real-time rt pcr, dr mohamed ibrahim

Modern Molecular Biology

Technique

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Dr. Mohamed Ibrahim Hasan

Nov. 2016

Application of Modern Technique Real-Time qPCR in Microbial

Identification

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Title of Lecture

Introduction

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Water borne viruses can be found in water with different morphology and types. According to ICTV, 25 type of viruses have diameter ranged between 18-120nm detected in aquatic system. Small size viruses have been much harder to get rid of, as they are extremely resistant to physical and chemical inactivation.

Viruses are submicroscopic in size, but they can have large effects on organisms. They replicate by taking over a cell’s DNA function. Bacteria and viruses are different from each other. For treatment, some antiviral medications are available, but antibiotics are not effective against them.

Introduction

A few examples of viruses that can infect people are influenza, rhinovirus (common cold), hepatitis, polio and norovirus, states Cotruvo. Some viruses can cause gastroenteritis, which describes illnesses that involved stomach or intestines inflammation. The viruses that can cause gastroenteritis can be present in contaminated water. They include: Rotavirus, Norovirus, Adenovirus

Thirty-three disease outbreaks related to drinking water were reported in 2010, a number that is underreported and less than years prior. Norovirus and hepatitis A were two of the illnesses reported.

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Adenovirus18-26nmssDNA

Rotavirus80nm

dsRNA

HAV30nm

(+) ssRNA

Enterovirus30nm

(+) ssRNA

Polyomavirus50nm

dsDNA

Alphacoronavirus120nm

(+) ssRNA

Zikavirus50nm

(+) ssRNA

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Waterborne Viruses

Norovirus38-40nm

(+) ssRNA

Virus Structure

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Virus Shape

Ebola VirusFilamentous

Influenza Virus Spherical

Lambda Phage Complex

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Virus GenomeNucleotideStructure

RNA & DNA

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Nitrogen bases (RNA/DNA)

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British Scientific Watson & Crick, Feb 28th 1953

Virus Replication

Influenza Virus

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Genome Types Group

IGroup

IIGroup

IIIGroup

IVGroup

VGroup

VIGroup

VII

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ssDNA(+)

dsRNA(+/-)

ssRNA(+)

ssRNA(-)

rRNA(+)

rDNA(+/-)

dsDNA(+/-)

mRNA

ssRNA (-) RT EnzymeRT EnzymeDNA (-)

cDNA

Central Dogma

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Transcription

Translation

CCTGAGCCAACTATTGATGAA

CCUGAGCCAACUAUUGAUGAA

DNA

RNA

Amino AcidsProteins

Detections Steps

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Sample Collection Samples are collecting in clean big containers (40 liters).

Sodium thiosulfate (one to three drops per 250ml) is add to all chlorinated water samples.

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Plastic Container

Samples stored in an ice cooler box and delivered immediately to the laboratory for analyses. Magnesium chloride 1M will add during collection of samples.

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Virus ConcentrationMembrane Filter

System

Filtration & Trapping virus

particles on membrane

Elution by 50 ml beef extract

buffer

Ultra-Filteration System

Nucleic Acid Purification

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QlAamp Viral RNA Mini

Spin Protocol

Lyse

Wash

Elute

Master Mix Amplification

QIAGENMaster Mix

kits

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Vortex

PCR Tubes

Polymerase Chain Reaction

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• PCR is the technique for generating the large quantities of specific DNA. PCR is a cell free amplification technique to

synthesizing multiple identical copies of the DNA.

• 1983: Karry Mullis conceived the idea of PCR and developed it in 1984. He was awarded the Nobel Prize for the

discovery of PCR in 1993.

Discovery & Principle of PCR

Mohamed.Ibrahim@Cleqm.org.egThermal Profile

PCR Types Anchore

d PCR

Asymmetric PCR

Allele specific

PCR

Assemble

PCR

Inverse PCR

Helicase depende

nt amplica

tion

Overlap extension PCR

Race PCR

Reverse Transcriptase PCR Real Time PCR

Multiplex PCR

Ligation-mediated

PCR

Inter-sequence specific PCR

)ISSR (

Methylation specifin

PCR

MiniprimerPCR

NestedPCR

Solid phase PCR

Touch down PCR

Semi NestedPCR

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Real-Time PCR

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Short History• Real-Time PCR is a advanced biotechnological instrument. The term

real-time denote it can monitor the progress of the amplification when

the process is going on.

• 1993: first real-time PCR detection experiments to show utility for DNA

quantization reaction took place using EtBr detection (illumination, CCD

camera detection).

• 1996: Taqman detection methods used, instead of EtBr for real-time

detection of PCR.

• July 1996: first real-time qPCR (sequence detection system) instrument

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• Highly sensitive and reliable methods for detection & quantification of difrernt

types of nucleic acid (ds/ssDNA - ds/ssRNA – cDNA).

• The detection occurs during the accumulation of the PCR product with each

cycle of amplification.

• The system allows monitoring of the PCR reaction during early exponential

phase.

• This technique based on detection of fluorescence emitted from a reporter

molecule in real-time.

• Real-time is much faster, easy to use and can detect low levels of nucleic acid in

different types of water samples

Advantages of real-time PCR

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Principle Real-Time PCR

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1. Primers

Primer & probe designing• 15 – 30 bp in length

• G/C content of 20-80%.

• Avoid primer dimers.

• The Tm should be within 2°C.

• Purify by gel electrophoresis or HPLC.

• Optimize concentrations by performing

of 50nM, 300nM and 900nM to forward

& Reverse primers.

2. Probes• 20 – 30 bp in length

• G/C content of 20-80%.

• Tm 7-10°C higher than primers.

• To maximize signal or reporter

vary the probe concentration

between 5-400nM .

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A. Reaction SetupResults

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B. Real time Monitoring

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C. QReal timeStandard curve

Gene copies (GC) per microliters

Tube 1 (Positive control)

2x105 GC µl-1

Tube 2 2x104 GC µl-1

Tube 3 2x103 GC µl-1

Tube 4 2x102 GC µl-1

Tube 5 20 GC µl-1

Tube 6 2 GC µl-1

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Thank you to every one here

Dr. Mohamed Ibrahim Hasan. Ph.D. in Microbiology, Ain Shams University

Mohamed_Ibrahim2515@yahoo.com