Publication Date

8-2010

Type of Culminating Activity

Thesis - Boise State University Access Only

Degree Title

Master of Science in Biology

Department

Biology

Major Advisor

Gregory Hampikian, Ph.D.

Abstract

New methods of bioterror detection are being developed from novel materials that simultaneously detect several biological threat gents. The recently developed material, Low Temperature Co-fired Ceramic (LTCC), is in many ways ideal for biosensors involved in bioterror detection. It is resistant to corrosion, allows co-manufacture with electric components, has a similar expansion coefficient to co-fired circuitry materials, can be autoclaved or baked, and is inexpensive to manufacture. However, the material has been known to inhibit some enzymatic reactions (through an undefined mechanism), including the most common DNA amplification enzyme Taq polymerase. Our laboratory is part of a team that is developing the first generation of biosensors made from LTCC. This study characterizes the effects of several possible causes of Taq polymerase inhibition, and demonstrates that the inhibition can be overcome by the addition of negatively charged proteins, which competively bind LTCC. Also described here is the first multiplex PCR assay that simultaneously detects very low levels of target sequences from three select bioterrorism agents in the presence of LTCC. The targets detected in this multiplex assay are from the Category A select agents B. anthracis, F. tularensis, and Y. pestis.

This new assay fulfills the two key requirements of bioterror detection: it is both sensitive and selective, reducing the risk of false negative and false positive results respectively. Three design features optimize correct analysis in this multiplex reaction. First, two gene targets are amplified from each of the three organisms, enhancing the sensitivity of the assay, and decreasing the likelihood of obtaining a false positive or false negative result. Second, the majority of the PCR targets are located on plasmids, which generally occur in higher copy numbers than chromosomal DNA, further increasing the sensitivity of the assay. Third, most of the gene targets are virulence factors, which can be used to discriminate bioterror agents from non-virulent close relatives. Simultaneous amplification from five of the six PCR targets was confirmed in the presence of extraneous environmental DNA, at a sensitivity of ten or fewer target molecules per reaction. One target could not be assayed due to bio-safety level restrictions on the Pasteur strain of B. anthracis.

A proposed model for LTCC inhibition of PCR is presented in the discussion. It is hypothesized that LTCC binds negatively charged proteins like DNA polymerase (the enzyme responsible for PCR amplification) impairing or preventing catalysis. In support of this model, LTCC is shown to bind the negatively charged protein ovalbumin. Furthermore, another negatively charged protein, bovine serum albumin (BSA), is shown to competitively bind to LTCC freeing ovalbumin; BSA addition also alleviates PCR inhibition by LTCC. In further support of the proposed model, the addition of negative chloride ions is also shown to overcome PCR inhibition, possibly though direct interaction with the putatively charged LTCC surface.

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