Multiplexed Detection of Pathogens using Fluorescence Resonance Energy Transfer in a Spatial Detection Format Multiplex PCR reaction Bruce Applegate Collaborators: Sergei Savikhin Pina Fratamico Wilfredo Dominguez Hanyoup Kim Alex Aimet (Proligo) Michael Kane Beacon detection Multiplex PCR primer and beacon binding sites in target genes from L. monocytogenes, Salmonella spp, and E. coli O157:H7 Multiplex PCR reaction 600bp INVA SIPB FIMY SPVC FLICH7 STX2 STX1 EAEA HLYC RFBE IAP HLYA PRFA INLA SIPC Analysis of multiplex PCR reactions Capillary Electrophoresis (traces poster) ROX standard 100bp JOE, FAM and NED tagged Primers for PCR PCR of multiplex PCR primer pairs Multiplex PCR One reaction with 12 genes Three reactions with four genes for each pathogen 1
spvc sipc sipb inva prfa hlya inla iap Capillary electrophoretic fragment analysis for multiplex PCR from S. Enteritidis with approximately four copies of genomic DNA in 25 µl. (The red peaks represent the size standard ROX. The green, blue, and black peaks represent the multiplex PCR products from the fluorescently labeled primers with JOE, FAM, and NED respectively. The X axis represents the electrophoresis time.) Capillary electrophoretic fragment analysis for multiplex PCR from L. monocytogenes with approximately four copies of genomic DNA in 25 µl. (The red peaks represent the size standard ROX. The green, blue, and black peaks represent the multiplex PCR products from the fluorescently labeled primers with JOE, FAM, and NED respectively. The X axis represents the electrophoresis time.) eaea rfbe stxi hlyc sipb inva sipc eaea prfa hlya rfbe stxi spvc inla iap hlyc Capillary electrophoretic fragment analysis for multiplex PCR from E. coli O157:H7 gene fragments with approximately four copies of genomic DNA in 25 µl. (The red peaks represent the size standard ROX. The green, blue, and black peaks represent the multiplex PCR products from the fluorescently labeled primers with JOE, FAM, and NED respectively. The X axis represents the electrophoresis time.) Capillary electrophoretic fragment analysis for multiplex PCR with approximately 4x10 3 copies of genomic DNA in 25 µl from E.coli O157:H7, S. Enteritidis, and L. monocytogenes. (The red peaks represent the size standard ROX. The green, blue, and black peaks represent the multiplex PCR products from the fluorescently labeled primers with JOE, FAM, and NED respectively. The X axis represents the electrophoresis time.) eaea mpcr results for E. coli O157:H7 inoculated ground beef rfbe stxii hlyc Fragment analysis using capillary electrophoresis of a positive multiplex PCR from E. coli O157:H7 inoculated beef. (The red peaks represent the size standard ROX. The green, blue, and black peaks represent the multiplex PCR products from the fluorescently labeled primers with JOE, FAM, and NED respectively. The X axis represents the electrophoresis time.) 2
mpcr results for S. enteritidis inoculated on lettuce sipc spvc sipb inva Fragment analysis using capillary electrophoresis of a positive multiplex PCR from S. Enteritidis inoculated lettuce. (The red peaks represent the size standard ROX. The green, blue, and black peaks represent the multiplex PCR products from the fluorescently labeled primers with JOE, FAM, and NED respectively. The X axis represents the electrophoresis time.) mpcr results for L. monocytogenes inoculated hotdogs prfa hlya inla iap Fragment analysis using capillary electrophoresis of a positive multiplex PCR from L. monocytogenes inoculated hot dog. (The red peaks represent the size standard ROX. The green, blue, and black peaks represent the multiplex PCR products from the fluorescently labeled primers with JOE, FAM, and NED respectively. The X axis represents the electrophoresis time.) Beacon Concept Complementary strand Beacon detection Target strand A B C Molecular beacon closed (A), open (B), and hybridized (C). 3
Fluorescence of iap (Listeria) in solution with cy-3 and cy-5 dye labeled 1 Spatial format detection Fluorescence (a. u.) 0.5 A B C - control 0 500 550 600 650 700 750 800 Wavelength (nm) 70 C (black, line) => 55 C (blue, line) => 40 C (red) => 55 C (blue, circle) => 70 C (black, circle) + control Arrayer (Genomic Solutions) Michael Kane Scanner (Genomic Solutions) Michael Kane Preliminary array experiments Preliminary array experiments Unhybridized beacon Hybridized beacon Target beacon eae Target beacon eae hybridized with complimentary oligo 4
Prototype Detector Microarray A. Results from 3 different microarrays hybridized with control (no DNA, top row), 1.0-1.5 ng/ul of DNA (1:50 dilution of the PCR products, middle row), and 5-10 ng/ul of DNA (1:10 dilution of PCR products, bottom row). Note that in the absence of target DNA, the microarray features appear red due to FRET signaling (B), and in the presence of target DNA the microarray features appear green due to the absence of FRET signaling (C). Simplified setup of the biosensor. ND: neutral density filter, TS: translation stage, FL: collimating lens, FW: interference filter wheel, PMT: photomultiplier. TS and FW are pc-controlled and the data from PMT are sent to pc and analyzed. Two interference filters of 570 nm and 670 nm are switched during each measurement. The picture of the working biosensor prototype with the cover open. All components of the system are housed in a dark box. The green line in the picture is following the path of the green exciting laser (532 nm) which is hitting the target MB spot. The close view of the MB arrayed slide with the laser on. During the measurement, the neutral filters will be inserted at the output of the laser to adjust the intensity of the excitation. The temporary manual translation stage for the adjustment of the vertical height of the slide is also seen. The screen shot of the biosensor controlling software. (A) Individual measurement control with the choice of spot position and filter. (B) The status box showing the position, filter choice and PMT measurement at each spot. (C) The automated scan result is shown here and real time analysis is presented. 5
Summary Individual four gene mpcrs were able to yield a detectable signal with as low as four copies of template chromosomal DNA Collective twelve gene mpcr was able to yield a detectable signal with 4E+03 copies of template chromosomal DNA Summary The method to extract the DNA from the samples was inefficient for L. monocytogenes in hotdogs Array detection of PCR products using the beacon approach was successful Prototype detection device has been completed and is currently being evaluated An enrichment step was necessary to yield a detectable signal in all three cases Acknowledgments Purdue University Agriculture Research Programs Center for Food Safety Engineering, Purdue University Purdue core sequencing facility National Aeronautics and Space Administration USDA/ARS cooperative agreement 1935-42000-035 Future work Evaluating array with multiple beacons Optimizing hybridization parameters Design and develop a hand held device for scanning array incorporating a peltier heating and cooling block Purdue Research Foundation Trask Fund 6