河鲀中河鲀毒素检测研究进展

河鲀毒素(tetrodotoxin,TTX)是一种毒性极强的天然小分子生物碱,无特效解毒剂,中毒潜伏期短,在水产品质量安全领域受到极大关注。本文总结了河鲀毒素的毒性、相关法规、化学性质和反应特点,并对河鲀毒素检测过程中样品前处理以及检测方法进行了总结,并详细综述了河鲀毒素常用的生物检测法、液相色谱荧光检测法、气相色谱串联质谱法、液相色谱串联质谱检测法和免疫及电化学检测方法应用情况,尤其对免疫检测与电化学技术结合的方法进行了分类分析。最后,本文分析了免疫分析过程中竞争型和非竞争型免疫的区别与优缺点,并对新型电化学免疫快速检测的方法开发进行了展望,以期为检测TTX等小分子化合物的新型检测技术的开发提供参考。     

COI及cytb基因对河鲀鱼鱼种鉴定的适用性研究

目的建立河鲀鱼DNA条形码鉴定方法,探讨细胞色素氧化酶亚基I(COI)及细胞色素b(cytb)基因对我国常见东方鲀属、兔头鲀属河鲀鱼鱼种鉴定的适用性。方法野捕河鲀鱼经形态学鉴定后,钓取COI及cytb基因序列并测序。从Gen Bank下载已有河鲀鱼参考序列,分别构建COI及cytb基因分子进化树,确定样品种属并与形态学鉴定比对。应用所建方法对中毒样品进行河鲀成分鉴定。结果 COI和cytb基因分子进化树将57份样品聚类到东方鲀属和兔头鲀属的9个鱼种,除棕斑兔头鲀和暗鳍兔头鲀(COI进化树)、暗纹东方鲀和晕环东方鲀(cytb进化树)外,2种条形码均能对其余鱼种进行有效区分。中毒样品经鉴定均含有月兔头鲀。结论所建立的DNA条形码方法可有效鉴定河鲀鱼鱼种,弥补形态学鉴定的缺陷。     

超高效液相色谱-串联质谱法检测河鲀毒素

目的建立准确、快速检测河鲀毒素(tetrodotoxin,TTX)的超高效液相色谱-串联质谱(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)的分析方法,了解市售烤鱼片、鱿鱼干制品和鱼丸中河鲀毒素污染状况。方法样品经2%乙酸/甲醇溶液超声离心提取后,通过MCX固相萃取柱进行净化处理,采用液相色谱串联质谱法进行检测。结果市售的107份样品中,鱿鱼干制品和鱼丸中没有检出TTX;64份烤鱼片样品中检出16份含有TTX,检出率为25.0%。结论部分烤鱼片中TTX呈阳性,建议生产厂家严把原料关,监管部门应加强对市售烤鱼片标签标识的监管。     

不食河鲀 不知鱼味

正千百年来,在民间一直流传着"不食河鲀不知鱼味,食了河鲀百鱼无味""一朝食得河鲀肉,终生不念天下鱼"。爱好美食的人,每年都有两个翘首期待的时节,金秋伺螃蟹,暮春候河鲀。所谓"拼死吃河鲀",绝不是一句空话。品味河正当时河鲀的常见品种有:虫纹东方鲀、横纹东方鲀、铅点东方鲀、月腹刺鲀、红鳍东方鲀、暗纹东方鲀等。     

液相色谱串联质谱法测定河豚毒素标准品

河鲀毒素(TTX)是河鲀鱼类体内合成的一种有毒的生物碱,是目前人类发现的自然界中毒性最大的神经毒素之一。本文在3家公司的质谱仪上分别建立了测定河豚毒素标准品的仪器方法,可为用不同公司生产的仪器测定河豚毒素提供参考。     

基于Cycling探针的实时荧光聚合酶链式反应检测河鲀鱼中掺杂的横纹东方鲀成分

摘 要：目的 开发基于Cycling探针实时荧光聚合酶链式反应(real-time fluorescent polymerase chain reaction，RT-PCR)用于检测河鲀鱼中横纹东方鲀成分。方法 基于细胞色素C氧化酶亚型I (cytochrome C oxidase subunit I, COI)基因为靶标设计RT-PCR引物及Cycling探针，开发横纹东方鲀成分检测方法，评价方法的特异性、扩增效率、灵敏度和稳定性。结果 横纹东方鲀成分与密切相关的其他8种河鲀鱼、4种其他鱼类物种DNA无交叉反应，具有很好的特异性；方法扩增效率为93.47%；方法重复18次均给出阳性报告的最小稀释点(limit of detection 6，LOD6)为14.64 pg/μL(相对应的质量含量为0.1%)，95%置信水平条件下检出限为34.41 pg/μL (11.59~119.05 pg/μL，LOD95%)，稳定性分析(P=0.152)表明该方法可转移到其他实验室以及在常规分析中使用。结论 本研究开发的Cycling探针RT-PCR方法可对河鲀鱼食品中掺杂0.1%的横纹东方鲀成分进行可靠追溯。 关键词：河鲀鱼; 食品真实性；横纹东方鲀；Cycling探针；实时荧光聚合酶链式反应     

河鲀鱼基质中河豚毒素标准样品的研制

目的 研制基于河鲀鱼基质的河豚毒素(tetrodotoxin,TTX)定量分析标准样品.方法 以野生河鲀鱼肝脏、卵巢、精巢和肌肉组织为基料,利用正己烷脱脂分离处理后,经两次冷冻干燥制备河鲀鱼基质的TTX标准样品,采用液相色谱-串联质谱法进行定性确证.样品分装成500瓶后,进行均匀性、稳定性检验和定值分析.结果 经确证该...     

免疫亲和柱净化-亲水液相色谱-串联质谱法测定水产食品中河鲀毒素

目的本文采用免疫亲和柱选择性吸附样品溶液中的河鲀毒素,建立了测定水产食品中河鲀毒素(TTX)的亲水液相色谱-串联质谱分析方法。方法样品以含1%乙酸的甲醇溶液提取,磷酸盐缓冲溶液稀释,再经免疫亲和柱富集和净化后进样分析。目标物以TSK-gel Amide-80亲水色谱柱(150 mm×2.0 mm,5μm)分离,乙腈-0.1%甲酸水溶液(含5 mmol/L乙酸铵)梯度洗脱,采用电喷雾离子源,选择反应监测(SRM)正离子模式检测,溶剂标准曲线校正,外标法定量。结果 TTX在1~1 000μg/L范围内线性良好,方法的检出限为1μg/kg,定量限为3μg/kg,在3~300μg/kg范围内加标回收率为73.6%~95.2%,相对标准偏差(RSD)为5.37%~10.7%。结论该方法可有效消除复杂基质样品中普遍存在的基质抑制效应,操作简便,色谱保留时间稳定,灵敏度高,准确度和重复性好,适用于烤鱼片、风味鱼干等水产食品中河鲀毒素的测定。     

河鲀毒素生物传感器的研究

基于河鲀毒素(TTX)对细胞膜Na+通道的阻断作用,建立一种检测TTX的简易组织生物传感器。组织生物传感器由pNa电极、青蛙膀胱膜、电位计等构成。在pNa电极顶端用两层醋酸纤维薄膜包裹,中间夹入青蛙膀胱膜,构成感受器。将电极插入pNa=4的NaCl溶液中,使电极输出信号趋于稳定。将TTX被测液注入感受器系统,测定感受器输出量的抑制值。每次测定只需5min。最低检出限为0.002MU/ml(3.56×10-3μg/ml)。用0.003%Na3N保存,可连续使用250h。以生物传感器和小鼠生物试验法比较,建立了工作曲线,便于在餐饮企业对原料鱼进行检测和应用。     

基质分散固相萃取净化-亲水液相色谱-串联质谱法检测织纹螺与贝类中河鲀毒素

目的针对海产品常见中毒原因分析需求,建立基质分散固相萃取净化-亲水液相色谱-串联三重四极杆质谱(HILIC-MS/MS)快速定性定量检测织纹螺和贝类中河鲀毒素的新方法。方法 1.0 g样品经0.1%乙酸溶液沸水浴提取后,用50 mg亲水亲油平衡填料(HLB)和5 mg石墨化碳黑(GCB)吸附剂吸附净化,最后经乙腈蛋白沉淀后过0.22μm聚四氟乙烯(PTFE)滤膜,亲水液相色谱柱(150 mm×2.0 mm,3μm)分离,电喷雾离子化,选择反应监测(SRM)模式检测,基质匹配外标法定量。结果在2.0~40.0 ng/ml浓度范围内,河鲀毒素呈现良好的线性关系,相关系数r~2≥0.999;以3倍基线噪声所对应的浓度为检出限时,河鲀毒素的方法检出限可达10.0μg/kg;在25、100和200μg/kg的加标水平时,方法回收率为74.2%~87.9%,相对标准偏差为2.3%~9.1%。应用本方法对浙江沿海地区市售织纹螺和贝类样品进行检测,15份织纹螺中有14份检出河鲀毒素,检出率为93.3%,含量范围为0.04~15.75 mg/kg,60份贝类样品均未检出河鲀毒素。结论该检测方法准确、快速、易操作,能满足典型海产品中河鲀毒素的公共卫生应急检测或日常监测要求。     

Tetrodotoxin poisoning due to smooth-backed blowfish Lagocephalus inermis and toxicity of L. inermis caught off the Kyushu coast, Japan

Food poisoning due to ingestion of a puffer fish occurred in Nagasaki Prefecture, Japan, in October 2008, causing neurotoxic symptoms similar to those of tetrodotoxin (TTX) poisoning. In the present study, we identified the species, toxicity, and toxins using the remaining samples of the causative puffer fish. The puffer fish was identified as smooth-backed blowfish Lagocephalus inermis by nucleotide sequence analysis of the 16S rRNA and cytochrome b gene fragments of muscle mitochondrial DNA. The residual liver sample showed toxicity as high as 1,230 mouse unit (MU)/g by bioassay and TTX was detected by liquid chromatography/mass spectrometry analysis. We therefore concluded that the food poisoning was due to TTX caused by consumption of the toxic liver of L. inermis. This is the first report that the liver of L. inermis caught in Japanese waters is strongly toxic, with levels exceeding 1,000 MU/g. In this context, we re-examined the toxicity of L. inermis collected off the coast of Japan. Of 13 specimens assayed, 12 were toxic, although the toxicity varied markedly among individuals and tissues. Because the intestine and ovary of L. inermis have been considered non-toxic, it is particularly noteworthy that these organs were determined to be toxic, with a maximum toxicity of 43.6 MU/g and 10.0 MU/g, respectively. Furthermore, kidney, gallbladder, and spleen, whose toxicity has been unknown, were frequently found to be weakly toxic with levels ranging from 10 to 99 MU/g. Therefore, further study is needed to re-examine the toxicity of smooth-backed blowfish L. inermis in the coastal waters of Japan.     

LC-MS/MS method for the determination of tetrodotoxin (TTX) on a triple quadruple mass spectrometer

Tetrodotoxin (TTX), often referred to as the ‘puffer fish’ poison, is a marine toxin and it has been identified as the agent responsible for many food poisoning incidents around the world. It is a neurotoxin that blocks voltage-gated sodium channels, resulting in respiratory paralysis and even death in severe cases. It is known to occur in many different species of fish and other organisms. The toxin is mainly found in the Southeast Asia region. Worryingly, TTX is starting to appear in European waters. It is suspected that this is a consequence of Lessepsian migration, also known as the Erythrean invasion. Therefore, straightforward and reliable extraction and analytical methods are now urgently required to monitor seafood of European origin for TTX. This paper provides a versatile, dependable and robust method for the analysis of TTX in puffer fish and trumpet shellfish using LC-MS/MS. A three-stage approach was implemented involving: (1) the screening of samples using fast multiple reaction monitoring (MRM) mass spectral analysis to identify quickly positive samples on a triple quadrupole mass spectrometer (QqQMS/MS), the API 3000; (2) a Fourier-transform (FT)-MS full-scan analysis of positive samples to collect qualitative data; and (3) a method with a longer chromatography run to identify and quantitate the positive samples using the QqQMS. The quantitative LC-QqQMS method delivered excellent linearity for solvent-based standards (0.01–7.5 µg ml^–1; R² ≥ 0.9968) as well as for matrix-matched standards (0.05–37.50 µg g^–1; R² ≥ 0.9869). Good inter-day repeatability was achieved for all the relevant analytes with %RSD values (n = 9) ranging from 1.11% to 4.97% over a concentration range of 0.01–7.5 µg ml^–1. A sample clean-up procedure for the puffer fish and trumpet shellfish was developed to ensure acceptable and reproducible recoveries to enable accurate and precise determination of TTX in a myriad of tissues types. Blank mackerel matrix was used for the TTX standard spiking studies in order to calculate the recoveries of the toxin during the extraction procedure. The recovery was 61.17% ± 5.42% for the extraction protocol. MS/MS studies were performed on a linear-trap quadruple-Orbitrap mass spectrometer (LTQ-Orbitrap) to obtain high-mass-accuracy data of the target analytes and their characteristic fragment ions in the puffer fish and trumpet shellfish samples. This facilitated identification of TTX and its associated analogues. These high-mass-accuracy studies facilitated the development of a rapid MRM-based quantitative method for TTX determination on the LC-QqQMS.     

河豚毒素及其发酵生产的研究进展

河豚毒素(tetrodotoxin,TTX) 常见于河豚体内,是一种毒性很强的氨基全氢化喹唑啉化合物。它是世界上最致命的生物毒素之一,同时也是对神经有选择性作用的强效麻醉药和戒毒良药。因此,科研人员在弄清TTX的产生途径及产生菌后,正在竭力研究其生产工艺及高产方式,以使其显著的医药学价值为人类所用。本文综述TTX 的起源、理化性质及其生物活性等基本特性,并重点介绍近年的研究方向和热点问题,即TTX 的产生菌和发酵生产等方面的研究。     

Liquid chromatography-tandem mass spectrometry determination of the toxicity and identification of fish species in a suspected tetrodotoxin fish poisoning

Suspected tetrodotoxin (TTX) poisoning was associated with eating unknown fish in April 2009 in Taiwan. After ingestion of the fish, symptoms of the victim included perioral paresthesia, nausea, vomiting, ataxia, weakness of all limbs, respiration failure, and death within several hours. The toxicity in the remaining fish was determined, with the mice exhibiting symptoms of neurotoxin poisoning. The implicated fish and deceased victim tissues were analyzed for TTX by liquid chromatography-tandem mass spectrometry. The urine, bile, cerebrospinal fluid (spinal cord), pleural effusion, and pericardial effusion of the victim contained TTX. In addition, the partial cytochrome b gene of the implicated fish was determined by PCR. The DNA sequence in the partial 465-bp cytochrome b gene identified the implicated fish as Chelonodon patoca (puffer fish). These results indicate that people should avoid eating unknown fish species from fish markets where harvested fish may include toxic species.     

High-resolution mass spectrometry analysis of tetrodotoxin (TTX) and its analogues in puffer fish and shellfish

Tetrodotoxin (TTX) is an emerging toxin in the European marine environment. It has various known structural analogues. It acts as a sodium channel blocker; the ability of each analogue to bind to the sodium channel varies with the particular structure of each analogue. Thus, each analogue will vary in its toxic potential. TTX analogues co-occur in food samples at variable concentrations. An LC-MS method was developed for the identification and quantitation of several analogues of TTX using an LTQ-Orbitrap XL mass spectrometer. The LTQ-Orbitrap XL mass spectrometer facilitates high mass accuracy measurement up to 100,000 full width at half maximum (FWHM). Using high resolution at 100,000 FWHM allows for the identification of TTX and its analogues in various matrices, including puffer fish and molluscan shellfish samples (Δ ppm = 0.28–3.38). The confirmation of characteristic fragment ions of TTX and its analogues was achieved by determining their elemental formulae via high mass accuracy. A quantitative method was then developed and optimised using these characteristic fragment ions. The limit of quantitation (LOQ) of the method was 0.136 µg g^–1 (S/N = 10) and the limit of detection (LOD) was 0.041 µg g^–1 (S/N = 3) spiking TTX standard into TTX-free mackerel fish extracts. The method was applied to naturally contaminated puffer fish and molluscan shellfish samples to confirm the presence of TTX and its analogues.     

从一起河鲀中毒调查探讨河鲀食用安全性的风险管理措施

目的通过一起河鲀中毒事件的流行病学调查和人群关于河鲀安全性的知识、态度、行为(KAP)调查,探讨河鲀食用安全的风险管理措施。方法通过病例搜索,调查病例家庭成员进食史及发病情况,采用回顾性队列研究分析发病的高危因素,制作调查问卷调查了解当地村民有关河鲀中毒的知识、态度和行为。结果共搜索到14例病例,其中1例死亡。症状主要为头晕(100%)、四肢无力(79%)、四肢肌肉麻痹和行走困难(71%)、口舌肢端麻痹(71%)等。食用河鲀是发病的危险因素,加工过程保留鱼肝共同煮食可增加中毒的危险(RR=1.8,95%CI=1.1~3.1)。KAP调查发现70%(33/47)的村民近3年曾进食过河鲀,其中88%(29/33)来源于市场或流动摊贩;98%(46/47)的村民知道河鲀有毒,94%(44/47)的村民知道国家法律禁止食用河鲀鱼,但仍有94%(44/47)的人认为其味道鲜美值得品尝。结论食用混杂于无毒河鲀中的有毒河鲀是导致本次食物中毒的主要原因。居民对河鲀安全性知晓率高但依从性差。     

Rapid screening of tetrodotoxin in urine and plasma of patients with puffer fish poisoning by HPLC with creatinine correction

A rapid and simple detection method for tetrodotoxin (TTX) in urine and plasma of patients with puffer fish poisoning was developed using commercially pre-packed solid-phase extraction (SPE) cartridges (C18 and weak cation exchange columns) and subsequent analyses by HPLC with UV detection. The detection limit of the standard TTX, TTX-spiked urine and plasma samples were all 10 ng/ml and the average TTX recovery in urine and plasma samples after SPE were 90.3 ± 4.0 and 87.1 ± 2.9%, respectively. It was noticed that the creatinine-adjusted urinary TTX levels obtained within the first 24 h of presentation apparently correlated much better with the severity of poisoning than the urinary TTX concentration without adjusting for variations in concomitant creatinine excretion.     

鼠李糖乳酸杆菌对河豚肝脏中河豚毒素毒性的影响

以野生河豚肝脏中的河豚毒素（tetrodotoxin,TTX）为研究对象,研究活化与热灭活处理的鼠李糖乳杆菌对其毒性的消减作用。将活化的与热灭活处理的鼠李糖乳杆菌作为材料对TTX进行脱除,采用酶联免疫吸附剂测定（enzyme-linked immunosorbnent assay,ELISA）、层析试纸检测和小鼠生物法分析TTX消减率的变化,采用气相色谱-质谱分析发酵处理后的肝脏二十碳五烯酸（eicosapentaenoic acid,EPA）与二十二碳六烯酸（docosahexaenoic acid,DHA）含量变化。竞争性ELISA结果显示加热灭活后的鼠李糖乳杆菌对TTX的消减率高达82.16%;而未经灭活处理的鼠李糖乳杆菌对TTX的消减率为70.05%,表明热灭活处理的鼠李糖乳杆菌消减效果更佳。此外,经鼠李糖乳杆菌发酵7 d后野生河豚肝脏的TTX消减率为93.27%,气相色谱-质谱检测结果显示发酵后肝脏内的EPA与DHA含量分别减少了11.93%、22.50%。综上所述,鼠李糖乳杆菌能消减河豚肝脏中的TTX,经鼠李糖乳杆菌发酵处理后消减效果更佳,本研究结果可为河豚内脏组织的毒性消减提供参考。     

基于食品防护的出口河豚鱼加工过程控制的研究

基于食品防护和HACCP原理对出口河豚鱼加工过程控制的研究,为出口河豚鱼加工企业科学化控制加工过程提供借鉴。应用危害分析的方法,对出口河豚鱼加工过程中的生物性、化学性和物理性的食品安全危害进行识别和分析,应用关键控制点判断树的方法确定关键控制点并制定控制措施。出口河豚鱼加工过程中的关键控制点如原料验收、挑选、排盘、金属探测可以通过实施HACCP计划加以控制;而对于人为蓄意破坏而引起的危害则通过实施食品防护计划包括企业内外部安全、加工过程安全、贮存和运输过程安全等11个方面加以控制。通过实施HACCP计划和食品防护计划,可对出口河豚鱼加工过程中食品安全危害进行有效地控制,从而确保出口河豚鱼的质量。     

养殖河豚鱼毒素的研究

为了探究养殖河豚鱼可食用性,本实验对养殖河豚鱼的食用安全进行了研究,提取河豚鱼的肝脏、肌肉、血液、性腺为研究对象,用液相色谱法测定TTX的含量。结果表明,养殖(红鳍东方豚、暗纹东方豚、菊黄东方豚、黄鳍东方豚)肝脏、血液无毒或弱毒,肌肉无毒,可作为食用鱼种,而野生的暗纹东方豚均有剧毒,不可食用。