Cryptosporidium, Giardia and Toxoplasma have been recorded worldwide in economically important edible shellfish, and are thus likely to present a significant public health risk. The development of an innovative, user-friendly diagnostic tool is required to improve food safety control. The Q3 system, a miniaturized platform which integrates amplification and detection process for realtime-PCR on a Lab-on-chip, was developed by STMicroelectronics for the detection of zoonotic protozoans in three shellfish species according to realtime-PCR protocols previously set up in our lab. Q3’s efficiency and applicability were investigated and compared with results obtained by standard realtime-PCR. Tanks of saltwater containing acclimated pathogen-free Mytilus galloprovincialis, Tapes semidecussatus and Ostrea edulis specimens were spiked with purified Cryptosporidium, Giardia and Toxoplasma cysts/oocysts at different concentrations (i.e., 103, 104 and 105). Untreated control shellfish were included in each test. At 24h and 72h post-contamination (p.c.), thirty specimens of each shellfish species were collected from each group. Haemolymph, gills and gastric glands were removed and stored in pools at -20°C. After DNA extraction, all samples were tested by standard realtime-PCR and Q3, and we evaluated the sensitivity, specificity, predictive values, repeatability and the concordance between standard realtime-PCR and Q3-system. A significant concordance between standard realtime-PCR and Q3-system CT (Threshold Cycle) values was registered for all the shellfish species at the highest parasite load (i.e. 105) at 72h p.c. for Toxoplasma and Giardia in M.galloprovincialis, for Toxoplasma and Giardia in O.edulis and for Cryptosporidium in T.semidecussatus (P<0.05). No significant differences were registered between the anatomical sites. Q3 demonstrated an ability to detect all investigated pathogens, that was similar to standard realtime-PCR, and a very high level of ability to detect Toxoplasma in M.galloprovincialis and Toxoplasma and Giardia in O.edulis. Currently, the Q3-system can be used efficiently to detect Toxoplasma from whole mussels and oysters, and to a lesser extent also from clams.
Efficiency of the Q3 lab-on-chip platform in detecting protozoan pathogens in bivalve mollusks
GIANGASPERO, ANNUNZIATA;Marangi, Marianna;NORMANNO, GIOVANNI GIUSEPPE;
2016-01-01
Abstract
Cryptosporidium, Giardia and Toxoplasma have been recorded worldwide in economically important edible shellfish, and are thus likely to present a significant public health risk. The development of an innovative, user-friendly diagnostic tool is required to improve food safety control. The Q3 system, a miniaturized platform which integrates amplification and detection process for realtime-PCR on a Lab-on-chip, was developed by STMicroelectronics for the detection of zoonotic protozoans in three shellfish species according to realtime-PCR protocols previously set up in our lab. Q3’s efficiency and applicability were investigated and compared with results obtained by standard realtime-PCR. Tanks of saltwater containing acclimated pathogen-free Mytilus galloprovincialis, Tapes semidecussatus and Ostrea edulis specimens were spiked with purified Cryptosporidium, Giardia and Toxoplasma cysts/oocysts at different concentrations (i.e., 103, 104 and 105). Untreated control shellfish were included in each test. At 24h and 72h post-contamination (p.c.), thirty specimens of each shellfish species were collected from each group. Haemolymph, gills and gastric glands were removed and stored in pools at -20°C. After DNA extraction, all samples were tested by standard realtime-PCR and Q3, and we evaluated the sensitivity, specificity, predictive values, repeatability and the concordance between standard realtime-PCR and Q3-system. A significant concordance between standard realtime-PCR and Q3-system CT (Threshold Cycle) values was registered for all the shellfish species at the highest parasite load (i.e. 105) at 72h p.c. for Toxoplasma and Giardia in M.galloprovincialis, for Toxoplasma and Giardia in O.edulis and for Cryptosporidium in T.semidecussatus (P<0.05). No significant differences were registered between the anatomical sites. Q3 demonstrated an ability to detect all investigated pathogens, that was similar to standard realtime-PCR, and a very high level of ability to detect Toxoplasma in M.galloprovincialis and Toxoplasma and Giardia in O.edulis. Currently, the Q3-system can be used efficiently to detect Toxoplasma from whole mussels and oysters, and to a lesser extent also from clams.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
