Alok Pandey

Post-doctoral Fellow

2006-contd.

CV

alokp06@msu.edu

Evaluation of Quantum Dosts as Surrogates/Recovery Efficiency of P22 from Fomites

1- Evaluation of Quantum Dots as Surrogates for Microorganisms

    • Quantum dots (QDs; of CdSe/ZnS) with varying properties are available commercially for various applications, and may be useful as surrogates for pathogens in air dispersal experiments.
    • Currently experimental evidence for use of QDs as surrogates of microorganisms is nonexistent. QDs have been used to detect single nucleotide polymorphisms, pathogens, and toxins in a multiplex manner.
    • It is possible to tag QDs with short signature sequences of DNA (oligonucleotide)
    • Some of the selected characteristics are to be evaluated for the suitability of QDs as surrogate of microorganisms i.e. Detection methods, size, toxicity and cost.
    Figure 1. Comet assay and set up

As nanoparticles have been used in various applications and were also found genotoxic in mammalian test system (e.g. fullerenes, Dhawan et al, 2006, Environmental Science and Technology, 40(23): 7394-7401), it is prudent to test the genotoxic potential of QDs in the mammalian cell system. The Comet assay has proved to be a versatile, simple and the most useful technique for the detection of a wide range of DNA damage and repair processes in single cells and has become a test of choice in preliminary screening of chemical entities for their genotoxic potential. The assay is an important tool for measurement of DNA damage at a point where it can even undergo repair and hence is an indicator of risk at an early stage. We evaluated DNA damage due to CdSe quantum dots under in vitro conditions on human lymphocytes using single cell gel electrophoresis assay (Comet assay). The lymphocytes separated from human blood were exposed to different concentrations of quantum dots in serum free medium, for 3 hrs and 6 hrs and analyzed for DNA damage.

 

2- Evaluation of Detection Limit for qPCR and Cultivatable Method Using P22 and Bacillus thuringiensis from Large Surfaces

We are currently working on an experimental evaluation of the detection limit of qPCR and cultivatable method using P22 and Bacillus thuringiensis recovered from various fomites.  Fomites of interest include acrylic, laminar, wood and stainless steel with surface areas of 0.01 m2, 0.1 m2, and 1m2.  The method of recovery is horizontal and vertical strokes on the fomite using the Fellowes Premoistened Surface Cleaning Wipes.  Samples to be detected by qPCR are processed for DNA extraction using the QIAamp DNA Mini Kit (Qiagen) and real-time PCR on Light Cycler 1.5 instrument (Roche).  The samples to be detected by cultivatable method are processed using the plaque assay by double agar layer (DAL) method using Salmonella typhimurium as the bacterial host cells for P22. Plate count method is used for Bacillus thuringiensis as cultivablemethod.

Acknowledgements

    • This project is supported from the Environmental Protection Agency (RD83236201-0) as part of the Center for Advancing Microbial Risk Assessment (CAMRA, http://camra.msu.edu/index.html )
    • American Red Cross Blood Services, Lansing, Michigan.