Microchimica Acta

, Volume 180, Issue 13–14, pp 1241–1248 | Cite as

Immunomagnetic nanoparticle based quantitative PCR for rapid detection of Salmonella

  • Padmavathy Bakthavathsalam
  • Vinoth Kumar Rajendran
  • Uttara Saran
  • Suvro Chatterjee
  • Baquir Mohammed Jaffar AliEmail author
Original Paper


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.

The method uses silica coated magnetic nanoparticles immobilized with anti-Salmonella antibody for immunomagnetic separation of Salmonella from beverages followed by detection by multiplex PCR (mPCR) and real time PCR (qPCR). This methodology contributes to rapid screening and accurate detection of Salmonella contaminations in beverages.


Immunomagnetic 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.

Supplementary material

604_2013_1052_MOESM1_ESM.doc (1.4 mb)
ESM 1 (DOC 1414 kb)


  1. 1.
    Valdés MG, González ACV, Calzón JAG, Díaz-García ME (2009) Analytical nanotechnology for food analysis. Microchim Acta 166: 1–19CrossRefGoogle Scholar
  2. 2.
    María Isabel Pividori MI, Salvador Alegret S (2010) Micro and nanoparticles in biosensing systems for food safety and environmental monitoring. An example of converging technologies. Microchim Acta 170:227–242CrossRefGoogle Scholar
  3. 3.
    Lu AH, Salabas EL, Ferdi S (2007) Magnetic nanoparticles: synthesis, protection, functionalization, and application. Angew Chem Int Ed 46:1222–1244CrossRefGoogle Scholar
  4. 4.
    Ashtari P, He X, Wang K, Gong P (2005) An efficient method for recovery of target ssDNA based on amino-modified silica-coated magnetic nanoparticles. Talanta 67:548–554CrossRefGoogle Scholar
  5. 5.
    Fu A, Hu W, Xu L, Wilson RJ, Yu H, Osterfeld SJ, Gambhir SS, Wang SX (2009) Protein-functionalized synthetic antiferromagnetic nanoparticles for biomolecule detection and magnetic manipulation. Angew Chem Int Ed Engl 48:1620–1624CrossRefGoogle Scholar
  6. 6.
    Chou TC, Hsu W, Wang CH, Chen YJ, Fang JM (2011) Rapid and specific influenza virus detection by functionalized magnetic nanoparticles and mass spectrometry. J Nanobiotechnology 9:52CrossRefGoogle Scholar
  7. 7.
    Huang C, Neoh KG, Wang L, Kang ET, Shuter B (2010) Magnetic nanoparticles for magnetic resonance imaging: modulation of macrophage uptake by controlled PEGylation of the surface coating. J Mater Chem 20:8512–8520CrossRefGoogle Scholar
  8. 8.
    Fuchigami T, Kitamoto Y, Namiki Y (2012) Size-tunable drug-delivery capsules composed of a magnetic nanoshell. Biomatter 2:313–320CrossRefGoogle Scholar
  9. 9.
    Li Z, He L, He N, Deng Y, Shi Z, Wang H, Li S, Liu H, Wang Z, Wang D (2011) Polymerase chain reaction coupling with magnetic nanoparticles-based biotin-avidin system for amplification of chemiluminescent detection signals of nucleic acid. J Nanosci Nanotechnol 11:1074–1078CrossRefGoogle Scholar
  10. 10.
    Tang Y, Zou J, Ma C, Ali Z, Li Z, Li X, Ma N, Mou X, Deng Y, Zhang L, Li K, Lu G, Yang H, He N (2013) Highly sensitive and rapid detection of Pseudomonas aeruginosa based on magnetic enrichment and magnetic separation. Theranostics 3:85–92CrossRefGoogle Scholar
  11. 11.
    Pappert G, Rieger M, Niessner R, Seidel M (2010) Immunomagnetic nanoparticle-based sandwich chemiluminescence-ELISA for the enrichment and quantification of E. coli. Microchim Acta 168:1–8CrossRefGoogle Scholar
  12. 12.
    Iqbal Z, Lai EPC, Avis TJ (2012) Development of polymer-modified magnetic nanoparticles and quantum dots for Escherichia coli binding test. Microchim Acta 176:193–200CrossRefGoogle Scholar
  13. 13.
    Roda A, Mirasoli M, Roda B, Bonvicini F, Colliva C, Reschiglian P (2012) Recent developments in rapid multiplexed bioanalytical methods for foodborne pathogenic bacteria detection. Microchim Acta 178:7–28CrossRefGoogle Scholar
  14. 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
  15. 15.
    Liu G, Yu X, Xue F, Chen W, Ye Y, Yang X, Lian Y, Yan Y, Zong K (2012) Screening and preliminary application of a DNA aptamer for rapid detection of Salmonella O8. Microchim Acta 178:237–244CrossRefGoogle Scholar
  16. 16.
    Wang H, Zhang C, Xing D (2011) Simultaneous detection of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes using oscillatory-flow multiplex PCR. Microchim Acta 173:503–512CrossRefGoogle Scholar
  17. 17.
    Salomonsson AC, Aspa’n A, Johansson S, Heino A, Ha¨ggblom P (2005) Salmonella detection by polymerase chain reaction after pre-enrichment of feed samples. J Rapid Meth Automat Microbiol 13:96–110CrossRefGoogle Scholar
  18. 18.
    Huang YF, Wang YF, Yan XP (2010) Amine-functionalized magnetic nanoparticles for rapid capture and removal of bacterial pathogens. Environ Sci Technol 44:7908–7913CrossRefGoogle Scholar
  19. 19.
    Lian W, Litherland SA, Badrane H, Tan W, Wu D, Baker HV, Gulig PA, Lim DV, Jin S (2004) Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles. J Anal Biochemistry 334:135–144CrossRefGoogle Scholar
  20. 20.
    Padmavathy B, Patel A, Vinoth Kumar R, Jaffar Ali BM (2012) Superparamagnetic nanoparticles based immunomagnetic separation - multiplex PCR assay for detection of Salmonella. Sci Adv Mat 4:1–7CrossRefGoogle Scholar
  21. 21.
    Marathe SA, Chowdhury R, Bhattacharya R, Nagarajan AG, Chakravorthy D (2012) Direct detection of Salmonella without pre-enrichment in milk, ice-cream and fruit juices by PCR against hilA gene. Food Control 23:559–563CrossRefGoogle Scholar
  22. 22.
    Rieger M, Schaumann GE, Mouvenchery YK, Niessner R, Seidel M, Baumann T (2012) Development of antibody-labelled superparamagnetic nanoparticles for the visualisation of benzo[a]pyrene in porous media with magnetic resonance imaging. Anal Bioanal Chem 403:2529–2540CrossRefGoogle Scholar
  23. 23.
    Feifel SC, Lisdat F (2011) Silica nanoparticles for the layer-by-layer assembly of fully electro-active cytochrome c multilayers. J Nanobiotechnology 9:59CrossRefGoogle Scholar
  24. 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
  25. 25.
    Spanova A, Rittich B, Karpiskova R, Cechova L, Skapova D (2000) PCR identification of Salmonella cells in food and stool samples after immunomagnetic separation. Bioseparation 9:379–384CrossRefGoogle Scholar
  26. 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. 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. 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. 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
  30. 30.
    Mercanoglu TB, Ben U, Aytac SA (2009) Rapid detection of Salmonella in milk by combined immunomagnetic separation-polymerase chain reaction assay. J Dairy Sci 92:2382–2388CrossRefGoogle Scholar
  31. 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. 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. 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
  34. 34.
    Hagren V, von Lode P, Syrjälä A, Korpimäki T, Tuomola M, Kauko O, Nurmi J (2008) An 8-hour system for Salmonella detection with immunomagnetic separation and homogeneous time-resolved fluorescence PCR. Int J Food Microbiol 125:158–161CrossRefGoogle Scholar
  35. 35.
    Li A, Zhang H, Zhang X, Wang Q, Tian J, Li Y, Li J (2010) Rapid separation and immunoassay for low levels of Salmonella in foods using magnetosome-antibody complex and real-time fluorescence quantitative PCR. J Sep Sci 33:3437–3443CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2013

Authors and Affiliations

  • Padmavathy Bakthavathsalam
    • 1
  • Vinoth Kumar Rajendran
    • 1
  • Uttara Saran
    • 1
  • Suvro Chatterjee
    • 1
  • Baquir Mohammed Jaffar Ali
    • 2
    Email author
  1. 1.AU-KBC Research CentreM.I.T Campus of Anna UniversityChennaiIndia
  2. 2.Centre for Green Energy TechnologyPondicherry UniversityPuducherryIndia

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