This document discusses waterborne viruses and their detection in water samples. It provides information on different types of viruses found in aquatic systems, including their size and morphology. Smaller viruses are more difficult to remove from water through physical and chemical treatment. The document then discusses the process of collecting and analyzing water samples to detect viruses, including concentrating the viruses, lysing the cells, amplifying the nucleic acids through PCR, and obtaining quantitative results in real-time. Key aspects of primer and probe design for PCR-based detection are also outlined.

2. Title of Lecture

4. 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.

5. Waterborne Viruses

11. Genome Types
Group I Group II Group III Group IV Group V Group VI Group VII
ssRNA (-)
RT EnzymeRT Enzyme
DNA (-)
cDNA

14.  Samples are collecting in clean big containers (40 liters).
 Sodium thiosulfate (one to three drops per 250ml) is add
to all chlorinated water samples.
 Samples stored in an ice cooler box and
delivered immediately to the laboratory
for analyses.
 Magnesium chloride 1M will add during
collection of samples.

15. Filtration & Trapping
virus particles on
membrane
Elution by 50
ml beef extract
buffer

16. Lyse
Wash
Elute

17. Vortex
PCR Tubes

19. Thermal Profile

20. PCR Types
Anchored
PCR
Asymmetric
PCR
Allele
specific
PCR
Assemble
PCR
Inverse
PCR
Helicase
dependent
amplication
Overlap
extension
PCR
Race PCR
Reverse Transcriptase PCR Real Time PCR
Multiplex
PCR
Ligation-
mediated
PCR
Inter-sequence
specific PCR
(ISSR)
Methylation
specifin
PCR
Miniprimer
PCR
Nested
PCR
Solid phase
PCR
Touch down
PCR
Semi Nested
PCR

23. • 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

25. • 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.
• 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 .

26. Results

28. Standard 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