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Volume 24
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10.3390/md24090314
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Article

Optimization and Intra-Laboratory Validation of the Neuro-2a Assay for Tetrodotoxin Detection in Mussels

by
Alessandra D’Arelli
1,
Silvio Sosa
1,
Sonia Dall’Ara
2,
Monica Cangini
2,
Michela Carlin
1,
Pietro Antonelli
3,4,
Aurora Dall’Occo
3,
Nicolas Scapin
5,
Giuseppe Arcangeli
5,
Carmen Losasso
3,
Aurelia Tubaro
1 and
Marco Pelin
1,*
1
Department of Life Sciences, University of Trieste, Via A. Fleming 22, 34127 Trieste, Italy
2
National Reference Laboratory for Marine Biotoxins, Fondazione Centro Ricerche Marine, V.le A. Vespucci 2, 47042 Cesenatico, Italy
3
Laboratory of Microbial Ecology and Genomics, Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Università 10, 35020 Legnaro, Italy
4
PhD National Programme in One Health Approaches to Infectious Diseases and Life Science Research, Department of Public Health, Experimental and Forensice Medicine, University of Pavia, 27100 Pavia, Italy
5
National Reference Center for fish, Mollusc and Crustacean Diseaseas, Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Università 10, 35020 Legnaro, Italy
*
Author to whom correspondence should be addressed.
Mar. Drugs 2026, 24(9), 314; https://doi.org/10.3390/md24090314
Submission received: 31 July 2026 / Revised: 2 September 2026 / Accepted: 4 September 2026 / Published: 8 September 2026
(This article belongs to the Section Marine Toxins)
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Abstract

Tetrodotoxin (TTX) is a potent marine neurotoxin responsible for severe seafood poisoning in humans, characterized by neurological symptoms that may be fatal. Originally identified as a natural contaminant of pufferfish (Tetraodontidae family), over the last few years TTX and its analogs have also been detected in other edible marine organisms, including mollusks, gastropods and crustaceans. Consequently, there is a need for rapid, sensitive, and reliable methods for TTX detection in seafood. In this study, a functional assay based on the use of mouse neuroblastoma Neuro-2a cells has been optimized and characterized for TTX detection in mussels. The assay is based on the toxin’s ability to block voltage-gated sodium channels, thereby counteracting the sodium-dependent cytotoxicity induced by veratridine and ouabain. The linear range of the TTX standard curve fell between 0.44 and 33 ng/mL, with limits of TTX detection (LOD) and quantitation (LOQ) of 0.132 ng/mL and 0.439 ng/mL, respectively, and good intra- and inter-day repeatability (RSDr= 15 and 11%, respectively). The assay also detected saxitoxin, which shares the same mechanism of action as TTX, but was less sensitive towards 4,9-anhydro-TTX. The minimum mussel extract dilution of 1:100 did not result in matrix-related interference, allowing accurate TTX quantitation, with a LOQ of 0.54 µg TTX equivalents/kg mussel meat. Given its sensitivity, the optimized Neuro-2a assay represents a promising tool for toxicity-based TTX quantitation in mussels before their consumption.
Keywords: tetrodotoxin; neuroblastoma cells; food safety; toxicity; detection method tetrodotoxin; neuroblastoma cells; food safety; toxicity; detection method

Share and Cite

MDPI and ACS Style

D’Arelli, A.; Sosa, S.; Dall’Ara, S.; Cangini, M.; Carlin, M.; Antonelli, P.; Dall’Occo, A.; Scapin, N.; Arcangeli, G.; Losasso, C.; et al. Optimization and Intra-Laboratory Validation of the Neuro-2a Assay for Tetrodotoxin Detection in Mussels. Mar. Drugs 2026, 24, 314. https://doi.org/10.3390/md24090314

AMA Style

D’Arelli A, Sosa S, Dall’Ara S, Cangini M, Carlin M, Antonelli P, Dall’Occo A, Scapin N, Arcangeli G, Losasso C, et al. Optimization and Intra-Laboratory Validation of the Neuro-2a Assay for Tetrodotoxin Detection in Mussels. Marine Drugs. 2026; 24(9):314. https://doi.org/10.3390/md24090314

Chicago/Turabian Style

D’Arelli, Alessandra, Silvio Sosa, Sonia Dall’Ara, Monica Cangini, Michela Carlin, Pietro Antonelli, Aurora Dall’Occo, Nicolas Scapin, Giuseppe Arcangeli, Carmen Losasso, and et al. 2026. "Optimization and Intra-Laboratory Validation of the Neuro-2a Assay for Tetrodotoxin Detection in Mussels" Marine Drugs 24, no. 9: 314. https://doi.org/10.3390/md24090314

APA Style

D’Arelli, A., Sosa, S., Dall’Ara, S., Cangini, M., Carlin, M., Antonelli, P., Dall’Occo, A., Scapin, N., Arcangeli, G., Losasso, C., Tubaro, A., & Pelin, M. (2026). Optimization and Intra-Laboratory Validation of the Neuro-2a Assay for Tetrodotoxin Detection in Mussels. Marine Drugs, 24(9), 314. https://doi.org/10.3390/md24090314

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