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BIOTECH PROJECT 1 Regione Veneto – RESEARCH LINE N. 14

EXPERTEAM. 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)

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BIOTECH PROJECT 1 Regione Veneto – RESEARCH LINE N. 14

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  1. EXPERTEAM 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.

  2. 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’:

  3. MAIN SECTORS OF ACTIVITIES • 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

  4. AIM OF THE PROJECT 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: • 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)

  5. ENTERIC VIRUS virus characterized by the affection of the respiratory and gastro-intestinal tissues in men and mammals in general Enterovirus Norwalk Epatite A Rotavirus Reovirus Adenovirus virus Poliovirus Coxsackievirus Echovirus

  6. DETERMINATION OF ENTEROVIRUSIN WATER Traditional method: cellular culture New biotech: RT-PCR • easy technique • fast • cheap • higly sensible specific • hard to do technique • long-time (10-30 gg. • expensive • not sensible enough

  7. TRADITIONAL METHOD SAMPLE POSITIVE SAMPLE Enterovirus CONENTRATION. & DECONTAMINATION citotossic effect IMMUNOFL. TECHNIQUE 1° STEP: CELLULAR CULTURE NEGATIVE SAMPLE: presence of enteric virus no enterovirus NO CITOTOSSIC EFFECT Problably no enterovirus Single strand of cells. BGM (Buffalo Green Monkey POS. SAMPLE citotossic effect IMMUNOFL. TECHNIQUE NEG. SAMPLE 2° STEP CELL. CULTURE no citotossic effect POS. SAPLE: CITOTOSSIC EFFECT: IMMUNOFL. TECHNIQUE NEG. SAPLE 3°STEP CELL. CULTURE no citotossic effect MIN. 10 DAYS TO MAX. 30 DAYS. NEG. SAMPLE

  8. METHODIC 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

  9. 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 CN CN CP CP 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 RT-PCR to analyze ENTEROVIRUS in waste-water sample just outside the purifier protocollo single step protocollo PCR nested

  10. PCR to analyze ADENOVIRUS in waste-water sample just outside the purifier protocollo single step protocollo PCR nested TYPING ADENOVIRUS(sierotype 40/41)

  11. OPTIMIZED RECORD Take sample Conc. sample Cell. colture Viral RNA extraction Reverse-transcription PCR (I° ampl.) PCR nested (II° ampl.) Electrofor. agar. gel ADENOVIRUS ENTEROVIRUS • Take sample • Conc. sample / • Viral RNA extraction • / • PCR (I° ampl.) • PCR nested (II° ampl.) • Electrofor. Agar. gel

  12. RESULTS

  13. CP CN CN CP B B CN 1 2 3 CP CN RT-PCR TO ANALYZE HAV IN WASTE-WATER SAMPLES JUST OUTSIDE THE PURIFIER RT-PCR TO ANALYZE REOVIRUS IN WASTE-WATER SAMPLE JUST OUTSIDE THE PURIFIER single step PCR nested single step PCR nested

  14. OPTIMIZED PROCEDURE HAV REOVIRUS • Take sample • Conc. sample • Viral RNA extraction • Reverse-transcription • PCR (I° ampl.) • PCR nested (II° ampl.) • Agarose gel electrophoresis • Take sample • Conc. sample • Viral RNA extraction • Reverse-transcription • PCR (I° ampl.) • PCR nested (II° ampl.) • Agarose gel electrophoresis

  15. Analysis of 39 waste-water samples taken just outside the purifiers in 11 different places along the April-August 2005 period *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 .

  16. CONCLUSIONS ADENOVIRUS DETECTION ADVANTAGES AS INDEX QUALITY COMPARE TO ENTEROVIRUS & HAV • > Sensibility • DNA INSTEAD OF RNA • NOT NECESSARY CELL CULTURE 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

  17. METHODIC PROCEDURES FOR QUANTITATIVE PCR

  18. REAL TIME PCR SYBR GREEN method Amplification plots of 5 dilutions (106,105,104,103,102copies of viral genoma) of cloned plasmidic DNA containing the sequence 5’ UTR of Enterovirus

  19. REAL TIME PCR TAQ-MAN method Amplification plots of 5 dilutions(106,105,104,103,102,10 copie di genoma virale) ofcloned plasmidic DNA containing the sequence 5’ UTR of Enterovirus

  20. Analysis on 2 concentrate water samples already positive for Enterovirus with Experteam kit“Enterovirus kit 1” • We have determinated: • The viral load with real time PCR • 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.

  21. Sequence obtained by amplified 5? NTR of sample 1 The Gene Bank analysis has determinated Sample 1: Coxsackievirus B1 Sample 2: even if we could not arrive to a specific genotyping we can exclude that it was an human enterovirus already described in literature

  22. REAL TIME PCR SYBR GREEN method Samples analysis Determination of Adenovirus in samples already positive by qualitative PCR Standard amplification plots CT = 27 → [500 copies/ml] CT = 34 → [5 copies/ml]

  23. Discussion 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

  24. DETERMINATION OF ADENOVIRUS IN SEDIMENT SAMPLE BY NESTED PCR

  25. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 INHIBITORIESOF PCR IN SEDIMENT • To avoid inhibitor effect in the Adenovirus amplification we tried 4 different procedures • Adding polivinilpolipirrolidone (PVPP) during extraction • adding PVPP to amplification mix • adding “T4 gene Protein” to amplification mix • Diluition from 1/10 to 1/10.000of extracted DNA • 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 Samples with addition of T4 Gene 32 Protein Diluition from 1:10 to 1:10000

  26. 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.

  27. RESEARCH CONCLUSIONS(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

  28. Analysis of drain water samples, taken just outside the depurators in 11 differentsitesand previously analyzed, adding determination of: Reovirus, Norwalk virus and Rotavirus - Negative sample + positive sample / not analyzed sample

  29. 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 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

  30. 50°C 53°C 55°C 57°C 60°C 50°C 53°C 55°C 57°C 60°C 50°C 53°C 55°C 57°C 60°C Marcatore 50°C 53°C 55°C 57°C 60°C 50°C 53°C 55°C marcatore SITE 4 9382 SITE 3 6497 SITE 3 9805 SITE 8 8843 SITE 7 4479 Sample problably positive, to be veryfied by capillary electrophoresis Sample positive, veryfied by capillary electrophoresis Norwalk virus Rotavirus

  31. Sequencing of rotavirus amplified

  32. 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

  33. E.Coli (UFC/100ml) NaClO → sodium hypochlorite CH3CO3H → peracetic acid

  34. 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

  35. Sample sites choose criteria 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 • -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 becauselocalized 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

  36. SA3 SA4 SA2 SA10 SA5 SA6 SA1

  37. SA7 SA9 SA8

  38. Instrument used for sediment samples Instrument used for sediment samples

  39. 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

  40. Results scheme sediment & water

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