麻痹性贝类毒素常规检测分析方法比较与研究进展

麻痹性贝类毒素作为贝类产品中一种毒性最强、分布广泛的毒素,不仅严重威胁人们的身体健康,而且会造成相当大的经济损失。因此其监测检测方法的研究与改进一直是人们的研究热点。本文分析评述了麻痹性贝类毒素的三种常规检测分析方法的优缺点以及最新研究进展,并探讨了小白鼠生物法、免疫测定法和色谱联用技术作为主要的检测方法由于原理不同,结合不同的研究需求其应用的领域。其中,小白鼠生物测定法虽然概括毒性有效,但是其灵敏度低、误差大、并且需要大量活体动物而逐渐被色谱技术和免疫测定法所取代,此外,神经细胞分析法、毛细管电泳技术和表面等离子体共振传感器技术等方法也逐渐得到应用。不管怎样,这些方法由于需要专业人员、成本高等问题仍需进一步完善。     

水产品中麻痹性贝类毒素（PSTs）检测技术的研究进展

麻痹性贝类毒素是一种分布范围广及危害较大的赤潮毒素。可经食物链的富集、传递作用,引发人体麻痹性中毒,大量发生的中毒事件,对人类健康和经济构成了严重威胁。目前,麻痹性贝类毒素常用检测技术主要是小鼠生物法、高效液相色谱法和酶联免疫试剂盒测试法,这些检测方法均有各自的优势,但麻痹性贝类毒素成分多且复杂、结构特殊,毒性又较强,这使得其监管检测较为困难,亟待建立快速简便、灵敏度高、特异性强的分析检测方法。本文基于麻痹性贝类毒素的基本性质,依据检测原理的不同论述了其生物检测技术、仪器分析技术和生化测试技术,并对各类技术的特点进行分析,提出建设性的意见,最后展望了未来麻痹性贝类毒素检测技术的发展趋势,以期为麻痹性贝类毒素检测监管提供借鉴。     

海产品中麻痹性贝类毒素快速检测技术研究进展

麻痹性贝类毒素是我国海洋赤潮中最常见的贝类毒素之一,分布最广,危害最大,事故发生率也最高,对人类健康构成了严重威胁,加强对该类毒素的检测监控成为保障海产品安全的重要措施。传统的检测方法主要有小鼠生物检测法、液相色谱法、液相色谱-串联质谱法和酶联免疫法,这些方法均有各自的优势,但在实际应用中还缺少用于现场检测的快速筛查技术。因此,开发快速、灵敏、准确、低成本的麻痹性贝类毒素检测技术具有重要的应用价值。本文主要介绍了麻痹性贝类毒素目前开发出来的快速检测方法,主要包括免疫层析技术和生物传感器技术,对各方法的特点迚行分析。最后对未来麻痹性贝类毒素快速检测技术在实际应用中面临的主要问题迚行了评述,幵对发展趋势迚行了展望。     

连云港海州湾麻痹性贝类毒素中毒分析

为分析连云港地区麻痹性贝类毒素中毒特征,对连云港海州湾织纹螺进行形态鉴定,收集近年麻痹性贝类中毒资料,应用小白鼠生物测定法检测螺肉中麻痹性贝类毒素。结果连云港海州湾存有4种织纹螺,其中以半褶织纹螺含麻痹性贝类毒素量高(1.6×103μg100g螺肉),并导致连云港地区10起、银川1起麻痹性贝类中毒;1992年还发生一起由泥螺引起的麻痹性贝类中毒。中毒者均表现为下行性神经麻痹症状,重者致死。鉴于海洋污染的严重性,为保障海洋贝类的食用安全,应对贝类进行毒素等安全指标的监测。     

舟山海域麻痹性贝类毒素污染情况及其2种检测方法比较

目的检测舟山东极与嵊泗枸杞2个海域养殖贻贝中的麻痹性贝类毒素(paralytic shellfish poison,PSP),比较小鼠生物测定法与酶联免疫分析法(ELISA)的测定结果。方法采用小鼠生物测定法与酶联免疫吸附法检测贝类中的麻痹性贝类毒素,并将2者的检测结果进行比较分析。结果 2种检测方法检测的麻痹性贝类毒素含量结果基本一致。5月份东极岛海域的厚壳贻贝中检出PSP((500±3.2)MU/100 g),超标率为5%;嵊泗枸杞海域贝类PSP含量较低,未超出安全食用标准。2个海域的紫贻贝PSP含量均未超出安全食用标准。结论小鼠生物法与ELISA方法的评价结果基本一致,其检测出的PSP结果可以为摄入PSP风险评估提供数据支撑。由于ELISA方法的检测成本较高,因此可采用小鼠生物法进行麻痹性贝类毒素风险监测。     

钦州湾7种经济贝类中麻痹性贝类毒素分析与安全评价

采用小鼠生物检测法和液相色谱-荧光检测法,分别对采自钦州湾的7种典型经济贝类中麻痹性贝类毒素(paralytic shellfish toxins,PSTs)组成成分与含量进行分析,同时参考我国渔政渔港监督管理局制订的贝类安全食用标准(400 MU/100 g或80μg/100 g STX_(eq))评价其食用安全性。结果如下,小鼠生物法分析表明小鼠在观察15 min内均不死亡,说明7种贝类中PSTs含量均小于400 MU/100 g或者不含有毒素;进一步对贝类样品进行液相色谱-荧光检测分析,表明7种贝类中可检测到麻痹性贝类毒素,毒素组成成分以高毒性的氨基甲酸酯类毒素为主如膝沟藻毒素4(gonyautoxin,GTX4)、膝沟藻毒素1(gonyautoxin,GTX1)、新石房哈毒素(neosaxitoxin,NEO)和石房蛤毒素(saxitxin,STX),其中异毛蚶中有最大毒素含量/毒性值,分别为0.27 nmol/g或13.1μg/100 g STX_(eq),低于安全标准80μg/100 g STX_(eq)。研究中有6种贝类可检测到毒素成分,检出率为86%。这说明,在钦州湾所采集的7种贝类中,尽管毒素含量低于食用安全标准规定的阈值,但多种贝类中仍可检测到麻痹性贝类毒素成分,因此其存在的安全性问题不容忽视,应加强该地区经济型贝类中麻痹性贝类毒素的监测,以防中毒事件的发生。     

南海海域养殖牡蛎中麻痹性贝类毒素分析

本研究在2013年3月采集湛江、阳江、钦州、北海、汕头、汕尾、深圳、台山等8个位于南海海域的养殖牡蛎,用小鼠生物(MBA)法和高效液相色谱(HPLC)法进行麻痹性贝类毒素检测。MBA毒性分析结果显示:8个区域养殖牡蛎的毒素含量为0MU/g-2.3MU/g,符合我国贝类食品安全限量要求;HPLC毒素成分分析结果显示:湛江、深圳、汕头、台山和阳江等5个海域的养殖牡蛎体内检测出痕量的麻痹性贝类毒素,湛江、深圳、汕头和阳江海域的养殖牡蛎中检出高毒性的STX和neo STX。本次麻痹性贝类毒素测试MBA法的检出率为25%,HPLC法的检出率为62.5%,HPLC法更适合于检测麻痹性贝类毒素含量低的样品。     

硅胶性能表征及其对麻痹性贝类毒素吸附研究

研究硅胶对麻痹性贝类毒素吸附性能情况,探究其作为毒素检测前处理材料的可能性。利用扫描电子显微镜、红外光谱分别表征硅胶形貌尺寸、官能团与化学键,使用差示扫描量热仪、热重分析仪分析硅胶的热稳定性,采用液相色谱-串联质谱研究硅胶对麻痹性贝类毒素标准溶液吸附情况。结果表明,所选用硅胶直径介于30～60μm,尺寸相对均一,富含硅羟基团,热稳定性好,在弱酸或中性条件下有效吸附麻痹性贝类毒素,并能在0.1%酸性条件下解吸附。鉴于硅胶自身特性及对麻痹性贝类毒素良好的吸附能力,有望用于动物性水产食品中麻痹性贝类毒素检测净化前处理,其吸附机理是表面的硅羟基与麻痹性贝类毒素分子中氨基、亚氨基之间形成氢键的作用。     

认识麻痹性贝类毒素

正织纹螺因含有麻痹性贝类毒素而被国家禁止食用。麻痹性贝类毒素是如何产生的?如何避免?既然称其为"贝类毒素",我们平时所吃的美味贝类都安全吗?麻痹性贝类毒素是一种生物毒素麻痹性贝类毒素并非来自贝类生物体本身,而是贝类摄食有毒藻类,并经其体内蓄积、放大、转化等过程形成的具有神经肌肉麻痹作用的赤潮生物毒素。人体若误食含有此类毒素的贝类     

贝类毒素大田软海绵酸的免疫分析检测技术研究进展

大田软海绵酸(okadaic acid, OA)是一种广泛存在于贝类等生物中的海洋生物毒素, 可引起人或动物的急性中毒, 对食品安全和海产养殖具有严重危害。因此建立快速、可靠、灵敏的OA检测技术具有重要意义。免疫分析检测技术基于抗原抗体的结合, 特异性强、灵敏度高、应用范围广, 是当前检测贝类毒素OA的主要手段。本文综述了近年来针对贝类毒素OA的免疫分析检测技术, 其中包括酶联免疫吸附检测、免疫层析检测、时间分辨荧光免疫检测和基于免疫传感器的检测技术等。本文着重阐述了不同免疫分析技术的原理及其OA检测的实际应用, 同时探讨了免疫分析技术在贝类毒素OA检测方面的挑战和发展趋势, 以期为开发性能更加优异的OA免疫检测技术提供研究思路。     

Keeping shellfish safe to eat: a brief review of shellfish toxins,and methods for their detection

Poisoning can result from the ingestion of shellfish contaminated with phycotoxins. Various types of poisoning may occur, each of which is caused by a toxin (or group of toxins) from a particular alga. Classically, the mouse bioassay has been used to detect shellfish toxins, but there is pressure, both ethical and regulatory, to move away from this. A number of techniques have been developed to replace the bioassay, including immunoassay, chromatography, pharmacological assay and tissue-culture tests. All have advantages and limitations. These methods and their potential are reviewed.     

Azaspiracid shellfish poisoning: unusual toxin dynamics in shellfish and the increased risk of acute human intoxications

A number of recent acute human intoxications in Europe from the consumption of Irish mussels have been attributed to the presence of a new class of toxins named azaspiracids. The study demonstrates that azaspiracids behave differently from other polyether toxins, and this accounts for most false-negative results in the mouse bioassay employed by regulatory agencies to detect azaspiracids. Typically, polyether toxins are concentrated in the digestive glands of shellfish, but this is not always the situation with azaspiracids. Liquid chromatography-mass spectrometry (LC-MS), especially multiple tandem MS methods, have been applied to demonstrate that azaspiracid (AZA1) and its methyl- and demethyl- analogues, AZA2 and AZA3 respectively, are distributed throughout shellfish tissues. Using conventional mouse bioassay protocols, only 0-40% of the total azaspiracid content of shellfish was used in the assay, which could directly account for false-negative results. It was also observed that the toxin profiles differed significantly in various mussel tissues with AZA1 as the predominant toxin in the digestive glands and AZA3 predominant in the remaining tissues.     

The problem of selective determination of PSP toxins in mussels

Levels of paralytic shellfish poisoning (PSP) toxins in shellfish are routinely determined by mouse bioassay. In order to improve the qualitative and quantitative determination of PSP toxins, chromatographic techniques with fluorescence detection have been developed. These HPLC methods and the HPLC/MS coupling were used to determine a second PSP toxin which was found, in addition to saxitoxin, in canned Spanish mussels. These canned mussels were rejected in 1986 by the German food control because PSP concentrations were too high. It has been shown that these samples contained mainly dc-saxitoxin.     

贝类毒素检测中两种筛选方法的比较研究

目的 采用传统小鼠生物法(MBA)和酶联免疫吸附法(ELISA)对贝类样品中四类毒素进行检测, 为不同要求下建立或选择准确的贝类毒素检测快速筛选方法提供参考。方法 分别采用MBA和ELISA检测腹泻性贝毒(DSP)和麻痹性贝毒 (PSP), 并采用ELISA检测记忆缺损性贝毒(ASP) 和神经性贝毒(NSP)。结果 对2009~2011年8种67份贝类样品进行检测, 结果表明: 两种测试方法在实际应用中对DSP、PSP检测结果不存在差异, 检测结果有很好的吻合性。使用ELISA法对自制ASP、NSP模拟阳性样品进行检测, 均测得ASP、NSP, 检测结果满意。结论 两种筛选方法在贝类毒素检测中均有其应用空间。实验室可根据不同情况选择合适的检测方法。     

Monitoring phytoplankton and marine biotoxins in production waters of the Netherlands: results after one decade

Shellfish products may be contaminated with marine biotoxins which, after consumption, may lead to human illness. The Netherlands has a regular monitoring programme for marine biotoxins and the possible toxic phytoplankton in shellfish production waters. The aim of the current study was to evaluate the presence of potential toxic phytoplankton species and marine biotoxins in Dutch production waters over the last decade, and to analyse the relationship between toxin levels and abundance of possible causative phytoplankton species. The results of the monitoring programme of the period 1999-2009 were used. The presence of Alexandrium spp. were negligible, but Pseudo-nitzschia spp. and phytoplankton causing diarrhetic shellfish poisoning (DSP toxin-producing phytoplankton) were present in nearly all three main production areas and years. The main DSP toxin-producing species was Dinophysis acuminata followed by D. rotundata and Prorocentrum lima. Toxins causing paralytic shellfish poisoning (PSP) and amnesic shellfish poisoning (ASP) were present in only a few individual shellfish samples, all at low levels. At the end of 2002, an episode of DSP toxicity was recorded, based on the rat bioassay results. Of the samples that were chemically analysed for DSP toxins in 2007 and 2008, about half of the samples in 2007 contained these toxins, although levels were low and no positive results were obtained using the rat bioassay. There was a slight positive correlation between concentrations of DSP toxin-producing phytoplankton and levels of DSP toxins in 2007. Increased DSP toxin levels were found up to 5 weeks after the peak in DSP toxin-producing phytoplankton. This positive, but weak, relationship needs to be confirmed in future research using more samples and chemical methods to quantify the presence of DSP toxins. If this relationship is further substantiated and quantified, it could be used within the current monitoring programme in the Netherlands to predict the risk areas regarding DSP toxicity in shellfish.     

Measurement of paralytic shellfish toxins in molluscan extracts: comparison of the microtitre plate saxiphilin and sodium channel radioreceptor assays with mouse bioassay, HPLC analysis and a commercially available cell culture assay

This study compared five methods of measuring paralytic shellfish toxins (PSTs) including the long-used mouse lethality bioassay, a commercially available cell culture test (MIST ® Quantification kit), HPLC analysis, and two newly developed radioreceptor assays utilizing mammalian sodium channels and saxiphilin. Methods were challenged with toxic shellfish extracts prepared according to the AOAC official method. The best correlations between predicted toxicity values being 0.9 or better, were those between HPLC analysis when compared with both radioreceptor assays and the mouse lethality bioassay, as well as that between the saxiphilin and the sodium channel radioreceptor assays. In all cases, statistically significant correlations existed between the toxicity measurements of the same extracts. The ratios between some methods were not unitary as measured by the slopes of the regression lines used for correlation analyses. HPLC analysis predicted more toxicity than all of the bioassays. The saxiphilin assay underestimated toxicity relative to the mouse bioassay, the MIST ® kit determinations and the sodium channel assay. The sodium channel assay predicted there to be less toxicity than the mouse bioassay and the MIST ® kit. Of all of the techniques used, the MIST ® kit correlation with the mouse bioassay was nearest to one. Each method possesses different virtues and it may be that a multi-method approach would harness the benefits of each method for various aspects of a shellfish testing regime.     

Comparative Toxicity and Paralytic Shellfish Poisoning Toxin Profiles in the Mussel Mytilus galloprovincialis and the Oyster Crassostrea gigas Collected from a Mediterranean Lagoon in Tunisia: A Food Safety Concern

Edible shellfish Mytilus galloprovincialis and Crassostrea gigas have been investigated for the paralytic shellfish poisons using mouse bioassay and high performance liquid chromatography with fluorescence detection. Paralytic shellfish poisons toxins were detected in mussels and oysters from September 2007 to May 2008. The level of paralytic shellfish poisons toxins in mussels reached the maximum in November with 832.9 μg saxitoxin-eq/100 g tissue. In oysters, toxins were detected with a maximum of 11.2 μg saxitoxin-eq/100 g tissue. The toxin high performance liquid chromatography profiles in mussels and oysters revealed the dominance of gonyautoxin 5 and N-sulfocarbamoyl-gonyautoxin-2 and -3 (C1-2), whereas GTX1-4, saxitoxin, and neosaxitoxin were found at low amounts. Overall, levels of paralytic shellfish poisons toxins were 20–70 times greater in mussels than in oysters. This is the first report on the qualitative and quantitative paralytic shellfish poisons content of M. galloprovincialis and C. gigas from a shellfish farming lagoon in Tunisia.