Superparamagnetic nanoparticles based immunomagnetic separation-Multiplex polymerase chain reaction assay for detection of Salmonella

We report a method that combines Immunomagnetic separation and polymerase chain reaction (IMS-PCR) to detect Salmonella sp. The IMS-PCR assay exploits selective extraction of bacteria using antibody coated superparamagnetic particles and multiplex PCR amplification that enable detection of Salmonella sp. The anti- Salmonella polysera used was raised in female BalbC mice using heat inactivated Salmonella whole cell. The common organosilane aminopropyltriethoxy silane (APTES) under reflux conditions amine functionalizes γFe ₂O ₃ nanoparticles enabling covalent attachment of anti-Salmonella polysera. We examine the efficiency to capture and detect target bacteria in suspension using immunomagnetic particles in two different attachment modes namely, random assembly and oriented assembly of antibodies. The functionalized γFe ₂O ₃ nanoparticles conjugated with Salmonella antibody specifically capture Salmonella sp. from bacterial suspension. Retrieved genomic DNA of captured bacteria were amplified using set of four primers specifically targeting the invA, fliC, viaB, and prt genes of Salmonella Typhi and Salmonella Paratyphi A by multiplex PCR. We find that site-directed attachment and random attachment of antibody was sensitive to 10 ⁷ and 10 ⁶ cells/ml respectively. Thus we demonstrate IMS-PCR with improved surface chemistry and covalent attachment of antibody was sensitive to 10 ⁶ and 10 ⁷ cells/ml respectively. Thus we demonstrate IMS-PCR with improved surface chemistry and covalent attachment of antibody to be rapid, specific and cost effective detection method for Salmonella species in clinical isolates.