a.selvapandiyan lbpua, obrr, cber, fda, bethesda, md july 13 2006 multiplex fluorescence-based pcr...
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A. SelvapandiyanLBPUA, 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
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Background:Background:
Transfusion blood safety has improved with pathogen testingIncreasing number of known potential infectious agents and emerging threats, including bioterrorism increases the burden of testingUrgent need for methods to streamline and consolidate testing
Aim:Aim:
Design a method that can dDesign a method that can detect rapidly for many pathogens simultaneously using Multiplex Fluorescence-based PCR
Performing in a convenient portable instrument SmartCycler
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PathogensPathogens Assay conducted withAssay conducted with
Bacillus anthracis pa gene B. anthracis (vaccine strain)
Yersinia sp. 16S rRNA Y. pseudotuberculosis
Leishmania sp. 18S rRNA L. donovani
Trypanosoma sp.
Internal control:Internal control:Human 18S rRNA
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How SYBR Green based fluorescence PCR worksHow SYBR Green based fluorescence PCR works
☹☹☹☹
☹☹
SYBR Green
Primer1) Denaturation
☺☼2) Hybridization
☺☼☺☼☺☼☺☼ ☉Polymerase
3) Extension
☺☼
Melt Derivative
Readout
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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 oC
Flu
ores
cen
ce
82oC 86oC 87.3oC 89.8oC
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.
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Multiplex fluorescence PCR on the DNA isolated from Multiplex fluorescence PCR on the DNA isolated from the blood spiked with the serially diluted the blood spiked with the serially diluted B. anthracis cells (200 cells (200 l blood)l blood)
B. anthraciscells
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 bloodFor this pathogen we could detect as low as 10 cells /200l blood.
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Cells/200 l
Flu
ore
sce
nce
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
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Multiplex PCR with Multiplex PCR with in vivoin vivo infected blood samples 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
5
5
5
4
2
0
0
1 (25%)(200-1000 cells/ml)
3 (66%)(1X104 –105 cells/ml)
2 (100%)(1X104 –105 cells/ml)
Mouse anthrax
Assay accurately detected pathogens in the samples from leishmaniasis patients as well as mice infected with B. anthracis.
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SummarySummary
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