CCID Spectrum
Volume 1, Number 4 September 2006

Editorís note: This is a technically detailed version of Dr. Brian Mahyís article about two research projects currently being funded through CCID.

Office of Extramural Research funds research on new technologies for infectious disease pathogen detection

By Brian W.J. Mahy, MA, PhD, ScD, DSc (Hon)

The Office of Extramural Research, NCID, soon to become part of the Extramural Research Program Office (ERPO) in the CCID Strategic Science and Program Unit (SSPU), is funding two groups engaged in state-of-the-art research to develop new approaches for the detection of pathogens causing infectious diseases. The funding for these research grants came from Coordinating Office for Terrorism Preparedness and Emergency Response (COTPER). The principal investigators of both groups recently gave seminars on their work in the Tom Harkin Global Communications Center.

TIGER technology
Dr. David Ecker and his team at the Ibis division of Isis Pharmaceuticals, Carlsbad, California, are using electrospray ionization mass spectrometry to deduce the base composition of PCR amplification products from highly conserved genomic regions to identify and determine the relative quantity of pathogenic bacteria present in patient respiratory samples. For example, Francisella tularensis, Yersinia pestis, Escherichia coli 0157, and Pseudomonas aeruginosa could all be detected and distinguished. Mass-based genotyping can also be used to detect viruses. It can distinguish SARS coronavirus from other human coronaviruses such as 229E and OC43; under development are refinements which could distinguish subtypes and strains of influenza and different orthopoxviruses.

Dr. Ecker has termed this biosensor method TIGER (Triangulation Identification for the Genetic Evaluation of Risk). A prerequisite of the method is knowledge of the base composition of the microbial DNA (or the RNA of an RNA virus) so that primers can be designed for multiplex PCR amplification from the sample. The PCR product (typically a mixture of amplicons of about 100 base-pairs) is then desalted and electrosprayed into a mass spectrometer. The spectral signals from the mass spectrometer can then be used to measure the precise weight of each of the nucleic acids present, and by comparison to a database of masses associated with known genetic variants to rapidly identify the species present in the sample.

Several CCID scientists are already collaborating with Ecker and his team, including Bernard Beall, Brandi Lombard, Kevin Tong, and Bob Wohlheuter. The week of September 25, the equipment for the TIGER biosensor technology will be established within CCID under the care of the Biotechnology Core Facility. The equipment, called the Ibis T5000, should be fully functional by mid-October.

Nanoparticle photonic sensors
Dr. Ashutosh Chilkoti and his team at Duke University are using nanoparticles, 10-100 nm diameter, prepared from noble metals, particularly gold, which display unique size, shape, and composition-dependent optical properties. The colors of these particles are due to surface plasmon resonance caused by the interaction of incident light with “free” surface electrons resulting in resonant excitation of an oscillating dipole. In fact, in ancient times the Romans used gold colloids to color glass. When a target analyte interacts with the nanoparticle surface, a spectral shift results as well as an increase in the intensity of the absorbance that can be measured.

As a further refinement, Dr. Chilkoti’s group developed a “chip” made by adding an optically transparent monolayer from self-assembled gold colloids on a glass surface. They then showed that these glass “chips” displayed colloidal surface plasmon resonance, and this could be detected in a regular spectrophotometer. These chips can be used by attaching biotin or streptavidin to the surface, and Chilkoti is developing a method for the detection of anthrax protective antigen using this method.

Recommended reading
Ecker DJ et al (2005) Rapid identification and strain-typing of respiratory pathogens for epidemic surveillance. Proc Natl Acad Sci USA 102, 8012-8017.

Nath N and Chilkoti A (2002) A colorimetric gold nanoparticle sensor to interrogate biomolecular interactions in real time on a surface. Anal Chem 74, 504-509.

About the author: Dr. Brian Mahy is acting director of the Office of Extramural Research, Strategic Science and Program Unit (SSPU, proposed).