14 Bioaccumulation and Removal Dynamics of Murine Norovirus in Manila Clams. . .
169
Quantification Standards and Viral Quantification
Quantification of MNV-1 was estimated by standard curves constructed with serial
dilutions of viral RNA, plotting the number of genome copies against the C t . This
quantification was corrected with the extraction and qRT-PCR efficiencies and
expressed as number of RNA viral genome copies per gram of digestive tissue
(RNA/g DT). A sample with a C t Ä 41, with no evidence of amplification in the
negative controls, was considered as positive. The number of RNA copies present
in each positive sample was estimated by comparing the C t value of the sample to
the standard curve. All samples were tested in duplicate and with a ten-fold dilution.
The final concentration was then adjusted based on the dilution factor used.
Statistics
ANOVA analysis compared the results of the number of copies of RNA/g DT
obtained among the different depuration times (T 0 –T 7 ) and between the two species
of molluscs. Moreover, post-hoc tests were–employed to determine the statistical
significance of bioaccumulation and the viral reduction detected for each depuration
using the Student-Newman-Keuls (SNK) and Dunnett’s tests. All statistical analyses
were permormed using the statistical package IBM SPSS v20.0.0 software.
Results
Physicochemical parameters of water did not significantly differ for each depuration
cycle, and showed normal levels for the depuration process. The average values
were: conductivity (mS/cm) 49; salinity 30.6‰; pH 8.1; water temperature 14.5
ı C
and dissolved oxygen 7.6 ppm. In addition to these parameters, the concentration of
ammonia, nitrites and nitrates were evaluated, showing no differences among trials.
Ammonia and nitrite concentrations ranged between 0 and 0.3 mg/l except in one
of the experiments where nitrite values reached to 0.8 mg/l. Nitrate concentrations
were between 0 and 50 mg/l.
Experimental depuration trials carried out with mussels, showed a quantification
range at the initial stage (T 0 ) between 4.7and 5.9 logRNA copies/g DT. However,
in one of them (trial 3) higher contamination levels were achieved at T 1 (24 h
of depuration) coinciding with the highest contamination value reached in this
trial (5.6 log RNA copies/g DT) (Table 14.1). All experiments with mussels
showed some reduction in viral quantification. At the end of the depuration (T 7 ),
results showed contamination values between 4.1 and 4.9 log RNA copies/g DT
(Table 14.1, Fig. 14.2). The average reduction for these trials was 0.8 log units
(74 % of reduction in RNA copies/g DT), being statistically significant (p < 0.05)
from 72 h (T 3 ) until the end of the depuration period (T 7 ).
169
Quantification Standards and Viral Quantification
Quantification of MNV-1 was estimated by standard curves constructed with serial
dilutions of viral RNA, plotting the number of genome copies against the C t . This
quantification was corrected with the extraction and qRT-PCR efficiencies and
expressed as number of RNA viral genome copies per gram of digestive tissue
(RNA/g DT). A sample with a C t Ä 41, with no evidence of amplification in the
negative controls, was considered as positive. The number of RNA copies present
in each positive sample was estimated by comparing the C t value of the sample to
the standard curve. All samples were tested in duplicate and with a ten-fold dilution.
The final concentration was then adjusted based on the dilution factor used.
Statistics
ANOVA analysis compared the results of the number of copies of RNA/g DT
obtained among the different depuration times (T 0 –T 7 ) and between the two species
of molluscs. Moreover, post-hoc tests were–employed to determine the statistical
significance of bioaccumulation and the viral reduction detected for each depuration
using the Student-Newman-Keuls (SNK) and Dunnett’s tests. All statistical analyses
were permormed using the statistical package IBM SPSS v20.0.0 software.
Results
Physicochemical parameters of water did not significantly differ for each depuration
cycle, and showed normal levels for the depuration process. The average values
were: conductivity (mS/cm) 49; salinity 30.6‰; pH 8.1; water temperature 14.5
ı C
and dissolved oxygen 7.6 ppm. In addition to these parameters, the concentration of
ammonia, nitrites and nitrates were evaluated, showing no differences among trials.
Ammonia and nitrite concentrations ranged between 0 and 0.3 mg/l except in one
of the experiments where nitrite values reached to 0.8 mg/l. Nitrate concentrations
were between 0 and 50 mg/l.
Experimental depuration trials carried out with mussels, showed a quantification
range at the initial stage (T 0 ) between 4.7and 5.9 logRNA copies/g DT. However,
in one of them (trial 3) higher contamination levels were achieved at T 1 (24 h
of depuration) coinciding with the highest contamination value reached in this
trial (5.6 log RNA copies/g DT) (Table 14.1). All experiments with mussels
showed some reduction in viral quantification. At the end of the depuration (T 7 ),
results showed contamination values between 4.1 and 4.9 log RNA copies/g DT
(Table 14.1, Fig. 14.2). The average reduction for these trials was 0.8 log units
(74 % of reduction in RNA copies/g DT), being statistically significant (p < 0.05)
from 72 h (T 3 ) until the end of the depuration period (T 7 ).