5. Outline
• Introduction
• Food born pathogen
• Microorganism
• Listeria monocytogenes
• Methods
• Real time PCR
• Lamp
5
6. Introduction
• Food-borne diseases are mainly caused by
pathogenic bacteria which are either transmitted
to humans from the animal reservoir or which
contaminate the food process line. Detection and
isolation of these bacteria from food are often
di•fficult due to the high number of
contaminating and indigenous bacteria and a low
number of the pathogenic bacteria of concern.
Food-borne illnesses are a major public health
problem, and a method for the rapid detection
and identification of food-borne pathogens is
needed by the food industry.
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7. • Food borne disease is any illness
resulting from the consumption of
food contaminated with one or more
disease-producing agents. These
include
bacteria, parasites, viruses, fungi and
their products as well as toxic
substances not of microbial origin.
7
8. different types of microorganisms contaminating a food
1- Gram-negative rods and coccobacilli: Acinetobacter, Aeromonas,
Alkaligenes, Citrobacter, Escherichia, Flavobacterium, Moraxella, Proteu
s,Pseudomonas, Salmonella, Yersinia, Shewanella, Enterobacter
2- Gram-positive rods:
Bacillus, Brochothrix, Clostridium, Corynebacterium ,Lactobacillus
and Listeria
3-Gram-positive cocci: Enterococcus, Lactococcus, Micrococcus,
Pediococcus andStaphylococcus
4- Molds, Mucor, Rhizopus and Thamnidium and
Penicillium, Cladosporium, Geotrichum and Sporotrichum.
5-About 6 genera of yeast are known to contaminate meat. The most
common is Candida spp.
8
9. Most common microorganisms which spoil food
Organism
- Clostridium perfringens(Contaminates poultry
meat and meat products, pies)
2- Salmonella(Contaminates poultry meat and meat
products, especially poultry.cream,milk and egg
products and salads)
3- Staphylococcus(Meat, eggs and fish products)
4- Yersinia(Contaminates meat and meat products,.
Contaminated water,seafood and raw milk)
5- Yeasts (Sweet, jellies)
9
17. Flow cytometry
• An optically based method for analyzing individual cells in
• complex matrixes.
• It is used to estimate the number ,size and shape of
• microorganisms.
• Sensitivity of the technique is very high (102 yeast cells;
• 102 -103 bacterial cells per ml can be detected within few
• minutes.
• Suitable for detecting low numbers of specific organisms
• in fluid.
• Use to enumerate viruses in sea water.
17
20. Enzyme Linked Immunosorbent Assays
(ELISA)
ELISA is most commonly capture the
target antigen
The captured antigen is then
detected using a second antibody
which may be conjugated to an
enzyme.
A wide range of enzyme-linked
immunosorbent assays (ELISA) are
commercially available, especially
for
21. Enzyme Linked Immunosorbent Assays
(ELISA)
• The technique generally requires the
target organism to be 106
cfu/ml, although a few tests report a
sensitivity limit of 104
• Immunoassays may be performed
either as the 'standard' ELISA tray
22.
23.
24. Advantages of ELISA
1. Rapid
2. Cost-effective
3. Sensitive
4. Easy to operate
5. Requires little training
6. Reduced waste disposal
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25. Molecular Methods for Microbial
Detection
PCR
unimplex
multiplex
RT-PCR
Real-time PCR
DNA fingerprinting
Pulsed Field Gel Electrophoresis (PFGE)
Lamp
25
26. 4/28/2012 Free Template from www.brainybetty.com 26
27. Polymerase chain Reaction
Advantages of PCR Problems of PCR
1-The specificity of the 1-Inability to distinguish
reaction between live and dead
2-The flexibility of the cells
method. 2- The presence of
3-The simplicity and speed of polymerase inhibitors in
the automated procedure. food samples
4-The ability to utilize minute 3- The accessibility of the
samples to produce a high target organisms
yield of amplified target
DNA
27
29. Multiplex PCR
• Amplification products
obtained by multiplex PCR.
M,DNA ladder; 1, negative
control; 2, 3 and 4 PCR with
E. coli; L.monocytogenes
and S.typhimurium DNA (100
pg each), respectively.
5, PCR with 100 pg DNA
from each of E. coli and
S.typhimurium , and 6, PCR
with100 pg DNA from each
of E.
coli, L.monocytogen, and
S.typhimurium
29
30. Realtime PCR
Real-time PCR
End point PCR
PCR
Higuchi Real-Time PCR
30
34. In 2000 , Notomi , T. et al. reported a new
method, termed LAMP, that amplifies DNA with high
specificity, efficiency and rapidity under isothermal
conditions . Except for the primer set,all reagents for
LAMP is available as a kit,called “Loopamp DNA
amplification.
The LAMP employs a Bst DNA polymerase and a set of
4 specially designed primers that recognize six
distinct Distinct sequences on the target DNA.
34
40. Two types of RT-LAMP procedures
_ RNA extraction → Reverse transcription → LAMP
(cDNA synthesis)
_ RNA sample → LAMP with reverse transcriptase
(Loopamp RNA amplification kit)
RNA extraction and reverse transcription procedures
have to be simplified in order to apply LAMP
detection
system to RNA viruses.
51. Fluorescent Detection
Calcein
Loopamp Fluorescent Detection Reagent
Only by mixing this reagent with
Loopamp DNA Amplification Kit or
Loopamp RNA
Amplification Kit (RT-LAMP) before the
reaction, results can be visually
observed.
About instructions for using this
reagent, refer to the package insert.
59. Listeria monocytogenes
Buffered
- Listeria Enrichment Broth
PALCAM
MOX AGAR- OXFORD AGAR , AGAR
-
IMPEDANCE, DNA- PCR RECOMBINENT DNA
-, DNA PROBE
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60. REFRENCE
1- karami A, Ranjbar R, Ahmadi Z, safari Z. Rapid Detection of Different
Serovares of Salmonell antrica by Multiplex PCR. Iranian J Pub1
Health. Vol. 36, No.2, 2007, pp.38-42
2- karami A, Morrovati S, Ahmadi Z, safari Z, khalilpour A. Devlopment
of an ultra rapid and simple multiplex polymerase chin reaction
technique for detection of salmpnella typhi. Saudi Med J. 2006: 27
(8): 1134-1138
3- Doran JL, Collinson SK, Burian J, Sarlos G, Todd EC, Munro CK et al.
DNA-based diagnostic tests for Salmonella species targeting agfA, the
structurral gene for thin, aggregative fimbiae, J Clin Microbiol. 1993;
31:2263-73.
4- Massi MN, Shirakawa T, Gotoh A, Bishnu A, Hatta M, Kawabata M.
Rapid diagnosis of typhoid fever by PCR assay using one pair of
primers from flagellin gene of Salmonella typhi. J Infect Chemother.
2003; 9:233-7.
5-Morel G. Raccurt M. PCR in situ light and electron. I microscopy
, 2005; 5.0I.E, Manual of standards for diagnosis tests and vaccines.
1992; 408-425.
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61. REFRENCE
6- HAND BOOK OF FROZEN FOODS, 1TH ED, MARCEL DEKKER, 2004
, PP.595-617
7-FOOD ADMINESTERATION MANUAL, MICROBIOLOGICAL REFERNCE
CERTERIA FOOR FOOD,VERSION 2, OCTOBER 1995, PP11-12
8-FSIS DIRECTIVE 102403, USDA/FSIS,2002, PP2-12
9-MIKE STRINGER &COLIN DENNIS, CHILLED FOODS,SECEND ED, CRC
PRESS,2000, PP166-169
10-DRAFT COMMISSION REGULATION OF MICROBIOLOGICAL CERITERIA
FOR FOODSTUFFS, BRUSSSELS c(2005), PP : 15-16
11-USDA FSIS CENTER OF FOOD SAFETY, DRAFT ASSESSMENT OF
RELATIVE RISK TO PUBLIC HEALTH FROM FOODBORNE Listeria
monocytogenes AMONG SELECTED CATEGORIES OF RTE . 2001
12-FOOD MODERN MICROBIOLOGY-2006
13- U.H. . HUI,PAUL COUNILLON PAUL, GUERRERO LEGARETTA, H.LIM
MIANG, MURRELL K.D., NIP WAI-KIT
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