Studies On The Identification And Rapid Detection Method Of Ansakis And Gnathostoma In Aquatic Product

Nematodes belongs to the genus Anisakis or Gnathostoma are important foodborne parasites,and the human cases caused by them are distributed worldwide,posing a great threat to human health.Larval Anisakis spp.mainly parasiticing in marine fish and larval Gnathostoma spp.mainly parasiticing in freshwater fish are main causative agents of anisakiasis and anthostomiasis,respectively,via accidentaly ingestion of raw or under-cooked fish infected by those larvae.The Food and Agriculture Organization of the United Nations(FAO)lists parasites as the first biohazard in aquatic products,including nematodes of genus Anisakis or Gnathostoma which are also important quarantine pests in our county.However the current detection methods for larval Anisakis spp.and Gnathostoma spp.are lagging behind,time-consuming and laborious,which are not suitable for the application to primary level quarantine station.Loop-mediated isothermal amplification(LAMP)and real-time PCR are rapid and accurate detection method with high specificity and sensitivity.In this case,we examined the fish in Guangzhou Airport Port,and established quick and accurate detection methods to provide new ideas for the prevention and control of those parasites.In this study,214 batches of marine fish from 36 countries and regions are examined at Guangzhou Airport Port and 10 batches were infected with larval Anisakis spp..Four Anisakis genotypes were identified: Anisakis simplex sensu stricto,A.pegreffii,A.typica and A.physeteris.19 batches of eels from Indonesia and Philippines were examined and 11 batches were infected with larval Gnathostoma spp..Gnathostoma spinigerum was the most prevalent species and the other was G.hispidum.At the same time,17 G.nipponicum AL3 and 2 G.spinigerum AL3 were isolated from loaches or eels of Zhangzhou city.According to the level of pathogenicity and harm in different species,we established LAMP methods for the detection of Anisakis simplex,A.typica and G.spinigerum.A series of validation experiments was carried out including specificity,sensitivity and actual samples test.The test results indicated that the LAMP approaches possessed high specificity without cross-reations observed.The capable detection of plasmid DNA in approaches was 1 fg/?L.The detection results of the actual samples by the LAMP methods are consistent with that of the traditional methods.We also established real-time PCR methods for the detection of G.spinigerum,as it is widespread pathogen in the Asian region.The specificity and repeatability were tested.Specific amplification products were obtained with G.spinigerum while no amplification products were detected with DNA of related parasites,demonstrating the specificity of the assay.The values of variation coefficients all remain in a low level(<0.2%)in the repeatability test.10-fold serial dilutions of plasmid were used as standard templates to generate standard curve,and the capable detection was less than 10 copies/?l.The data regarding the occurrence of larval Anisakis spp.and larval Gnathostoma spp.in imported fish provided the basis for the relevant departments to formulate relevant policies.The LAMP and real-time PCR detection methods of Anisakis and Gnathostoma can provide more accurate and rapid identification methods for the grassroots.Because they overcome the shortcomings of the traditional identification method of long cycle and tedious steps,getting rid of the dependence of complex instruments and professionals.