Studies On The Identification Method Of Anisakid Nematodes In Marine Fish

Anisakids were often found in most of marine fishes around the world. Human anisakiasis is caused by eating some larvae of the nematodes of anisakidae. It has been reported that six nematodes of anisakids such as Anisakis simplex, A. typica, A. physeteris, Psetwloterranova decipiens, Contracaccum spp. and Hysterothylacium spp. can be infected by the people. Because Anisakias are marine natural focal pathogens, with the increasing popularity of eating undercooked or raw fish dishes, human anisakiasis has shown a tendency to spread around the world. The infection of anisakiasis pathogen has important public health significance. In 1993, anisakiasis was listed in the"prohibited the entry of animal infectious diseases and parasitic diseases list of The People's Republic of China". Due to the great varieties of anisakis and their different pathogenic to human, the accurate method to identify the species of Anisakis is the premise and foundation in the study of anisakis. It also has the great significance in the prevention and treatment of anisakiasis infected by human and animal.In recent years, the anisakis larvae infected by the marine fishes which imported to Xiamen and sold in Xiamen supermarket were investigated. It showed that the anisakis are often found in the fishes of Decapterus maruadsi, Taius tumifrons and Trachurus japonicus, and Muraenesox cinereus, Trichiurus haumela and Priacanthus macracanthus have the high infection rates too. The parasitization rate of A. simplex in Decapterus maruadsi and Trachurus japonicus can be reached 100%. In this study, the preliminary identification of the anisakis larvae was carried out by using morphological method. The sequences of the internal transcribed spacer (ITS) of Anisakis nematodes were amplified by using of conserved primers. The amplicons were purified, reclaimed and cloned to pGM-T Vector, and then the positive clones were sequenced. According to the rusult of ITS sequences, A. simplex, A. tipica, Raphidascaris trichiuri and Contracaccum sp were identified. The amplicons digested by HinfⅠand RsaⅠselected according to the analysis of ITS sequences, followed by agarose gel electrophoresis analysis. It has been proved that the PCR-RFLP method was accurate, specific and simple to identify those nematodes above.A. simplex is the dominant species of anisakis in marine fishes in China, and also the major pathogen of human anisakiasis. So it is important to prevent this Anisakis. According its ITS gene sequence, a set of specific primers were designed to amplify the special DNA sequence by the technique of LAMP and fluorescent quantitative PCR using SYBR GreenⅠ. Moreover, the concentration of Mg2+, Betaine, dNTPs and primers were optimized. The sensitivity and specificity of the method were also tested. Then 7 samples of A. simplex were validated by LAMP. The optimal reaction system contained 0.8 mmol/L dNTPs, 10mmol/L Mg2+, 0.8mmol/L betaine, 0.2μmol/L exogenous primers, 1.6μmol/L inner primers and 8U Bst DNA polymerase. The optimal reaction temperature was 62℃and reaction time was 60 min. The detection limit for A. simplex DNA template was 10 copies/μl and no cross reactions with A. tapica, Contracaccum sp, Raphidascaris trichiuri were found. All of the 7 parasites of A. simplex were positive in the LAMP test. The r values of the standard curve from the fluorescent quantitative PCR test using SYBR GreenⅠachieved 0.999, it was specific and no amplicons with the nematodes of A. typica, Contracaccum sp or Raphidascaris trichiuri were detected. It can detect as low as 10 copies/μl of the DNA template. The two techniques to identify the parasite of A. simplex are sensitive and specific, and the LAMP test is more rapid, economic and effective.A.typica is also the major pathogen of Anisakiasis, but it has few reports in our country. In this paper, the method of real-time fluorescent quantitative PCR using SYBR GreenⅠto detect A. typica was established. It had also been proved as a rapid, specific, sensitive and quantifiable method to identify the pathogen of A. typica.The technique of LAMP and real-time fluorescent quantitative PCR established in this study to detect and identify the nematodes of anisakids in marine fishes are firstly reported in the world. The methods of PCR and PCR-RFLP to detect the nematodes of anisakids had also been studied, and the rapid detection techniques for the four anisakids A. simplex, A. tipica, Raphidascaris trichiuri and Contracaccum sp had also been established .