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

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

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

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

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.

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.

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

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.

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