Development Of Biological Methods For Detection Of Anisakid Larvae In Seafood

Anisakid larvae, belonged to the family of Anisakidae, are usually regarded as one of the most important pathogenic parasites in seafood. Humans could get the disease called anisakiasis by ingestion the third-phase larvae of these nematodes when eating sashimi and other undercooked, salted or smoked fishery products, such as fish and squid. The larvae, which are the second class animal parasites forbidden by our country, have been found a lot in the imported fish by the entry-exist inspection and quarantine bureau. Now with the fast increasing consumption of raw fish products and the prevalence of tourism, anisakiasis has become a worldwide disease. The risk of people getting anisakiasis is increasing, and the potential hazards of anisakid larvae should arouse enough attention.At present, candling and UV illumination are two commonly exploited techniques in factories for on-site visually inspecting of parasites, but the efficiency of both the methods for detection of parasites embedded deeply in fish muscles were proved very poor. In this study, enzymatic degradation, indirect competitive ELISA and direct competitive ELISA were developed for detection of anisakid larvae in seafood. Main results were listed as following:1. In the study of enzymatic degradation, the digestive efficiency was calculated based on the weight changes of fish muscles during enzymatic degradation. When the ratio of fillets to digestive solutions was 1:10 (g/mL), the optimal setting-off pH was found to be 1.1, the optimal temperature was about 37℃and the enzyme activity should be controlled at 8U/mL to reach best digestive effect. After enzymatic degradation, the nematodes were collected and then identified by multiplex PCR instead of traditional morphological methods, which was then combined with enzymatic process to construct a new analytical system for detection of anisakid larvae in sea foods. The established method was preliminarily validated with real fish samples, and proved to be suitable for confirmatory analysis of Anisakis simplex and Pseudoterranova decipiens in these fishery products. 2. For the development of indirect competitive ELISA, mouse polyclonal antibodies against crude extracts of Anisakis simplex were produced, purified and exploited as receptors. The ELISA procedure was optimized and evaluated in terms of specificity, sensitivity and precision. A broad selectivity of exploited antibodies was observed to Anisakis simplex and Pseudoterranova decipiens, and the lowest detection limits for them were estimated to be 46.9ng/mL and 78.7ng/mL, respectively. The developed immunoassay was tested with fish samples. Within spiking concentrations from 0.5 larvae/100g to 2.0 larvae/100g, the determination recovery for Anisakis simplex and Pseudoterranova decipiens were found in the range of 65.2%～99%, and relative standard deviations were less than 15%.3. Based on specific polyclonal antibodies which were raised against crude extracts of Anisakis simplex purified by Protein A affinity chromatography and labeled with horseradish peroxidase, a direct competitive ELISA was developed and validated for detection of anisakid larvae in seafood. The established method exhibited a broad selectivity to Anisakis simplex and Pseudoterranova decipiens, and the lowest detection limit to them was estimated to be about 0.5 parasite/100g in food matrix. Using Pseudopleuronectes yokohamae, Scomberomrus niphonius and Ommastrephes bartrami as samples and within spiking concentrations from 2 larvae/100g to 10 larvae/100g, the determination recovery for Anisakis simplex and Pseudoterranova decipiens ranged from 77.8% to 107.0%, with relative standard deviations all less than 20%.4. Enzymatic degradation and direct competitive ELISA were used to detect anisakid larvae in southernhake fillets, which had already been inspected by candling and UV illumination. The results showed that anisakid larvae could be detected in all the samples which were positive in candling and UV illumination inspection. When direct competitive ELISA was performed,10 samples considered containing no parasites after candling and UV illumination inspection showed negative results. However, when enzymatic degradation was used to detect another 20 samples considered containing no parasites after candling and UV illumination inspection,2 samples showed positive results and 18 samples showed negative results.5. After the characters and application scope analyzed, the procedure for detection of anisakid larvae was propsed. That is, first, the parasites in seafood are detected and removed by candling and UV illumination, then immunological method and enzymatic degradation is respectively used for rapid screening and confirmatory analysis of the anisakid larvae in seafood.