methods of bacterial identification polymerase chain

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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