methods of bacterial identification polymerase chain
TRANSCRIPT
Methods of bacterial identification
Polymerase Chain Reaction
(PCR),MALDI-TOF and Antimicrobial
susceptibility tests
Assistant Prof. Dr. Ayad almakki
Department of Clinical Laboratory Science
College of Pharmacy
5 th stage
Lab training
University of Basra
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What is PCR ?
It’s a means of selectively amplifying a particular segmentof DNA (DNA amplification in vitro)
Each cycle of amplification doubles the amount of DNA inthe sample
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PCR applications
PCR is now a common and often main technique used inmedical and biological research labs for a variety ofapplications
1. Disease detection
2. Mutation analysis
3. Forensic medicine
4. Scientific research
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Identification of bacteria by PCR
samples
Cultures
Amplification by PCR (16S rRNA gene V3 region)
rRNA gene sequencing
Data banks (GenBank and RDPII databases)
Identification of the main member of the bacterial community studied :(Operational Taxonomic unit)
DNA extraction
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Identification of bacteria by PCR
samples
Cultures
Amplification by PCR (16S rRNA gene V3 region)
rRNA gene sequencing
Data banks (GenBank and RDPII databases)
Identification of the main member of the bacterial community studied :(Operational Taxonomic unit)
DNA extraction
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Basic Components of PCR
1- Target DNA (DNA template)The product of our DNA extraction
2- pair of primers (Oligonucleotide primers) Anneal to single-stranded DNA template Provide initiation site for extension of new DNA Forward primerAnneals to DNA anti-sense strand Reverse primerAnneals to DNA sense strand
Primers act as starting point for the DNA polymerase
3- dNTP‘ s (deoxynucleotidetriphosphates)Nucleotides (Adenine,Cytosine,Guanine,Thymine)building blocks for new DNA strands
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Basic Components of PCR
4- Taq polymerase (DNA polymerase)Enzyme that extends growing DNA strand complementary to DNA template
This enzyme is heat-tolerant isolated from Thermus aquaticus
5- Mgcl2
Provides ions needed for enzyme reaction
Cofactor of the enzyme
6- Buffer solutionMaintains optimal pH for enzyme
7- ThermocycleReactions are done in tubes or 96 well microtiter plates
Machine that changes temperatures
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Performing PCR1-Prepare reaction mixture in PCR tube :
10 x reaction buffer……………………….. 5 μldNTPs (12.5 mM) ………………………….. 1 μ lForward primer 10 pmol/ml………… 1 μ lReverse primer 10 pmol/ml……. 1 μ lTaq DNA polymerase…………………… 0.5 μ lsterile destilled water…………………… 39.5 μ l
total reaction mixture……………………… 50 ul
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Performing PCRDesign program for specific amplification in thermal
cycler (PCR machine).Ex. PCR program for NDM gene (35 cycles)
1st denaturation 94 C for 4 minutescycle denaturation 94 C for 45 secondsprimer annealing 55 C for 45 secondsprimer extension 75 C for 1.5 minutesthe last cycle is extended 72 C for 5 minutes
2-Load PCR mixture on thermal cycler and incubate in design program condition as above.
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Overview of PCRThe three main steps of PCR The basis of PCR is temperature changes and the effect that these
temperature changes have on the DNA.
In a PCR reaction, the following series of steps is repeated 20-40 times
Note: 25 cycles usually takes about 2 hours and amplifies the DNA fragment of interest 100,000 fold
Step 1: Denature DNAAt 94C, the DNA is denatured (i.e. the two strands are separated)
Step 2: Primers AnnealAt 40C- 55C, the primers anneal (or bind to) their complementary sequences on the single strands of DNA
Step 3: DNA polymerase Extends the DNA chainAt 75C, DNA Polymerase extends the DNA chain by adding nucleotides to the 3’ ends of the primers.
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PCR animation
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3-Analyze PCR product by gel electrophoresis.
Performing PCR
Basic principles :
1- Separation of molecule mixtures based on the different of their charge, melecular weight (size) & shape.
2-DNA is negatively charge move from cathode to anode pole in electric field.
3-The movement is proportional to their molecular weight
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Performing PCREquipments : 1- Electrophoretic chamber
2- Electrophoretic tray3- power supply
Method :1- Prepare 0.8% agarose in 1 x TAE (Tris acetic acid EDTA) buffer melt it by boilling to 100 C for a minute after cool , mixed with ethidium bromide .
2-Poor the gel to electrophoretic tray that have been prepared with comb to create wells on one side end of the gel.
3-Allow the gel to solidify in room temperature.
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Performing PCR
4-Put the gel in electrophoretic chamber filled with 1 x TAE buffer the gel is soaked (covered) in buffer.
5-Mix DNA samples with tracking dye (bromophenol blue + xylene xianol) & load to the well by pippetting gently.
6-Connect the chamber to power supply & set the voltage at 100 V for an hour.
7-Remove the gel DNA seperation is examined under UV transilluminator
8-Take the picture using polaroid camera.
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Performing PCR
Co
ntro
l -ve
Co
ntro
l +ve
E. coli
E. coli
ladd
er
Negative control reaction (Blank reaction)
Control for contamination Contains all reagents except DNA template
Positive control reaction
Control for sensitivity Contains all reagents and a known target-containing DNA template
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Identification of bacteria by PCR
samples
Cultures
Amplification by PCR (16S rRNA gene V3 region)
rRNA gene sequencing
Data banks (GenBank and RDPII databases)
Identification of the main member of the bacterial community studied :(Operational Taxonomic unit)
DNA extraction
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Identification of bacteria by PCR
rRNA gene sequencing
Beckman CEQ 2000, 8 capillary
DNA sequence
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Identification of bacteria by PCR
samples
Cultures
Amplification by PCR (16S rRNA gene V3 region)
rRNA gene sequencing
Data banks (GenBank and RDPII databases)
Identification of the main member of the bacterial community studied :(Operational Taxonomic unit)
DNA extraction
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Identification of bacteria by PCR
Ribosomal Database Project (RDP)
Data banks (GenBank and RDPII databases)
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Identification of bacteria by PCRData banks (GenBank and RDPII databases)
National center for Biotechnology information (NCBI)
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Identification of bacteria by PCRData banks (GenBank and RDPII databases)
National center for Biotechnology information (NCBI)
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Identification of bacteria by PCR
samples
Cultures
Amplification by PCR (16S rRNA gene V3 region)
rRNA gene sequencing
Data banks (GenBank and RDPII databases)
Identification of the main member of the bacterial community studied :(Operational Taxonomic unit)
DNA extraction
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Identification of bacteria by PCRIdentification of the main member of the bacterial community studied :
(Operational Taxonomic unit)
241993 Nobel Prize in Chemistry (Kary B. Mullis)
Croxatto et al.,201225
Identification of bacteria by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry
(MALDI-TOF MS)
matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry (MALDI-TOF MS)
An ion source to ionize and transfer sample molecules ions into a gas phase
A mass analyser that separate ions according to their mass-to-charge ration (m/z)
A detection device to monitor separated ions
Croxatto et al.,201226
Ionization of ribosomal proteins
Croxatto et al.,201227
Croxatto et al.,201228
Resistance study
1-Liquid media (dilution)
2-Solid media (diffusion)
Antimicrobial susceptibility tests:
Minimal inhibitory concentration (MIC)
Antibiotics supplemented Selective media
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Liquid media (dilution)- Broth microdilution
Antimicrobial susceptibility test
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1 2 3 4 5 6 7 8 9 10 11 12
StrainT(-) 128 64 32 16 8 4 2 1 0.5 0.25
T(+
)
A 1
IMP
B 1
C 2
D 2
E 3
F 3
G 4
H 4
10
0 u
l IM
P (
25
6 m
g/L)
+
10
0u
l MH
B C
X2
10
0 u
l IM
P
(25
6 m
g/L)
10
0 u
l IM
P
(25
6 m
g/L)
Cascade dilutions in serial two-fold dilutions
using a 100 ul by micropipette beginning at the
3 column up to column 11
100 ul of distilled water
100 ul bacterial suspension
Bacterial growthMIC
Inhibition
0 0.25 0.5 1 2 4 8 16 mg/L
Liquid media (dilution)
Antimicrobial susceptibility test
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Antimicrobial susceptibility tests
Solid media (diffusion)-Disk diffusion
-E-testsPlastic strips with a predefined gradient of one antibiotic
One strip per antibiotic Wide range of antibiotics Easy to use
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Summary
Bacterial resistance to antibiotics analyzed mainly by Minimal inhibitory concentration (MIC)
MALDI-TOF used to identify bacterial species due to its rapidity and low cost
The main function of PCR is amplification and analysis of the DNA from ( a single cell, hair follicle, or spermatozoa )
As few as 20 cycles would yield ~10 6 times the amount of target DNA initially present
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