BIOTECH PROJECT 1 Regione Veneto – RESEARCH LINE N. 14(with collaboration of department of enviromental science, University of Venice & department of Biology, University of Padua)

Viral pollution in water & sediments: use of PCR to detect analytical presence of adenovirus as quality index.

EXPERTEAMEXPERTEAM

Experteam founded in 1996 by biologists Angelo de Bortoli & Federica Schiavon, started their business with a series of innovative molecular biology kits to detect virus, bacteria, protozoa in the genetic and tumor diseases field

• research & development

• production

• marketing of diagnostic kits

ATTIVITA’:

• Genetic Diagnostic (microdelection chromosome Y in oligo e azoospermic patients, X fragile, coagulation factors, haemochromatosis)

• Viral Diagnostic (HPV, CMV)

• Bacteria & Parasites (m. tuberculosis e complex, b. burgdorferi, ch. pneumoniae, p. carinii, t. gondii, g. lamblia)

• Oncology Diagnostic (linfomi B e T, traslocazioni cromosomiche)

• Detection of micro-organism in food (salmonella e listeria)

• Enviromental Monitoring & Diagnostic

• Organization and Coordination of tutoring lessons

MAIN SECTORS OF ACTIVITIES

• The risk to get diseases by bathing uses of water (D.P.R. 155 del 1988: attuazione della direttiva 76/160/CEE relativa alla qualità delle acque di balneazione)

• The index quality of water based on presence of micro-organism and virus

• The efficiency of depuration systems

• The determination of a particular viral & bacteria genotype (traceability)

Research & development for production and marketing of diagnostic kits based on molecular biology methods to detect virus, bacteria and protozoa in lagoons, rivers and waste-waters to analyze:

AIM OF THE PROJECT

ENTERIC VIRUS ENTERIC VIRUS

Poliovirus EchovirusCoxsackievirus

Rotavirus Reovirus

EnterovirusEpatite ANorwalkvirus

Adenovirus

virus characterized by the affection of the respiratory and gastro-intestinal tissues in men and mammals in general

DETERMINATION OF ENTEROVIRUSDETERMINATION OF ENTEROVIRUSIN WATERIN WATER

Traditional method: cellular culture

• hard to do technique

• long-time (10-30 gg.

• expensive

• not sensible enough

New biotech: RT-PCR

• easy technique

• fast

• cheap

• higly sensible specific

SAMPLE

CONENTRATION. & DECONTAMINATION

1° STEP: CELLULARCULTURE

NO CITOTOSSICEFFECTProblably no enterovirus

IMMUNOFL. TECHNIQUE

POSITIVE SAMPLEEnterovirus

NEGATIVE SAMPLE:presence of enteric virus no enterovirus

citotossiceffect

no citotossiceffect

IMMUNOFL. TECHNIQUE

POS. SAMPLE

NEG. SAMPLE

CITOTOSSIC EFFECT:

no citotossiceffect

IMMUNOFL. TECHNIQUE

POS. SAPLE:

NEG. SAPLE

2° STEP CELL.CULTURE

3°STEP CELL.CULTURE

NEG. SAMPLE

TRADITIONAL METHODTRADITIONAL METHOD

Single strand of cells. BGM (Buffalo Green Monkey

MIN. 10 DAYS TO MAX. 30 DAYS.

citotossiceffect

METHODIC STEPSMETHODIC STEPSFOR DETERMINATION OF ENTEROVIRUS, ADENOVIRUS & HAV

by PCR REACTION

• Bibliografy research

• Optimization of DNA, RNA extraction procedure

• Optimization of retro transcription-amplification phase (primers, annealing t°, etc)

Finding and analysis positive samples

• Finding and analysis of enviromental samples

• Comparison between traditional methods and PCR

RT-PCR to analyze ENTEROVIRUS in waste-water RT-PCR to analyze ENTEROVIRUS in waste-water sample just outside the purifiersample just outside the purifier

protocollo single step protocollo PCR nested

1 2 3 4 5 6 7 8 CN

CP 9 10 11 12 13 14 15 16

1 2 3 4 5 6 7 8 CN

CP 9 10 11 12 13 14 15 16

protocollo single step protocollo PCR nested

PCR to analyze ADENOVIRUS in waste-water PCR to analyze ADENOVIRUS in waste-water sample just outside the purifiersample just outside the purifier

TYPING ADENOVIRUSTYPING ADENOVIRUS(sierotype 40/41)(sierotype 40/41)

OPTIMIZED RECORDOPTIMIZED RECORD

• Take sample

• Conc. sample

• Cell. colture• Viral RNA extraction

• Reverse-transcription

• PCR (I° ampl.)

• PCR nested (II° ampl.)

• Electrofor. agar. gel

ENTEROVIRUS ADENOVIRUS

• Take sample

• Conc. sample

/

• Viral RNA extraction

• /• PCR (I° ampl.)

• PCR nested (II° ampl.)

• Electrofor. Agar. gel

SAMPLES

SUPERFICIAL WATER EC

ENTEROVIRUS (I° P BGM) ADENOVIRUS (TQ)

IF PCR PCR PCR 40/41

9721 POS POS POS POS POS

9552 NEG   NEG POS POS

9554 NEG   NEG POS POS

1397 NEG NEG POS POS

1398 NEG NEG NEG  

1728 NEG NEG POS NEG

2213 NEG NEG NEG  

2219 NEG NEG POS NEG

2389 NEG NEG POS POS

2558 NEG NEG NEG  

0485 POS POS POS POS / 

0321 POS POS POS POS  /

WASTE-WATER          

0706 POS POS NEG POS NEG

8970 NEG   NEG POS NEG

9761 NEG   NEG POS POS

2405 NEG NEG POS POS

2450 NEG NEG POS POS

RESULTS

TOT. SAMPLES

17

TOT. POSITIVE 3

ENTEROVIRUS

TOT. POSITIVE 14 ADENOVIRUS

single step PCR nested

CP CN B CP CN

RT-PCR TO ANALYZE HAV IN RT-PCR TO ANALYZE HAV IN WASTE-WATER SAMPLES JUST WASTE-WATER SAMPLES JUST OUTSIDE THE PURIFIEROUTSIDE THE PURIFIER

RT-PCR TO ANALYZE REOVIRUS IN RT-PCR TO ANALYZE REOVIRUS IN WASTE-WATER SAMPLE JUST WASTE-WATER SAMPLE JUST OUTSIDE THE PURIFIEROUTSIDE THE PURIFIER

single step PCR nestedCN CP B

1 2 3CN

OPTIMIZED PROCEDUREOPTIMIZED PROCEDURE

• Take sample

• Conc. sample

• Viral RNA extraction

• Reverse-transcription

• PCR (I° ampl.)

• PCR nested (II° ampl.)

• Agarose gel electrophoresis

HAV REOVIRUS

• Take sample

• Conc. sample

• Viral RNA extraction

• Reverse-transcription

• PCR (I° ampl.)

• PCR nested (II° ampl.)

• Agarose gel electrophoresis

ANALYZEDSAMPLES

POSITIVEADENOVIRUS

POSITIVEENTEROVIRUS

POSITIVEHAV

20 TQ 12 (60%) 1 (5%) 0 (0%)

19 IP 1 (5,3%)* 2 (10,5 %)* 0 (0%)

TOT. 39 13 (33,3%) 3 (7,7%) 0 (0%)

*I campioni positivi agli enterovirus sono stati analizzati mediante PCR specifica per determinare se si trattava di poliovirus, coxsackie o echovirus, l’analisi ha escluso la presenza di poliovirus .

Analysis of 39 waste-water samples taken just outside Analysis of 39 waste-water samples taken just outside the purifiers in 11 different places along the April-the purifiers in 11 different places along the April-

August 2005 periodAugust 2005 period

CONCLUSIONS

The Adenovirus detection, as indicators of faecal presence, results easier and more sensible compare to Enterovirus , while the HAV detection is not significant because none of the samples has resulted positive to this detection

• > Sensibility• DNA INSTEAD OF RNA• NOT NECESSARY CELL CULTURE

ADENOVIRUS DETECTION ADVANTAGES AS ADENOVIRUS DETECTION ADVANTAGES AS INDEX QUALITY INDEX QUALITY

COMPARE TO ENTEROVIRUS & HAV COMPARE TO ENTEROVIRUS & HAV

METHODIC PROCEDURESMETHODIC PROCEDURESFOR QUANTITATIVE PCRFOR QUANTITATIVE PCR

Amplification plots of 5 dilutions (106,105,104,103,102 copies of viral genoma) of cloned plasmidic DNA containing the sequence 5’ UTR of Enterovirus

REAL TIME PCR SYBR GREEN methodREAL TIME PCR SYBR GREEN method

Amplification plots of 5 dilutions(106,105,104,103,102,10 copie di genoma virale) of cloned plasmidic DNA containing the sequence 5’ UTR of Enterovirus

REAL TIME PCR TAQ-MAN methodREAL TIME PCR TAQ-MAN method

We have determinated:1) The viral load with real time PCR2) The specific type (poliovirus, coxsackie o echovirus) by sequencing

The Sybr Green Real Time PCR procedure detected virus presence in 5000 concentrate samples of 0.2 and 0.3 UFP\ml

the sequence analysis needed a new procedure to amplified changeable regions amongst different types of Enterovirus. The 3 analyzed & sequenced regions are: 5’NTR, VP1-2C, VP1-2.

Analysis on 2 concentrate water samples Analysis on 2 concentrate water samples already positive for Enterovirus withalready positive for Enterovirus with

Experteam kit“Enterovirus kit 1” Experteam kit“Enterovirus kit 1”

Sequence obtained by amplified 5? NTR of

sample 1

The Gene Bank analysis has determinatedSample 1: Coxsackievirus B1Sample 2: even if we could not arrive to a specific genotyping we can exclude that it was an human enterovirus already described in literature

Determination of Adenovirus in samples already positive by qualitative PCR

Standard amplification plots

Samples analysis

CT = 27 → [500 copies/l]CT = 34 → [5 copies/l]

REAL TIME PCR SYBR GREEN methodREAL TIME PCR SYBR GREEN method

DiscussionDiscussion

Comparison between qualitative PCR & RT-PCR to quantify Enterovirus and Adenovirus, demonstrated qualitative PCR more sensible than RT-PCR. We have to consider that all the quality procedures ” used were “nested” while the RT procedures were single step.

Comparison between quantitative Taqman & Sybr Green proved more sensibility for Syber green. Further trials must be done using more nucleic acids in the amplification mix

DETERMINATION OF ADENOVIRUS DETERMINATION OF ADENOVIRUS IN SEDIMENT SAMPLE IN SEDIMENT SAMPLE

BY NESTED PCRBY NESTED PCR

INHIBITORIESINHIBITORIES OF PCR IN SEDIMENTOF PCR IN SEDIMENT

To avoid inhibitor effect in the Adenovirus amplification we tried 4 different procedures

1. Adding polivinilpolipirrolidone (PVPP) during extraction

2. adding PVPP to amplification mix

3. adding “T4 gene Protein” to amplification mix

4. Diluition from 1/10 to 1/10.000 of extracted DNA 1 2 3 4 5 6 7

9 10 1112 13 1415 168

Diluition from 1:10 to 1:10000

Samples with addition of T4 Gene 32 Protein

Better results we achieve with procedures 3 e 4:

• with addition of “T4 gene Protein” we do not need diluition of extracted DNA

• diluition procedures don’t need further reactive. However we don’t need “a priori” the ideal diluition for every sample

Utilization of “FastPrep Instrument” (product of Q-Biogene) has made easier, more sensible & repeatable DNA & RNA extraction from sediment compare to other procedures used until now.

RESEARCH CONCLUSIONSRESEARCH CONCLUSIONS(Sept. 04 – Dec. 05)(Sept. 04 – Dec. 05)

• Improved of 6 kits with qualitative PCR (enterovirus, poliovirus, adenovirus, adenovirus 40 & 41, HAV and reovirus) and 2 kit in quantitative PCR (enterovirus e adenovirus)

• Determination by PCR of adenovirus result better as index of enviromental contamination because more sensible, faster and easier , both in concentrate water and sediment, to investigate for virus and micro-organism

• stated the complex of the molds (concentrate water and sediment) we must pay attention during DNA & RNA extractions, especially for Taq polimerase inhibitors presence

• Italian labs taking care of enviromental monitoring showed strong interest for our activities, therefore, we can feel that our products & procedures developed in this field will achieve remarkable succes

Analysis of drain water samples, taken just outside the Analysis of drain water samples, taken just outside the depurators in 11 differentdepurators in 11 different sites and previously

analyzed, adding determination of: Reovirus, Norwalk virus and Rotavirus

- Negative sample + positive sample / not analyzed sample

The analysis of reovirus have been made both on TQ sample (water sampletrated only with decontamination and concentration) and on BGM sample (water sample treated with a further cellular colture with BGM cells): none of the samples has resulted positive.

The analysis of norwalk and rotavirus has been made on TQ sample only because those virus don’t grow up on BGM cells colture

SITE 3

sampleData

prelievo

NORWALK

Nested

6497 TQ 05/07/05 ?

SITE 8

sampleData

prelievo

NORWALK

Nested

20508843 TQ 02/08/05 ?

SITE 7

sampleData

prelievo

ROTAVIRUS

Nested

20504479 TQ 27/04/05 +

The analysis of site 7 revealed positivity to rotavirus, data confirmed by capillary electrophoresis

The analysis of site 3 & 8 revealed positivity to norwalk virus, data confirmed by capillary electrophoresis

Norwalk virus

Sample problably positive, to be veryfied by capillary electrophoresis

50°C

53

°C55

°C57

°C60

°C50

°C

53°C

55°C

57°C

60°C

50°C

53

°C55

°C57

°C60

°CM

arca

toreSITE 4

9382SITE 36497

SITE 39805

Rotavirus

50°C

53

°C55

°C57

°C60

°C50

°C

53°C

55°C

mar

cato

reSITE 88843

SITE 74479

Sample positive, veryfied by capillary electrophoresis

Sequencing of rotavirus amplified

Analysis of results achieved and comparison between positivity of various analyzed virus to Escherichia coli ( analysis made by ARPAV for us) with the different kind of disinfection

E.Coli (UFC/100ml)UFC: units forming colony

NaClO → sodium hypochlorite CH3CO3H → peracetic acid

E.Coli (UFC/100ml)

The results confirmed adenovirus as the best index of viral contamination of water and that the better disinfection system is based on sodium hypochlorite,

An interesting analysis about the site number 4; we detect a very high presence of E. coli and a positivity to Enterovirus as well (the only positive site) and at the same time positivity to adenovirus. The disinfection system for this site was UV ray

Sample sites choose criteria

-Sito SA1: Mulino Stucky - Canale dei Lavraneri-Sito SA2: Rialto - Canal Grande-Sito SA3: Ospedale Fatebenefratelli - Rio di Zecchini-Sito SA4: Ospedale SS. Giovanni e Paolo - Fondamenta nuove-Sito SA5: Santa Marta – Rio delle Terese-Sito SA6: Ca’ Giustiniani - Rio delle Eremite-Sito SA7: Marghera - Canale Vittorio Emanuele-Sito SA8: Fusina - Naviglio del Brenta-Sito SA9: Marghera (zona industriale) - Canale dei Petroli-Sito SA10: Arsenale - Canale di San Pietro

• Siti SA3, SA4 e SA10 problably positive because nearby a hospital• Siti SA2, SA5 e SA6 problably positive because localized in canals with drain water• Siti SA1 e SA7 problably negative because localized in canals with high water-exchange• Siti SA8 e SA9 we have considered these sites because near the industrial area of Marghera and Fusina

We have choosen 10 sample sites in the lagoon, in any site we took 10 litres of water & 100 gr. of sediment, based on posssible traces of entero virus

SA3

SA4

SA2

SA1

SA5

SA6

SA10

SA9SA7

SA8

Instrument used for sediment samples

Instrument used for sediment samples

We conserved The water samples taken at +4°c 2 up to 2 days.the samples have been treated with decontaminant and concentrated, after that, we inoculated a single strate of BGM cell. Colture. we have considered positive to entero virus all the samples with a citopathic effect after a single cellular passage (10 days)

The sediment samples taken by any sites (100 gr.) have been conserved at -20°cfor a maximum of 2 days. We have done extraction by fastDNA Spin kit for soil and the FastPrep instrument (Qbiogene). The RNA extraction by FastRNA pro soil Direct kit and FastPrep instrument (Qbiogene)

The DNA and RNA sample extracted have been analyzed by Experteam for qualitative determination of different entero virus

Results scheme sediment & waterSito H2O

Citopathic effect

Sediment Adenovirus

Sediment Enterovirus

Sediment HAV

Sediment Reovirus

Sediment Rotavirus

Sediment Norwalk

virus

SA1 - + - + - + -SA2 - + - - - + -SA3 +/- + +/- - - - -SA4 + + - + - + -SA5 - + - - - - -SA6 + + - - - + -SA7 - + - - - - -SA8 - + - - - + -SA9 - + - - - - -

SA10 - + - - - + -