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

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Presentation on theme: "BIOTECH PROJECT 1 Regione Veneto – RESEARCH LINE N. 14 (with collaboration of department of enviromental science, University of Venice & department of."— Presentation transcript:

1 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. EXPERTEAM

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 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

4 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

5 ENTERIC VIRUS PoliovirusEchovirusCoxsackievirus Rotavirus Reovirus Enterovirus Epatite ANorwalk virus Adenovirus virus characterized by the affection of the respiratory and gastro-intestinal tissues in men and mammals in general

6 DETERMINATION OF ENTEROVIRUS IN 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

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

8 METHODIC STEPS METHODIC STEPS FOR 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 RT-PCR to analyze ENTEROVIRUS in waste-water sample just outside the purifier protocollo single stepprotocollo PCR nested CN CP CN CP

10 protocollo single step protocollo PCR nested PCR to analyze ADENOVIRUS in waste-water sample just outside the purifier 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 ENTEROVIRUS ADENOVIRUS Take sample Conc. sample / Viral RNA extraction / PCR (I° ampl.) PCR nested (II° ampl.) Electrofor. Agar. gel

12 SAMPLES SUPERFICIAL WATER EC ENTEROVIRUS (I° P BGM)ADENOVIRUS (TQ) IFPCR PCR 40/ POS 9552NEG POS 9554NEG POS 1397NEG POS 1398NEG 1728NEG POSNEG 2213NEG 2219NEG POSNEG 2389NEG POS 2558NEG 0485POS / 0321POS / WASTE-WATER 0706POS NEGPOSNEG 8970NEG POSNEG 9761NEG POS 2405NEG POS 2450NEG POS RESULTS TOT. SAMPLES 17 TOT. POSITIVE 3 ENTEROVIRUS TOT. POSITIVE 14 ADENOVIRUS

13 single stepPCR nested CPCNB CPCN 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 stepPCR nested CNCPB 123CN

14 OPTIMIZED 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

15 ANALYZED SAMPLES POSITIVE ADENOVIRUS POSITIVE ENTEROVIRUS POSITIVE HAV 20 TQ12 (60%)1 (5%)0 (0%) 19 IP1 (5,3%)* 2 (10,5 %)*0 (0%) TOT (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, lanalisi ha escluso la presenza di poliovirus. Analysis of 39 waste-water samples taken just outside the purifiers in 11 different places along the April- August 2005 period

16 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 INDEX QUALITY COMPARE TO ENTEROVIRUS & HAV

17 METHODIC PROCEDURES FOR QUANTITATIVE PCR

18 Amplification plots of 5 dilutions (10 6,10 5,10 4,10 3,10 2 copies of viral genoma) of cloned plasmidic DNA containing the sequence 5 UTR of Enterovirus REAL TIME PCR SYBR GREEN method

19 Amplification plots of 5 dilutions(10 6,10 5,10 4,10 3,10 2,10 copie di genoma virale) of cloned plasmidic DNA containing the sequence 5 UTR of Enterovirus REAL TIME PCR TAQ-MAN method

20 We have determinated: 1)The viral load with real time PCR 2)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: 5NTR, VP1-2C, VP1-2. Analysis on 2 concentrate water samples already positive for Enterovirus with Experteam kitEnterovirus kit 1 Experteam kitEnterovirus kit 1

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 Determination of Adenovirus in samples already positive by qualitative PCR Standard amplification plots Samples analysis C T = 27 [500 copies/ l] C T = 34 [5 copies/ l] REAL TIME PCR SYBR GREEN method

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 INHIBITORIESOF PCR IN SEDIMENT INHIBITORIES OF 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/ of extracted DNA 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 dont need further reactive. However we dont need a priori the ideal diluition for every sample

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

29

30 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 dont grow up on BGM cells colture SITE 3 sample Data prelievo NORWALK Nested 6497TQ05/07/05? SITE 8 sample Data prelievo NORWAL K Nested TQ02/08/05? SITE 7 sample Data prelievo ROTAVIRU S Nested TQ27/04/05+ 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

31 Norwalk virus Sample problably positive, to be veryfied by capillary electrophoresis 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 SITE SITE SITE Rotavirus 50°C 53°C 55°C 57°C 60°C 50°C 53°C 55°C marcatore SITE SITE Sample positive, veryfied by capillary electrophoresis

32 Sequencing of rotavirus amplified

33 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

34 NaClO sodium hypochloriteCH 3 CO 3 H peracetic acid E.Coli (UFC/100ml)

35 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

36 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

37 SA3 SA4 SA2 SA1 SA5 SA6 SA10

38 SA9 SA7 SA8

39 Instrument used for sediment samples

40 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

41 Results scheme sediment & water Sito H 2 O Citopathic effect Sediment Adenovirus Sediment Enterovirus Sediment HAV Sediment Reovirus Sediment Rotavirus Sediment Norwalk virus SA SA SA3 +/ SA SA SA SA SA SA SA


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