Transcript DETERMINAZIONE MEDIANTE PCR (POLYMERASE CHAIN REACTION…

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.
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
ATTIVITA’:
• research & development
• production
• marketing of diagnostic kits
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
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)
ENTERIC VIRUS
virus characterized by the affection of the respiratory and
gastro-intestinal tissues in men and mammals in general
Norwalk Epatite A
virus
Poliovirus
Enterovirus Rotavirus Adenovirus
Reovirus
Coxsackievirus
Echovirus
DETERMINATION OF ENTEROVIRUS
IN WATER
Traditional method:
cellular culture
New biotech: RT-PCR
• hard to do technique
• easy technique
• long-time (10-30 gg.
• fast
• expensive
• cheap
• not sensible enough
• higly sensible specific
TRADITIONAL METHOD
SAMPLE
POSITIVE SAMPLE
Enterovirus
CONENTRATION. &
DECONTAMINATION
IMMUNOFL.
TECHNIQUE
citotossic
effect
1° STEP: CELLULAR
CULTURE
NEGATIVE SAMPLE:
presence of enteric virus
no enterovirus
NO CITOTOSSIC
EFFECT
Problably no enterovirus
POS. SAMPLE
Single strand
of cells. BGM
(Buffalo Green
Monkey
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
MIN. 10 DAYS TO
MAX. 30 DAYS.
no citotossic
effect
NEG. SAMPLE
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
RT-PCR to analyze ENTEROVIRUS in waste-water
sample just outside the purifier
protocollo single step
protocollo PCR nested
1 2 3 4 5 6 7 8 CN
1 2 3 4 5 6 7 8 CN
CP 9 10 11 12 13 14 15 16
CP 9 10 11 12 13 14 15 16
PCR to analyze ADENOVIRUS in waste-water
sample just outside the purifier
protocollo single step
TYPING ADENOVIRUS
(sierotype 40/41)
protocollo PCR nested
OPTIMIZED RECORD
ENTEROVIRUS
ADENOVIRUS
• Take sample
• Take sample
• Conc. sample
• Conc. sample
• Cell. colture
• Viral RNA extraction
• Reverse-transcription
/
• Viral RNA extraction
• PCR (I° ampl.)
•
/
• PCR (I° ampl.)
• PCR nested (II° ampl.)
• PCR nested (II° ampl.)
• Electrofor. agar. gel
• Electrofor. Agar. gel
RESULTS
SAMPLES
ENTEROVIRUS (I° P BGM)
ADENOVIRUS (TQ)
SUPERFICIAL WATER
EC
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
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
/
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
NEG
WASTE-WATER
TOT. SAMPLES
17
TOT. POSITIVE
ENTEROVIRUS
3
TOT. POSITIVE
ADENOVIRUS
14
RT-PCR TO ANALYZE HAV IN
WASTE-WATER SAMPLES JUST
OUTSIDE THE PURIFIER
single step
CP CN B
PCR nested
CP CN
RT-PCR TO ANALYZE REOVIRUS IN
WASTE-WATER SAMPLE JUST
OUTSIDE THE PURIFIER
single step
CN CP B
PCR nested
CN
1
2
3
OPTIMIZED PROCEDURE
HAV
REOVIRUS
• Take sample
• Take sample
• Conc. sample
• Conc. sample
• Viral RNA extraction
• Viral RNA extraction
• Reverse-transcription
• Reverse-transcription
• PCR (I° ampl.)
• PCR (I° ampl.)
• PCR nested (II° ampl.)
• PCR nested (II° ampl.)
• Agarose gel electrophoresis
• Agarose gel electrophoresis
Analysis of 39 waste-water samples taken just outside
the purifiers in 11 different places along the AprilAugust 2005 period
ANALYZED
SAMPLES
POSITIVE
ADENOVIRUS
POSITIVE
ENTEROVIRUS
POSITIVE
HAV
20 TQ
12 (60%)
1 (5%)
0 (0%)
19 IP
1 (5,3%)*
2 (10,5 %)*
0 (0%)
13 (33,3%)
3 (7,7%)
0 (0%)
TOT.
39
*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 .
ADENOVIRUS DETECTION ADVANTAGES AS INDEX
QUALITY
COMPARE TO ENTEROVIRUS & HAV
• > Sensibility
• DNA INSTEAD OF RNA
• NOT NECESSARY CELL CULTURE
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
METHODIC PROCEDURES
FOR QUANTITATIVE PCR
REAL TIME PCR SYBR GREEN method
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 TAQ-MAN 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
Analysis on 2 concentrate water samples
already positive for Enterovirus with
Experteam kit“Enterovirus kit 1”
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: 5’NTR, VP1-2C, VP1-2.
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
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]
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
DETERMINATION OF ADENOVIRUS
IN SEDIMENT SAMPLE
BY NESTED PCR
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 1/10.000 of 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
1 2 3 4 5 6 7
Samples with addition of
T4 Gene 32 Protein
8 9 10 11 12 13 1415 16
• diluition procedures don’t need further reactive.
However we don’t need “a priori” the ideal diluition for
every sample
Diluition from 1:10 to 1:10000
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 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
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
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 7
Data
prelievo
sample
ROTAVIRU
S
Nested
20504479
TQ
27/04/05
+
SITE 3
sample
6497
TQ
Data
prelievo
NORWALK
05/07/05
?
Nested
SITE 8
Data
prelievo
sample
NORWAL
K
Nested
20508843
TQ
02/08/05
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
SITE 3
6497
SITE 3
9805
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 4
9382
Rotavirus
Sample problably positive, to be
veryfied by capillary electrophoresis
SITE 8
8843
SITE 7
4479
50°C
53°C
55°C
57°C
60°C
50°C
53°C
55°C
marcatore
Norwalk virus
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
E.Coli (UFC/100ml)
NaClO → sodium hypochlorite
CH3CO3H → peracetic acid
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
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 because localized in canals with drain water
• Siti SA1 e SA7 problably negative because localized in canals with high waterexchange
• Siti SA8 e SA9 we have considered these sites because near the industrial area of
Marghera and Fusina
SA3
SA4
SA2
SA10
SA5
SA6
SA1
SA9
SA8
SA7
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 & water
Sito
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