used for selective concentration of target bacteria from beverages for subsequent use in PCR detection. PCR also enables differentiation of Salmonella typhi and Salmonella paratyphi A using a set of four specific primers. In addition, IMS—PCR can be used as a screening tool in the food and beverage industry for the detection of Salmonella within 3–4 h which compares favorably to the time of several days that is needed in case of conventional detection based on culture and biochemical methods.
KeywordImmunomagnetic separation Salmonella PCR Multiplex detection Biofunctionalized nanoparticles
We acknowledge Central Instrumentation Facility, Pondicherry University for SEM imaging. We thank Dr. Vaidehi, SMF Hospital for providing the clinical isolates. BP thanks Council of Scientific and Industrial Research (CSIR) for the award of Senior Research Fellowship. This work was partially supported by KBC Research Foundation.
- 14.Benoit PW, Donahue DW (2003) Methods for rapid separation and concentration of bacteria in food that bypass time-consuming cultural enrichment. J Food Prot 66:1935–1948Google Scholar
- 24.Wang S, Wen S, Shen M, Guo R, Cao X, Wang J, Shi X (2011) Aminopropyltriethoxysilane-mediated surface functionalization of hydroxyapatite nanoparticles: synthesis, characterization, and in vitro toxicity assay. Int J Nanomedicine 6:3449–3459Google Scholar
- 26.Beuchat LR, Mann DA (2008) Survival and growth of acid-adapted and unadapted Salmonella in and on raw tomatoes as affected by variety, stage of ripeness, and storage temperature. J Food Prot 71:1572–1579Google Scholar
- 27.Bai Y, Song M, Cui Y, Shi C, Wang D, Paoli GC, Shi X (2013) A rapid method for the detection of foodborne pathogens by extraction of a trace amount of DNA from raw milk based on amino-modified silica-coated magnetic nanoparticles and polymerase chain reaction. Anal Chim Acta. doi: 10.1016/j.aca.2013.05.043 Google Scholar
- 28.Yang Y, Xu F, Xu H, Aguilar ZP, Niu R, Yuan Y, Sun J, You X, Lai W, Xiong Y, Wan C, Wei H (2013) Magnetic nano-beads based separation combined with propidium monoazide treatment and multiplex PCR assay for simultaneous detection of viable Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes in food products. Food Microbiol 34:418–424CrossRefGoogle Scholar
- 29.Wang L, Li Y, Mustaphai A (2007) Rapid and simultaneous quantitation of Escherichia coli 0157:H7, Salmonella, and Shigella in ground beef by multiplex real-time PCR and immunomagnetic separation. J Food Prot 70:1366–1372Google Scholar
- 31.Moreira AN, Conceição FR, Conceição Rde C, Ramos RJ, Carvalhal JB, Dellagostin OA, Aleixo JA (2008) Detection of Salmonella typhimurium in raw meats using in-house prepared monoclonal antibody coated magnetic beads and PCR assay of the fimA gene. J Immunoassay Immunochem 29:58–69Google Scholar
- 32.Notzon A, Helmuth R, Bauer J (2006) Evaluation of an immunomagnetic separation-real-time PCR assay for the rapid detection of Salmonella in meat. J Food Prot 69:2896–2901Google Scholar
- 33.Mercanoglu B, Griffiths MW (2005) Combination of immunomagnetic separation with real-time PCR for rapid detection of Salmonella in milk, ground beef, and alfalfa sprouts. J Food Prot 68:557–561Google Scholar