HUMAN ASSOCIATED VIRUS

QUANTIFICATION IN SINGAPORE WATER

DISTRIBUTION SYSTEM BY QPCR

Presented by:

Prannoy Chowdhury

Singapore Stanford Partnership(SSP)

NTU- Student ( MS Environmental Engineering)

Supervisors:

Dr. Gao Ping Ping (CAWT)

Dr. Karina Gin (NTU)

INTRODUCTION

Standard indicator of fecal contamination

- Total coliforms,
- faecal coliforms and
- enterococci

They may underestimate the risk of virus

associated waterborne diseases
Virus outbreaks reported in spite of compliance
with water treatment procedures
Adenoviruses are now considered as better
indicators of fecal contamination

OBJECTIVE

To investigate the presence of human associated

Adenovirus contamination in Singapore
distribution water system.
Generate quantified database for Adenovirus
pollution in Singapore waters
( Raw water and treated water)

Advise management strategies to reduce or

eliminate the potential Adenovirus sources

WHY ADENOVIRUSES?

Resistant to UV / Chlorine disinfection.

Relatively stable and long survival rates.

Detected in Drinking water supplies.

Sufficient concern for public health

(gastroenteritis, pneumonia etc.).
Placed by USEPA in its contaminant candidate
list for drinking water (2006).

LITTLE ABOUT ADENOVIRUSES

o Genome : Double Stranded DNA
o Capsid : 252 capsomers : 240 hexon forming
faces + 12 penton at vertices

pathmicro.med.sc.edu/mhunt/adeno-diag.jpg

ADENOVIRUS IDENTIFICATION

Usual method
- Virus isolation in cell culture
- Followed by antigen/antibody detection
- Visualization by electron microscope
Disadvantage : Laborious and time consuming

Molecular biology techniques

- Based on DNA identification using PCR
Advantage : Improved speed and sensitivity

VIRUS QUANTIFICATION

Conventional PCR vs. QPCR

Conventional PCR
Detection only/ poor quantification
Post PCR processing required
End point detection

QPCR
2x detection possible
Online detection /quantification

WORK PLAN
1

VIRUS RECOVERY FROM DRINKING

WATER SUPPLY

Filter 1000-2000 liters of drinking water through

10 inch 1MDS filter
Virus present in the sample will be adsorbed by
electrostatic interactions with the filter
Charged Virus particles

Influent

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Counter charged membrane

Effluent

VIRUS RECOVERY FROM DRINKING

WATER SUPPLY
CONTD....
Cartridge Filter

Pre-filter Module
1 MDS Filter in Filter Housing

Regulator Module

HCl Injector

Discharge Module

VIRUS RECOVERY FROM DRINKING

WATER SUPPLY
CONTD....

Tangential Flow Filter (TFF)

www.pall.com

VIRUS CONCENTRATION

Virus elution from filter

Elute the MDS filter with 1 liter Glycine-beef
extract buffer at pH 9.5

Add sodium thiosulphate for flock formation

Ultracentrifugation at 2,500 g, 15 min @ 4C

Dissolve precipitate in sodium phosphate solution

Centrifuge at 4,000-10,000 g. Store

supernantent (30ml)
Work Plan

DNA ISOLATION

DNA isolation kit to be used

www.slic2.wsu.edu:82/.../pages/Chap9.html

DNA QUANTIFICATION

Correlation between DNA quantity and number

of Viruses.
Principle
PCR and Real Time PCR (Q-PCR)

DETECTION (TAQMAN-QPCR)

Principle

RT-QPCR

Principle

PROBES AND PRIMERS

Primer Sequences
5-GACTCYTCWGTSAGYTGGCC-3
and 5-CCCTGGTAKCCRATRTTGTA-3

Probe sequence
FAM-AACCAGTCYTTGGTCATGTTRCATTGTAMRA
FAM - 6-carboxyfluorescein
TAMRA - 6-carboxytetramethylrhodamine
Real-Time PCR Quantification of Human Adenoviruses, Samuel Choi and Sunny C. Jiang, 2005

TIME MANAGEMENT

Virus Recovery and Concentration

Mid February Mid April
DNA Isolation and detection(QPCR)
Mid April Mid May
Data Analysis and Inferences
Mid May June

ANY SUGGESTIONS?