Detection of microorganism by BAXPCR

Detection of microorganism by BAX-pcr
Rolf Hugen 19-04-2007


Enterobacter sakazakii ?
Salmonella?
E. coli 0157:H7 ?

Listeria monocytogenes ?
Listeria spp


Structure of DNA

Snyder, L. and Champness, W., Molecular Genetics
of Bacteria (Washington, D.C.: ASM Press, 1997)

•

Double string molecule of
antiparallel ordered
Polynucleotide chains

•

Adenine

Thymine

•

Cytosine

Guanine


Opening of DNA (Denaturation)

•

neuen PCR_system


Annealing of primer


Chain extention

DNA
Polymerase

DNA
Polymerase


PCR: DNA amplification


Animation

pcranimatie .gif

BAX-PCR

Culture and PCR

Sample

Enrichment

Lysation
Amplification
Detection

Results


Sample preparation steps

ã

5 àl enrichment in 200 àl Lyses-buffer

ã

Lyses step
Salmonella

Listeria

20at 37°C

60´at 55°C.

stopping of the reaction at 95°C; then cooling.

•

No further DNA operating steps

ã

Pipetting 50àl in the PCR tubes

ã

Place the samples in BAX and start the program


The BAX-PCR tube

Content of the tubes:
Primer
Nucleotide
Polymerase
Fluorescing dye
Supplements
Internal control

A few pipetting steps so little chance of contamination

Melting point of DNA and fluorescents signal

>temperature

signal

no signal


DNA melt curve

1000

6000
5000
4000
3000
2000
1000
0

800
600
400
200
0

70

75

80

85

90

95

Bax detection

70

75

80

85

90

95

Transformed result


Internal control

Results


Speed of the test

BPW 1d

ND 1 d

BAX 0,5 d

BPW 1 d

ND 1 d

TSA 2 d

BAX-PCR 2,5 d
TSA 1 d

API 1 d

FLMV
6d


Choice of primer

•

PCR primer most be specific.

•

Molecular typing methods were used to search for specific
dna sequences.

•

With the RiboPrinter the first steps were made for primer
designing


Typing of micro-organism by the
RiboPrinter

genetic „Fingerprint“
Characterising and identifying of
micro-organism by analysing the
rRNA gene


Specificity of the primer for the Salmonella
BAX PCR

•
•

RiboPrinting of 1572 Salmonella strains

Demonstrating of the specificity with:
• 194 Salmonella strains
• 35 strains of other species

Specificity = 100%


Specificity of the primer for the Listeria
BAX PCR

•
•

RiboPrinting of 1572 L. monocytogenes strains
Demonstrating of the specificity with
• 196 L. monocytogens strains
• 78 strains of other species

specificity = 100%


AOAC certificate

Certification Report
AOAC Research Institute
Performance Tested MethodSM
100201
ofSalmonella
the
method = Detection

99,4%
BAXTMAccuracy
System with
Automated
PCR Assay for
Screening Salmonella
manufactered by
Qualicon, Icn


AOAC certificate

Certification Report
AOAC Research Institute
Performance Tested MethodSM
070202
BAXTMAccuracy
System with
Automated
of the
method =Detection
98%
L. monocytogens
PCR Assay for
Screening L. monocytogenes
manufactered by
Qualicon, Icn


International certificates

AOAC International Official Method
Salmonella #2003.09; L. Monocytogenes #2003.12

AOAC-RI Performance Tested Method
Salmonella #100201; Listeria monocytogenes #070202; E. Coli O157:H7
#010401

USDA-FSIS Adoption
Salmonella #MLG 4C.00; Listeria monocytogenes #MLG 8A.00

Health Canada Certification
Salmonella #MFLP-29; Listeria monocytogenes #MFLP-28
E. coli O157:H7 #MFLP-30; Enterobacter sakazakii #MFLP-27

AFNOR Certification
Salmonella #QUA 18/3-11/02

NordVal Certification
Salmonella #2003-20-5408

Brazil MAPA Official Reference Method
Salmonella #41

Japanese Ministry of Health, Labour and Welfare
Listeria; Listeria monocytogenes


Questions?