1. A.Selvapandiyan LBPUA, OBRR, CBER, FDA, Bethesda, MD July 13 2006 Multiplex fluorescence-based PCR assay to detect pathogens in blood Multiplex fluorescence-based PCR assay to detect pathogens in blood

2. Background:  Transfusion blood safety has improved with pathogen testing  Increasing number of known potential infectious agents and emerging threats, including bioterrorism increases the burden of testing  Urgent need for methods to streamline and consolidate testing Aim:  Design a method that can d  Design a method that can detect rapidly for many pathogens simultaneously using Multiplex Fluorescence-based PCR  Performing in a convenient portable instrument SmartCycler

3. PathogensAssay conducted with Pathogens Assay conducted with Bacillus anthracispa geneB. anthracis (vaccine strain) Yersinia sp.16S rRNAY. pseudotuberculosis Leishmania sp. 18S rRNAL. donovani Trypanosoma sp. Internal control: Human18S rRNA

4. How SYBR Green based fluorescence PCR works ☹ ☹ ☹ ☹ ☹ ☹ SYBR Green Primer 1) Denaturation ☺ ☼ 2) Hybridization ☺ ☼ ☺ ☼ ☺ ☼ ☺ ☼ ☉ Polymerase 3) Extension ☺ ☼ Melt Derivative Readout

5. Fluorescence-based Multiplex PCR Assay for Simultaneous Detection of Bacterial and Parasitic Pathogens 75 20 40 60 80 85 90 95 0 A B BA LD YP human 100 200 300 400 bp LD (163 bp) BA ( 99 bp) YP (121 bp) HS (141 bp) Temperature o C Fluorescence 82 o C 86 o C87.3 o C89.8 o C Selvapandiyan et al., 2005 J. Mol. Diag. 7:268-275 This study outlines the development of a multiplex PCR for the simultaneous detection of the bacterial and parasitic pathogens.

6. Multiplex fluorescence PCR on the DNA isolated from the blood spiked with the serially diluted cells (200  l blood) the blood spiked with the serially diluted B. anthracis cells (200  l blood) B. anthracis cells 1,000 100 10 0 81.37 Selvapandiyan et al., 2005 J. Mol. Diag. 7:268-275  The Tm peak height seems as a novel measure to quantitate pathogns in blood  For this pathogen we could detect as low as 10 cells /200  l blood.

7. Cells/200  l Fluorescence Quantitation graph showing the relationship between the height of fluorescence peak and number of pathogen cells in blood Selvapandiyan et al., 2005 J. Mol. Diag. 7:268-275 For all the pathogens we could detect as low as 50 cells/ ml of blood

8. Multiplex PCR with in vivo infected blood samples Source of blood Sample Total number of samples Number of PCR positive Patients with VL Post treated VL patients 11 3 11 (50 –1000 cells/ml) 0 Human leishmaniasis Blood sample by hour Total number of samples Number of PCR positive 0 12 24 36 48 5554255542 0 1 (25%) (200-1000 cells/ml) 3 (66%) (1X10 4 –10 5 cells/ml) 2 (100%) (1X10 4 –10 5 cells/ml) Mouse anthrax Assay accurately detected pathogens in the samples from leishmaniasis patients as well as mice infected with B. anthracis.

9. Summary This study outlines the development and evaluation of a single-tube multiplex real-time PCR for the simultaneous detection of the bacterial and parasitic pathogens. For all the three pathogens we could detect as low as 50 cells /ml blood. Leishmania primers recognized DNA sequences from other Leishmania sp as well. Similarly Yersinia primers could identify sequences from Y. enterocolitica. Assay accurately detected pathogens in the blood samples from leishmaniasis patients as well as mice infected with B. anthracis. This assay takes less than one and half hours and hence could be useful for rapid identification purposes. Selvapandiyan et al., 2005 J. Mol. Diag. 7:268-275