Single-Laboratory Validation of the Neogen Qualitative Lateral Flow Immunoassay for the Detection of Paralytic Shellfish Toxins in Mussels and Oysters.

Detection of paralytic shellfish toxins (PSTs) in bivalve shellfish by analytical methods is complicated and costly, requiring specific expertise and equipment. Following extensive blooms of Alexandrium tamarense Group 1 in Tasmania, Australia, an investigation was made into commercially available screening test kits suitable for use with the toxin profiles found in affected bivalves. The qualitative Neogen rapid test kit, with a modified protocol to convert gonyautoxins GTX1&4 and GTX2&3 into neosaxitoxin and saxitoxin (STX), respectively, with higher cross-reactivities, was the best fit-for-purpose. This validation study of the test kit and the modified protocol was undertaken following AOAC INTERNATIONAL guidelines for the validation of qualitative binary chemistry methods. The validation used four different PST profiles representing natural profiles found in Australia and in Europe: two in a mussel matrix and two in an oyster matrix. The test kit was shown to have appropriate selectivity of the toxin analogs commonly found in bivalve shellfish. The matrix and probability of detection (POD) study showed that the rapid test kit used with the modified protocol was able to consistently detect PST at the bivalve regulatory level of 0.8 mg STX⋅2HCl eq/kg, with a POD estimated via the binomial logistic regression of 1.0 at 0.8 mg STX⋅2HCl eq/kg in all tested profiles in both matrixes. The POD at 0.4 mg STX⋅2HCl eq/kg was 0.75 and 0.46 for the two toxin profiles in an oyster matrix and 0.96 and 1.0 for the two toxin profiles in a mussel matrix. No significant differences in the PODs of the PSTs at the regulatory level were found between production lots of the test kits. The results suggest the method is suitable to undergo a collaborative validation study.

Detection of Paralytic Shellfish Toxins in Mussels and Oysters Using the Qualitative Neogen Lateral-Flow Immunoassay: An Interlaboratory Study.

Paralytic shellfish toxins (PSTs) in bivalve molluscs represent a public health risk and are controlled via compliance with a regulatory limit of 0.8 mg saxitoxin (STX)⋅2HCl equivalents per kilogram of shellfish meat (eq/kg). Shellfish industries would benefit from the use of rapid immunological screening tests for PSTs to be used for regulation, but to date none have been fully validated. An interlaboratory study involving 16 laboratories was performed to determine the suitability of the Neogen test to detect PSTs in mussels and oysters. Participants performed the standard protocol recommended by the manufacturer and a modified protocol with a conversion step to improve detection of gonyautoxin 1&4. The statistical analysis showed that the protocols had good homogeneity across all laboratories, with satisfactory repeatability, laboratory, and reproducibility variation near the regulatory level. The mean probability of detection (POD) at 0.8 mg STX⋅2HCl eq/kg using the standard protocol in mussels and oysters was 0.966 and 0.997, respectively, and 0.968 and 0.966 using the modified protocol. The estimated LOD in mussels was 0.316 mg STX⋅2HCl eq/kg with the standard and 0.682 mg STX⋅2HCl eq/kg with the modified protocol, and 0.710 and 0.734 mg STX⋅2HCl eq/kg for oysters, respectively. The Neogen test may be acceptable for regulatory purposes for oysters in accordance with European Commission directives in which the standard protocol provides, at the regulatory level, a probability of a negative response of 0.033 on 95% of occasions. Its use for mussels is less consistent at the regulatory level due to the wide prediction interval around the POD.

Comparative performance of four immunological test kits for the detection of Paralytic Shellfish Toxins in Tasmanian shellfish.

Blooms of the toxic dinoflagellate Alexandrium tamarense (Group 1) seriously impacted the Tasmanian shellfish industry during 2012 and 2015, necessitating product recalls and intensive paralytic shellfish toxin (PST) product testing. The performance of four commercial PST test kits, Abraxis™, Europroxima™, Scotia™ and Neogen™, was compared with the official AOAC LC-FLD method for contaminated mussels and oysters. Abraxis and Europroxima kits underestimated PST in 35-100% of samples when using standard protocols but quantification improved when concentrated extracts were further diluted (underestimation ≤18%). The Scotia kit (cut off 0.2-0.7 mg STX-diHCl eq/kg) delivered 0% false negatives, but 27% false positives. Neogen produced 5% false negatives and 13% false positives when the cut off was altered to 0.5-0.6 mg STX-diHCl eq/kg, the introduction of a conversion step eliminated false negatives. Based on their sensitivity, ease of use and performance, the Neogen kit proved the most suitable kit for use with Tasmanian mussels and oysters. Once formally validated for regulatory purposes, the Neogen kit could provide shellfish growers with a rapid tool for harvesting decisions at the farm gate. Effective rapid screening preventing compliant samples undergoing testing using the more expensive and time consuming LC-FLD method will result in significant savings in analytical costs.