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Title: A HANDHELD REAL TIME THERMAL CYCLER FOR BACTERIAL PATHOGEN DETECTION

Author
item HIGGINS, JAMES
item NASARABADI, SHANAVAZ - L.L. NAT'L LAB, CA
item KARNS, JEFFREY
item SHELTON, DANIEL
item COOPER, MARY - NAT'L VET. SRVCS LAB
item GBAKIMA, AIAH - MORGAN STATE UNIV.
item KOOPMAN, RONALD - L.L.NAT'L LAB, CA

Submitted to: Biosensors and Bioelectronics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/19/2002
Publication Date: 5/1/2003
Citation: Higgins, J.A., Nasarabadi, S., Karns, J.S., Shelton, D.R., Cooper, M., Gbakima, A., Koopman, R.P. 2003. A handheld real time thermal cycler for bacterial pathogen detection. Biosensors and Bioelectronics. p. 115-1123.

Interpretive Summary: The Handheld Advanced Nucleic Acid Analyzer (HANAA) was used to detect E. coli and Bacillus anthracis Ames DNA. The HANAA runs four samples at a time and can be run off battery power. The E. coli samples were taken from a stream in Baltimore, Maryland. They were plated, grown up overnight and then the colonies were added to Instagene matrix to extract the DNA. Small amounts of this DNA was used in PCR on the HANAA. E. coli was also spiked into raw water samples to make dilutions. DNA was also extracted from these samples using the Instagene matrix. Samples for testing of Bacillus anthracis were taken from mailrooms in Washington DC. These samples were grown up overnight and then the colonies were used on Instagene matrix. This DNA was used in PCR on the HANAA. Real time PCR on E.coli using the HANAA gave positive results in a short period of time. Use of SYBR Green as the probe in the PCR did give false positive results on the HANAA. Positive results from the Bacillus anthracis DNAs were seen at later times than those of E. coli, in most cases. The HANAA proved to be an effective way to test samples in a situation where battery powered PCR is necessary, as in out in the field or in response to some biological attack.

Technical Abstract: The Handheld Advanced Nucleic Acid Analyzer (HANAA) is a portable real time thermal cycler unit that weighs under 1 kg and uses silicon and platinum-based thermalcycler units to conduct rapid heating and cooling of plastic reaction tubes. Two light emitting diodes (LED) provide greater than 1 mW of electrical power at wavelengths of 490nm (blue) and 525nm (green), allowing detection of the dyes FAM and JOE / TAMRA. Results are displayed in real time as bar graphs, and up to three, 4-sample assays can be run on the charge of the 12V portable battery pack. The HANAA was evaluated for detection of defined E. coli strains, and wild-type colonies isolated from stream water, using PCR for the lac Z and Tir genes. PCR reactions using SYBR Green dye allowed detection of E. coli ATCC 11775 and E. coli O157:H7 cells in under 30 minutes of assay time; however, background fluorescence associated with dye binding to nonspecific PCR products was present. DNA extracted from 3 isolates of Bacillus anthracis Ames, linked to a bioterrorism incident in Washington DC in October 2001, were also successfully tested on the HANAA using primers for the vrrA and capA genes. Positive results were observed at 32 and 22 minutes of assay time, respectively. A TaqMan probe specific to the aroQ gene of Erwinia herbicola was tested on the HANAA and when 500 cells were used as template, positive results were observed after only 7 minutes of assay time. Background fluorescence associated with the use of the probe was negligible. The HANAA is unique in offering real time PCR in a handheld format suitable for field use; a commercial version of the instrument, offering six reaction chambers, is expected to be available in Summer 2002.