INL BIOTECHNOLOGY

Science

Results

Yellowstone National Park

has been the focus for INL’s

field tests, given the large

number of bison and elk

within its boundaries.

Application of this new

equipment and associated

techniques allows INL to

Detecting PCR Pathogens in Real Time

Brucellosis in such wildlife species as bison and elk is endemic and is a concern for the

cattle industry. INL researchers are developing a blood test to detect active

Brucella infection in bison, other wildlife, and cattle. The detection of pathogens has

traditionally been accomplished using a polymerase chain reaction (PCR) followed by

electro-phoretic gel separation of amplified target DNA. The approach typically requires

about 3 hours for assay results. Our development of real-time PCR allows the amplification

process to be monitored during each cycle of the PCR process, rather than following

completion. This decreases the time required for detection to approximately 30 minutes and

also enables quantification of starting target DNA. Probes and melt analysis of products can

be used to increase specificity and determine the purity of a PCR product. INL’s acquired

field-portable real-time PCR instrument, called RAPID, for Ruggedized Advanced Pathogen

Identification Device, is used to optimize assays to detect Brucella. INL is also developing

and testing reliable primer sets for strain identification. These tools will greatly aid in the

study to determine the true potential for interspecies transfer of Brucella in the environment.

Continued on next page

determine on-site the infection of bison and elk. The

information will aid in

decisions regarding the need

for destruction of animals.

Furthermore, real-time PCR

will allow the longstanding

question of transfer between

species to be addressed. In

addition, a multi-plex PCR/

multiple probes assay holds

the promise of allowing

simultaneous detection and

discrimination of several

organisms in a sample.

In general, real-time PCR

potentially applies to forensics and national securityrelated needs. Available field

technology to meet law

enforcement and international

needs at border

check points will aid in

controlling the spread of

pathogens and other undesirable materials.
INL BIOTECHNOLOGY

05-GA50193-56

For more information

INL is a U.S. Department of Energy

national laboratory operated by

Battelle Energy Alliance

Frank Roberto, Ph.D.

(208) 526-1096

Francisco.Roberto@inl.gov

Deborah Newby, Ph.D.

(208) 526-7779

Deborah.Newby@inl.gov

Continued from front

Selected Publications/

Presentations

D.T. Newby and F.F.

Roberto, “Real-time PCR

assay for field diagnosis of

Brucella abortus in wildlife

populations in Yellowstone

National Park.” Brucellosis

2003 International Research

Conference, University of

Navarra, Pamploma, Spain,

September 15-17, 2003.

D.T. Newby, T.L. Hadfield

and F.F. Roberto, 2003

“Real-time PCR detection of

Brucella abortus: a comparative study of SYBR Green I,

5’-exonuclease, and hybridization probe assays.” Appl.

Environ. Microbiol. 69:4753-

4759.

D.T. Newby and F.F.

Roberto, “Rapid detection of

pathogens: DNA diagnosis for

Brucella.” 58th Northwest

Regional Meeting of the

American Chemical Society,

Montana State University,

Bozeman, MT, June 12-14,

2003.

F.F. Roberto, and D.T.

Newby, “Application of DNA

diagnostics for Brucella to

recent outbreaks in the

Greater Yellowstone Area.”

ASM Biodefense Meeting,

Baltimore, MA, March 9-12,

2003.

Don Maiers

Manager,

Biological Sciences

(208) 526-6991

Donald.Maiers@inl.gov

Technical Contact

Management Contact

D.T. Newby, H.G.

Silverman, T. Hadfield and

F.F. Roberto, “Real-time

PCR Detection of Brucella

abortus: a Comparative Study

of SYBR Green I, Taqman,

and Hybridization Probe

Assays,” American Society

for Microbiology, 102nd

General Meeting, Salt Lake

City, UT, May 2002, Poster

Q-381.

F. F. Roberto and D. T.

Newby, “DNA Technology—

An Introduction to Real-time

PCR,” Hispanic Youth

Symposium (Scientific/

Engineering Workshop), Sun

Valley, Idaho, April 2001.

D. T. Newby and F.F.

Roberto, “Application of the

Ruggedized Advanced

Pathogen Identification

Device (RAPID) for Brucella

detection,” INEEL Biotechnology Seminar Series, July

2001.

F. F. Roberto, H. G.

Silverman, M. Tsang, R.

Rodriguez, “A PCR Diagnostic System for Brucella in

Wildlife Species,” USGS

Bison Research Initiative,

1998–2000, Yellowstone

National Park, Bozeman,

Montana.