real-time rt pcr, dr mohamed ibrahim
TRANSCRIPT
Application of Modern Technique Real-Time qPCR in Microbial
Identification
Title of Lecture
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.
Adenovirus18-26nmssDNA
Rotavirus80nm
dsRNA
HAV30nm
(+) ssRNA
Enterovirus30nm
(+) ssRNA
Polyomavirus50nm
dsDNA
Alphacoronavirus120nm
(+) ssRNA
Zikavirus50nm
(+) ssRNA
Waterborne Viruses
Norovirus38-40nm
(+) ssRNA
Genome Types Group
IGroup
IIGroup
IIIGroup
IVGroup
VGroup
VIGroup
VII
ssDNA(+)
dsRNA(+/-)
ssRNA(+)
ssRNA(-)
rRNA(+)
rDNA(+/-)
dsDNA(+/-)
mRNA
ssRNA (-) RT EnzymeRT EnzymeDNA (-)
cDNA
Central Dogma
Transcription
Translation
CCTGAGCCAACTATTGATGAA
CCUGAGCCAACUAUUGAUGAA
DNA
RNA
Amino AcidsProteins
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.
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.
Virus ConcentrationMembrane Filter
System
Filtration & Trapping virus
particles on membrane
Elution by 50 ml beef extract
buffer
Ultra-Filteration System
• 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
[email protected] 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
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
(ABI 7700)[email protected]
• 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
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 .
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
Thank you to every one here
Dr. Mohamed Ibrahim Hasan. Ph.D. in Microbiology, Ain Shams University