Human Associated Virus Quantification In Singapore Water Distribution System By QPCR

Human Associated Virus Quantification In Singapore Water Distribution System By QPCRPresented by: Prannoy Chowdhury Singapore Stanford Partnership(SSP) NTU- Student ( MS Environmental Engineering)

Supervisors: Dr. Gao Ping Ping (CAWT) Dr. Karina Gin (NTU) INTRODUCTIONStandard 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

?OBJECTIVETo 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

pathmicro.med.sc.edu/mhunt/adeno-diag.jpg Genome : Double Stranded DNA

Capsid : 252 capsomers : 240 hexon forming faces + 12 penton at verticesADENOVIRUS IDENTIFICATIONUsual 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 Virus Recovery From Drinking Water SupplyFilter 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

.......................InfluentEffluentCharged Virus particlesCounter charged membraneVirus Recovery From Drinking Water Supply contd....

Regulator ModuleDischarge ModulePre-filter Module1 MDS Filter in Filter HousingHCl InjectorCartridge FilterVirus Recovery From Drinking Water Supply contd....Tangential Flow Filter (TFF)

www.pall.comVirus ConcentrationVirus 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 PlanDNA IsolationDNA isolation kit to be used

www.slic2.wsu.edu:82/.../pages/Chap9.htmlDNA QuantificationCorrelation between DNA quantity and number of Viruses.

Principle

PCR and Real Time PCR (Q-PCR) Detection (TaqMan-QPCR) Principle

RT-QPCR Principle

Probes and PrimersPrimer Sequences

5-GACTCYTCWGTSAGYTGGCC-3 and 5-CCCTGGTAKCCRATRTTGTA-3

Probe sequence

FAM-AACCAGTCYTTGGTCATGTTRCATTG- TAMRA FAM - 6-carboxyfluorescein TAMRA - 6-carboxytetramethylrhodamine

Real-Time PCR Quantification of Human Adenoviruses, Samuel Choi and Sunny C. Jiang, 2005Time managementVirus 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?

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