Study On Novel Methods For Nucleic Acid Detection Of Anisakid Nematodes In Seafood

Anisakiasis is a serious foodborne parasitic disease caused by accidental ingestion of marine fish or squid containing the third-stage larvae of anisakid nematodes.It is listed as a second-class parasitic disease prohibited from entry in China.With the prevalence of raw seafood eating habits,anisakiasis is on the rise.The current industry standard for anisakid nematodes detection in China include morphological identification and PCR sequencing analysis.However,these methods are time-consuming and labour-intensive,highly dependent on well-trained personnel and sophisticated equipment.What’s more,they cannot be applied to point-of-care testing（POCT）.Nucleic acid detection has the advantages of high sensitivity and specificity,and plays an increasingly important role in the detection of foodborne parasites.Therefore,it is of great significance to construct detection methods of nucleic acid with high sensitivity and excellent specificity for the prevention and control of anisakiasis.In this study,111 anisakid nematodes were isolated from four marine fishes with high infection rate of anisakid nematodes.PCR combined with restriction fragment length polymorphism（RFLP）was used to identify the obtained anisakid nematodes.A total of four species of anisakid nematodes were obtained,including A.pegreffii,A.simplex,A.typica and Hysterothylacium,accounting for 83.78%（93/111）,0.90%（1/111）,0.90%（1/111）and 14.41%（16/111）,respectively.The internal transcribed spacer（ITS）region of ribosomal DNA of anisakid nematodes was used as the target sequence to design fluorescent quantitative PCR（qPCR）primers and Taq Man-MGB probes.The recombinant standard plasmid of anisakid nematodes was constructed,and the reaction conditions of qPCR assay were optimized.Finally,it was applied to detect marine fish artificially contaminated with anisakid nematodes.The results showed that the sensitivity of the assay was 1.29×10² copies/μL,and it had excellent specificity and reproducibility.The detection limit of anisakid nematodes was 1 mg/kg in marine fish.In this study,a Taq Man-MGB qPCR method（Taq Man-MGB qPCR）for the detection of anisakid nematodes in seafood was successfully constructed,which was suitable for laboratory quantitative detection.To develop a simple and convenient method for on-site detection of anisakid nematodes in seafood,the primers of recombinase polymerase amplification（RPA）were designed using the ITS region of the ribosomal DNA of anisakid nematodes as the target sequence.The reaction temperature,primers concentration and amplification time of RPA were optimized,and SYBR Green I（SG）dye was used to visualize the RPA amplification products.The sensitivity and specificity of the assay were evaluated by employing the constructed recombinant standard plasmid of anisakid nematodes,finally the assay was applied in the detection of artificial simulation of contaminated samples.The results showed that the sensitivity of the assay for detecting the recombinant standard plasmid was 1.29×10² copies/μL,without non-specific reaction to other related parasites.In addition,the detection limit of anisakid nematodes was 5 mg/kg in marine fish.The detection could be completed within 20 min.What’s more,the results could also be obtained by the naked eye without relying on any precision instruments.In this study,a visual method for rapid detection of anisakid nematodes in seafood based on RPA and SG（RPA-SG）was successfully established,which is appropriate for POCT.Recently,increasing demands for POCT have been put forward for nucleic acid detection.CRISPR/Cas associated biosensors have been shown to have great potential in sensing applications due to their high sensitivity and high base resolution.Two-dimensional nanomaterials,e.g.,molybdenum disulfide nanosheets（MoS₂ NSs）with unique optical,electrical,and electrochemical properties and low preparation cost,which could be employed to address the need for well-designed fluorescence donors and acceptors for single-stranded DNA（ss DNA）for the biosensors of nucleic acid detection based on the CRISPR/Cas system.In this study,we explored the differences in the adsorption capacity of MoS₂ NSs for ss DNA of various lengths and the compatibility of the fluorescence resonance energy transfer（FRET）system of MoS₂NSs-FAM-ss DNA with the CRISPR/Cas12a system.Then some reaction conditions were optimized.Finally,the developed method was applied to the detection of anisakid nematodes in seafood.The results showed that the sensitivity of the constructed MoS₂NSs-CRISPR/Cas12a assay was 1.29×10² copies/μL with the aid of RPA,and with good specificity and reproducibility.In addition,the assay could achieve visual detection within 35 min.The probe cost was saved by about 90.7%using the MoS₂NSs-FAM-ss DNA,and the detection time was reduced by 20 min.In this study,a universal nucleic acid detection platform,namely,MoS₂ NSs-CRISPR/Cas12a was successfully fabricated,which not only provides strong technical support for on-site detection of anisakid nematodes,but also provides a new idea for the detection of foodborne parasites.