Study On Rapid Tracing Identification And Toxin Content Determination Of Common Pufferfish Poisoning

Objective:DNA barcoding technology was used for species identification of pufferfish,and an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for detection of tetrodotoxin(TTX)was established to conduct species identification and toxin detection of the collected pufferfish samples,so as to realize the effective link between gene,species,and toxin,and provide technical support for rapid traceability,identification and clinical treatment of pufferfish food poisoning.Methods:1 Pufferfish samples collection and morphological identification: Marine biology experts were commissioned to collect puffer fish in the Yellow Sea,the East China Sea,and the South China Sea from the autumn of 2019 to the spring of 2020,and were invited to preliminarily identify their species through morphology.2 Application of DNA barcoding technology in species identification of pufferfish: The collected samples were thoroughly cleaned,and the fish body was dissected into skin,muscle,liver,and ovary.After the tissues were fully homogenized,put them into a clean container,and the coded marks were packed and stored for later use.DNA was extracted from 50 mg pufferfish muscle tissue by kit method,and mitochondrial COI gene was amplified by PCR.The product was purified and sequenced.MUSIC algorithm was used for sequence alignment,and the 655 bp sequence of the 5 ’end of the gene was obtained after manual deletion.BLASTN tool was used for sequence alignment to determine the fish species.The K2 P substitution model was used to construct the adjacent tree,and the preliminary phylogenetic analysis was carried out to determine the family and genus.The simulated gastric juice was prepared.1.0 g of homogenous muscle tissue was put into 5.0 ml of simulated gastric juice and reacted in a 37℃ water bath at a constant temperature.The practicability of the technique in tracing the toxic source of pufferfish poisoning was investigated.3 Establishment of a laboratory method for detection of TTX(UPLC-MS/MS):According to the national standard(GB 5009.206-2016)liquid chromatography-tandem mass spectrometry,and combined with the actual laboratory conditions,the chromatographic column was adjusted.3.1 The establishment of instrument detection method: Waters ACQUITY UPLC BEH Amide(100 mm × 2.1 mm,1.7 mm)hydrophilic column was used as the separation column.The mobile phase was acetonitrile and 0.1% formic acid solution containing 5mmol/L ammonium acetate,gradient removed 8min,and monitored by ion spray source and selective reaction monitoring(SRM)positive ion mode.The retention time was used for qualitative analysis and the external standard method for quantitative analysis.3.2 TTX separation and extraction method: Accurately weigh 3.00 g homogenous sample,use acetic acid methanol(1:99,v/v)solution as extraction solvent,extract in 50℃water bath ultrasonic environment for 20 min,centrifuge and take the supernatant,and repeat the operation once.The supernatant was purified by TTX immunoaffinity column with p H6.5 ～ 7.5.The supernatant was eluted with primary water and methanol acetate(2:98,v/v)solution.The eluate was dried by nitrogen,and then redissolved with 0.1% formic acid water acetonitrile solution(1:1,v/v),ultrasonically,and through 0.22 μm organic phase microporous membrane.3.3 Methodological evaluation: the linear relationship,correlation coefficient,accuracy,stability,peak time,and reproducibility of the established method was verified to determine the detection limit of the method.The recovery and precision of TTX were calculated.3.4 Sample detection: TTX content of all puffer venom fish samples was detected by the above established method.Results:1 Through morphological identification,46 samples of 18 species of 6 genera were collected.2 The 46 pufferfish samples collected belonged to 19 species,7 genera,3 families.All the samples were clustered by species name on the adjacent tree,and the branches formed within the genus had high support rate,which was consistent with the morphological identification results.Mitochondrial COI gene was detected in the muscle tissue residue of puffer venom fish digested by simulated gastric juice.After amplification,purification and sequencing of the gene,the species alignment results were completely consistent with the corresponding species of undigested samples.3 The limit of detection and limit of quantitation of the UPLC-MS / MS method was1μg/kg and 3μg/kg,respectively.The correlation coefficient of the standard working curve was 0.99981,the precision was 0.16% ～ 9.52%,and the recoveries were 86.72% ～ 104.39%.TTX was detected in all samples.The skin toxin content of Canthigaster papua(86277.51 μg/kg)was the highest,and that of Ostracion solorensis(101.46 μg/kg)was the lowest.Takifugu niphobles(104335.80 μg/kg)was the highest,and Ostracion solorensis(87.45μg/kg)was the lowest.The liver toxin content of Takifugu alboplumbeus(75107.21μg/kg)was the highest,and that of Ostracion immaculatus(45.31 μg/kg)was the lowest.The highest content of ovarian toxin was found in Arothron hispidus(88335.30 μg/kg)and the lowest in Takifugu obscurus(73.55 μg/kg).The highest weighted average toxin content in Takifugu was Takifugu niphobles(81904.41 μg/kg),Takifugu xanthopterus(2115.38 μg/kg)was the lowest.Canthigaster papua(81874.89 μg/kg)was the most toxic among the three species of Canthigaster.The highest weighted average toxin content in Ostracion was Ostracion cubicus(5733.74 μg/kg).Arothron hispidus(73996.97 μg/kg)was the most toxic among the three species of Arothron.Conclusion:In this study,655 bp of the mitochondrial COI gene was used to trace and identify common puffer species.This method has the advantages of trace,high efficiency,and accuracy.The established UPLC-MS/MS method has the advantages of high signal-to-noise ratio and high sensitivity,which can realize the high-throughput analysis and trace detection of TTX.Two methods were used for species identification,TTX content and distribution detection and analysis of common pufferfish samples collected.The effective link between gene,fish species,and toxin was realized,which laid a foundation for the establishment of pufferfish species information and toxin content database.