Efficiency of the Q3 lab-on-chip Real Time-PCR platform for detecting protozoan pathogens in bivalve mollusks
The zoonotic protozoan parasites Toxoplasma gondii, Cryptosporidium parvum and Giardia duodenalis have been recorded worldwide in economically important edible shellfish, and are thus likely to represent a significant public health risk. Therefore, an innovative, user-friendly diagnostic tool is required in order to improve food safety control. The Q3 system is a miniaturized platform whose efficiency and applicability were investigated and compared with results obtained using standard Real-Time PCR. Tanks of saltwater containing acclimated Mytilus galloprovincialis, Ruditapes philippinarum and Ostrea edulis specimens were spiked with purified Cryptosporidium, Giardia and Toxoplasma cysts/oocysts at different concentrations (i.e., 103, 104 and 105). We then collected 30 specimens for each shellfish species from each group at 24 h and 72 h post-contamination. After DNA extraction, we tested all samples by Real-Time-PCR and Q3, and evaluated the sensitivity, specificity, predictive values, repeatability and concordance between the two systems. Concordance between Real-Time-PCR and Q3 was very good (p < 0.01), especially for Toxoplasma in M. galloprovincialis at both 24 h and 72 h after contamination, and in O. edulis at 72 h. The ability of Q3 to detect all the investigated pathogens was similar to that of Real-Time-PCR, and Q3 was efficient in detecting Toxoplasma in both M. galloprovincialis and O. edulis. This is the first study concerning the use of lab-on-chip technology in a food matrix, and in edible marine mollusks in particular.
KeywordsProtozoans Shellfish Real-Time PCR Lab-on-chip efficiency Food safety
The study was funded by “New Strategies for Improvement of Food Safety: Prevention, Control, Correction” (S.I.Mi.S.A.)—PON02_00186_3417512—PON Ricerca e Competitività 2007–2013” (PO Puglia FESR 2007e2013 Asse I, Linea 1.2dPO Puglia FSE 2007e2013 Asse IV). The authors wish to thank Anna Lass of the Department of Tropical Parasitology, Medical University of Gdansk (Poland), and Anja Joachim of the Institute of Parasitology, University of Wien (Austria) for providing Toxoplasma oocyst strains, Tiziana Caradonna for her helpful work in the lab, Tommaso Marazia for his outstanding assistance in aquarium system management, and Alessandra Barlaam and Sarah Christopher for the English revision of the MS.
AG, GN and DO conceived the study design; LP performed in vitro culture; FF and MC developed the Q3 system and trained for the Q3 analysis; MM, MSL and GA collected samples and performed the Real Time and Q3 analysis; GC performed the statistical analysis; AG, GN, MM, LP, MC and DO interpreted the data and wrote the paper. All authors contributed to editing the manuscript. All authors read and approved the final version of the manuscript.
- Aksoy U, Marangi M, Papini R, Ozkoc S, Bayram Delibas S, Giangaspero A (2014) Detection of Toxoplasma gondii and Cyclospora cayetanensis in Mytilus galloprovincialis from Izmir Province coast (Turkey) by Real time PCR/High-Resolution Melting analysis (HRM). Food Microbiol 44:128–135CrossRefGoogle Scholar
- Biava M, Colavita F, Marzorati A, Russo D, Pirola D, Cocci A, Petrocelli A, Delli Guanti M, Cataldi G, Kamara TA, Kamara AS, Konneh K, Cannas A, Coen S, Quartu S, Meschi S, Valli MB, Mazzarelli A, Venditti C, Grassi G, Rozera G, Castilletti C, Mirazimi A, Capobianchi MR, Ippolito G, Miccio R, Di Caro A (2018) Evaluation of a rapid and sensitive RT-qPCR assay for the detection of Ebola Virus. J Virol Methods 252:70–74CrossRefGoogle Scholar
- Coupe A, Howe L, Burrows E, Sine A, Pita A, Velathanthiri N, Vallée E, Hayman D, Shapiro K, Roe WD (2018) First report of Toxoplasma gondii sporulated oocysts and Giardia duodenalis in commercial green-lipped mussels Perna canaliculus in New Zealand. Parasitol Res 117:1453–1463CrossRefGoogle Scholar
- Eurostat (2015) http://ec.europa.eu/eurostat/statistics-explained/index.php/Aquaculture_statistics/. Accessed 13 Dec 2018
- FAO (2015) Fisheries and aquaculture statistics http://www.fao.org/3/a-i7989t.pdf/. Accessed 13 Dec 2018
- FAO-WHO (2014a) http://apps.who.int/iris/bitstream/10665/112672/1/9789241564700_eng.pdf/. Accessed 13 Dec 2018
- FAO-WHO (2014b) http://www.fao.org/tempref/codex/Meetings/CCFFP/ccffp34/Shellfish%20Sanitation%20Initiation%20meeting%20Report%20%20final%20for%20CCFFP.pdf. Accessed 13 Dec 2018
- Marziliano N, Notarangelo MF, Cereda M, Caporale V, Coppini L, Demola MA, Guidorossi A, Crocamo A, Pigazzani F, Boffetti F, Del Giudice F, Orsini F, Pirola D, Cocci A, Manzalini C, Casu G, Bianchessi M, Ardissino D, Merlini PA (2015) Rapid and portable, lab-on-chip, point-of-care genotyping for evaluating clopidogrel metabolism. Clin Chim Acta 451:240–246CrossRefGoogle Scholar
- Putignani L, Mancinelli L, Del Chierico F, Menichella D, Adlerstein D, Angelici MC, Marangi M, Berrilli F, Caffara M, di Regalbono DA, Giangaspero A (2012) Investigation of Toxoplasma gondii presence in farmed shellfish by nested-PCR and real-time PCR fluorescent amplicon generation assay (FLAG). Exp Parasitol 127:409–417CrossRefGoogle Scholar