基于RNA适配体检测和分析鱼组织中残留的MG和LMG

第28卷第6期大连海洋大学学报Vol.28No.6 2013年12月JOURNAL OF DALIAN OCEAN UNIVERSITY Dec.2013文章编号:2095-1388(2013)06-0515-07黄颡鱼免疫球蛋白M基因的克隆与组织表达分析叶仕根,费阳春,李强,李华(大连海洋大学农业部北方海水增养殖重点实验室,辽宁大连116023)摘要:根据GenBank中登录的免疫球蛋白M(IgM)基因编码氨基酸的保守序列以及鱼类密码子的偏好性设计简并引物,提取黄颡鱼Peltebagrus fulvidraco脾脏总RNA,经RT-PCR扩增首次获得了黄颡鱼IgM的部分序列(675bp)㊂以β-actin为内参基因,通过Real-time PCR法分析了黄颡鱼IgM基因的组织分布特点㊂结果表明,在黄颡鱼肝胰脏㊁脾脏㊁头肾㊁中肾㊁鳃㊁肌肉㊁皮肤和肠道中均检测到IgM基因的表达,头肾和脾脏是IgM表达的主要部位,肾脏㊁鳃㊁皮肤㊁肠道和肝胰脏等组织中表达量居中,肌肉中表达量最低㊂经鮰爱德华菌Edwardsiella ictaluri胞外产物免疫后,头肾㊁脾脏和肝胰脏中IgM基因表达呈现不同的变化规律㊂关键词:黄颡鱼;IgM;克隆;组织分布中图分类号:Q786 文献标志码:A 免疫球蛋白(Immunoglobulins,Ig)是有颌类脊椎动物所特有的介导体液免疫的重要效应分子,在脊椎动物特异性免疫系统中起着关键作用㊂Ig 分子包括重链和轻链两部分,根据重链恒定区化学结构的差异,可将Ig划分为多种类型㊂不同动物所拥有的Ig种类不同,目前已报道的硬骨鱼类Ig 有IgM㊁IgD㊁IgZ/T和IgM-IgZ嵌合体等几种类型[1-5]㊂有报道指出,病原菌感染可明显提高鳜鱼IgM的表达水平,而IgD和IgZ无明显变化[6],内毒素刺激则可增加鲤IgM-IgZ嵌合体基因的转录[5],这说明不同类型的Ig有不同的免疫应答机制㊂在这些Ig分子中,IgM存在于所有有颌类脊椎动物中,其重链代表了从软骨鱼类到哺乳类的一个连续进化的过程[7]㊂同时,IgM也是鱼类最先表达的抗体,在体液免疫尤其是抵抗细菌性抗原侵扰方面起着重要作用[8-9]㊂目前,已有多种鱼类IgM 重链基因被克隆鉴定,诸如模式鱼类斑马鱼Danio rerio[10]和一些重要的经济鱼类[11-17]㊂这些研究结果显示,不同鱼类的IgM重链之间存在基因数目差异和序列特异性,同时不同鱼类IgM基因的组织分布也存在差异㊂黄颡鱼Peltebagrus fulvidraco是重要的经济鱼类之一,然而对其IgM基因的研究至今尚未见报道㊂本研究中,作者根据GenBank数据库中登录的黄颡鱼近缘物种的IgM重链序列信息,通过设计简并引物克隆IgM重链基因的部分片段,同时采用Re⁃al-time PCR方法研究黄颡鱼IgM重链基因在各组织中的表达情况以及经胞外产物(OMPs)免疫刺激后的变化规律,以期为黄颡鱼的抗感染机制和免疫防治技术研究提供理论依据,对了解鱼类免疫应答的分子机制以及免疫系统的起源与进化等具有重要意义㊂1　材料与方法1.1　材料试验用黄颡鱼体质量为35~50g,购自辽宁营口某黄颡鱼养殖场,驯养一周后用于试验㊂Trizol总RNA提取试剂盒㊁M-MLV反转录酶㊁荧光定量PCR试剂盒SYBR®Green SuperMix-UDG 试剂盒等购自上海Invitrogen公司;pMD18-T载体㊁Taq DNA聚合酶㊁DNA Marker均购自宝生物(大连)工程有限公司;引物由北京六合华大基因科技股份有限公司合成㊂1.2　方法1.2.1　IgM重链基因核心序列的扩增　黄颡鱼暂　收稿日期:2013-03-13　基金项目:国家 十二五”科技支撑计划项目(2011BAD13B03)　作者简介:叶仕根(1976-),男,副教授㊂E-mail:yedlsy@ 通信作者:李华(1958-),女,教授㊂E-mail:lihua@养一周后,随机挑选2尾,取其脾脏,按照试剂盒说明书方法提取黄颡鱼脾脏总RNA㊂选用经电泳和紫外分析检测条带清晰㊁完整性好的RNA用于后续的反转录和PCR扩增㊂取4μL(约2μg)总RNA悬液,使用M-MLV反转录酶和oligo(dT) 18于37℃下进行反转录,所得cDNA于-20℃下保存备用㊂根据NCBI中已登录的鱼类免疫球蛋白重链恒定(CH)区氨基酸保守序列设计简并引物[13],正向引物F′为5′CCNACNCARACNGAY⁃ATHGAY3′,反向引物R′为5′NYTNACNTGYTAY⁃GTNAARGA3′,其中N为G㊁A㊁T或C;Y为T 或C;R为G或A,W为A或T㊂在此基础上,参照鱼类密码子偏好性[18]对引物做进一步修改,去掉一些鱼类较少使用的密码子,针对性地降低引物的简并度㊂修改后的正向引物IgM-F为5′CCAAC⁃CCAAACAGAMATAGAC3′,反向引物IgM-R为5′TCTTTWACATAGCAAGTCAGG3′,引物简并度由1 536㊁4096下降至2㊂以cDNA第一链为模板进行PCR扩增,将所得目的条带回收纯化并与PMD-18T载体连接,转化至DH5α中,经菌落PCR和酶切鉴定后,送北京六合华大基因科技股份有限公司测序㊂1.2.2　序列分析　参照李颖等[19]的方法,将测序得到的序列在NCBI网站(http://www.ncbi.nlm. /blast)进行相似性搜索和比对,并提交GenBank㊂采用Expasy网站的Prosite在线工具(ht⁃tp:///prosite.html)进行编码蛋白质的保守结构域分析,用Clustal X1.8和DNA6.0程序进行氨基酸序列同源性分析和作图㊂1.2.3　IgM重链基因组织表达量的检测　随机选取3尾黄颡鱼,分别取肝胰脏㊁脾脏㊁头肾㊁肾脏㊁鳃㊁肌肉㊁皮肤和肠等8个组织,液氮速冻后按上述方法提取各组织总RNA并反转录得到cDNA㊂根据 1.2.1”和 1.2.2”节中获得的IgM重链基因序列设计实时定量PCR引物,正向引物IgM-F1为5′AGAGCCAGAAGTGAGCATTA 3′,反向引物IgM-R1为5′CTTGGCAGGTGTATGT⁃GG3′㊂根据黄颡鱼β-actin基因的序列(GenBank 登录号:EU161066.1)设计实时定量PCR内参引物,正向引物ACTIN-F为5′GATCCGGTATGTG⁃CAAGGCT3′,反向引物ACTIN-R为5′TGC⁃CAGATCTTCTCCATATCA3′(表1)㊂引物合成后,以cDNA第一链为模板进行PCR扩增,经E.B染色,用15g/L琼脂糖电泳检验其特异性㊂表1　基因克隆与组织表达分析所用引物序列Tab.1　Sequences of the primers used in the gene cloning and tissue expression引物primer 序列sequence产物长度length/bp 用途application IgM-F5′CCAACCCAAACAGAMATAGAC3′675IgM基因克隆的上游简并引物IgM-R5′TCTTTWACATAGCAAGTCAGG3′675IgM基因克隆的下游简并引物IgM-F15′AGAGCCAGAAGTGAGCATTA3′216IgM Real-time PCR上游引物IgM-R15′CTTGGCAGGTGTATGTGG3′216IgM Real-time PCR下游游引物ACTIN-F5′GATCCGGTATGTGCAAGGCT3′203ACTIN Real-time PCR上游引物ACTIN-R5′TGCCAGATCTTCTCCATATCA3′203ACTIN Real-time PCR下游引物 荧光定量PCR参照刘俊等[20]的方法,并作适当修改㊂采用2 △△CT法计算基因在各组织中的表达量:　△△CT=(CT IgM x-CT actin x)-(CT IgM0-CT actin0),其中:CT为样品管中荧光强度达到特定阈值时的扩增循环数;x表示8个待测组织中的任一组织; 0表示8个待测组织中相对表达量最低的组织㊂用SYBR Green qPCR试剂盒,在Step one定量PCR仪上进行实时定量扩增㊂首先将反转录获得的cDNA 模板以及引物浓度进行优化,将CT值调整为20左右㊂扩增反应程序为:95℃下预变性2min;95℃下变性30s,62℃下退火30s,共进行40个循环㊂每个样品均进行IgM和β-actin表达量的测定㊂每个样本同时设立3个平行管,每次反应均设置熔解曲线分析,以验证扩增反应的特异性㊂反应完成后,系统软件将自动给出每个样本扩增的IgM和β-actin基因的CT值,在此基础上进一步进行基因表达差异的分析㊂1.2.4　鮰爱德华菌OMPs免疫对IgM基因表达的影响　取大连海洋大学农业部北方海水增养殖重点实验室分离保存的黄颡鱼病原菌鮰爱德华菌A86,经扩大培养后,参照李强等[21]的方法提取OMPs㊂调整OMPs浓度为1mg/mL,与弗氏完全佐剂等体积混合后腹腔注射免疫黄颡鱼30尾(100μL/尾)㊂分别于免疫接种后第4㊁8㊁14㊁21和28天随机选取3尾鱼,取其肝胰脏㊁脾脏和头肾组织,液氮速冻后按照上述方法提取各组织总RNA 并反转录得到cDNA㊂另随机选取3尾未免疫鱼,取其肝脏㊁脾脏和头肾组织作为免疫前对照㊂采用IgM-F1/R1进行IgM基因表达的实时定量扩增,615大连海洋大学学报 第28卷ACTIN-F /R 用作内参基因扩增引物,试验方法和数据计算同 1.2.3”节㊂2　结果2.1　脾脏RNA 质量和定量PCR 引物特异性验证黄颡鱼脾脏总RNA 经甲醛变性凝胶电泳分析,可见28S RNA 和18S RNA 两条清晰条带,亮度在2∶1左右,用核酸紫外分析仪检测样品的OD 260nm /OD 280nm 值为1.8~2.0,表明RNA 质量较好,无蛋白质㊁酚等污染(图1)㊂对黄颡鱼IgM 基因表达分析引物IgM-F1/R1和内参基因β-actin 引物ACTIN-F /R 扩增产物的熔解曲线分析表明,均呈现单一峰值(84.38㊁84.97℃),说明PCR 产物单一,无非特异性扩增,引物特异性强(图2)㊂图1　黄颡鱼脾脏总RNA 琼脂糖凝胶电泳Fig.1　Agarose gel electrophoresis of the total RNA inspleen of the yellow catfish2.2　黄颡鱼IgM 重链基因恒定区的基因克隆以脾脏总RNA 反转录得到的cDNA 为模板,以IgM-F /R 为引物进行PCR 扩增,扩增产物经凝胶电泳检测得到一条约为700bp 且与预期扩增片段大小相符的条带(图3)㊂条带经切胶回收后连接pMD-18T 载体,再经PCR 扩增和酶切鉴定初步确认后送交测序,最终获得一段675bp 的序列并提交GenBank (GenBank 登录号:JQ067604.1)㊂2.3　黄颡鱼IgM 重链基因恒定区的序列分析分析发现,黄颡鱼IgM 重链基因恒定区序列共编码225个氨基酸残基(GenBank 登录号:AEY79771),包含4个保守的半胱氨酸残基(图4,第15㊁74㊁120㊁172位,加框表示)㊂进一步分析发现,黄颡鱼IgM 重链基因恒定区第15㊁74㊁120㊁172位半胱氨酸残基的分布符合IgC (免疫球蛋白恒定区)结构域的特征(C-X(n)-C,X 为任意氨基酸残基),可分别形成两个二硫键,组成两个包含89个氨基酸残基的IgC 结构域(图4,第1~89位和第98~176位氨基酸残基,下划线表示)㊂经Blast 搜索和序列比对分析显示,其编码氨基酸与瓦式黄颡鱼㊁大鳍鳠㊁斑点叉尾鮰㊁草鱼和斑马鱼等鱼类IgM 重链基因编码氨基酸序列的相似度为56%~88%(表2,图5),与鱼类IgM 基因具有较高同源性㊂表2　黄颡鱼IgM 部分序列与其他鱼类IgM 氨基酸序列的一致性和相似度比较Tab.2　Similarity and identity of deduced amino acid se⁃quence of IgM in yellow catfish Peltebagrus ful⁃vidraco ,with other fishes 物种 species一致性/%identity相似度/%similarity黄颡鱼Peltebagrus fulvidraco --瓦氏黄颡鱼Pelteobagrus vachellii 7588大鳍鳠Hemibagrus macropterus 6275斑点叉尾鮰Ictalurus punectatus 4867草鱼Ctenopharyngodon idella 3956斑马鱼Danio rerio37562.4　黄颡鱼IgM 基因的组织表达以β-actin 为内参基因,黄颡鱼肝胰脏㊁脾脏㊁头肾㊁肾脏㊁鳃㊁肌肉㊁皮肤和肠道等8个组织中IgM 基因表达情况如图6所示㊂从图6可见,IgM基因在黄颡鱼各检测组织中均有表达,但各组织间的表达水平存在较大差异㊂各组织中表达量由高到低依次为头肾㊁脾脏㊁肾脏㊁鳃㊁皮肤㊁肠道㊁肝胰脏和肌肉,头肾和脾脏中IgM 基因的表达量明显高于其他组织,分别为表达量最低的肌肉组织的10.3和10.1倍;肾脏㊁鳃㊁皮肤㊁肠和肝胰脏中IgM 基因的表达量居中,分别为肌肉组织的5.3㊁3.7㊁3.0㊁2.7和2.2倍㊂2.5　OMPs 免疫对黄颡鱼IgM 基因表达的影响由于样品保存不当(从液氮取出时,装有样品的EP 管因温差过大炸裂),免疫前的黄颡鱼肝胰脏㊁脾脏和头肾组织样品未能用于IgM 基因表达的定量检测㊂因此,各组织IgM 基因表达变化情况均采用免疫后(第4天)首次取样为初始表达水平,并以此为基准进行比较㊂以β-actin 为内参基因,经OMPs 免疫后,黄颡鱼肝胰脏㊁脾脏和头肾组织(使用专用冻存管保存于液氮中)IgM 基因的表达情况如图7所示㊂从图7可见:黄颡鱼肝胰脏㊁脾脏和头肾中IgM 基因的表达呈现出不同的变化规律,与脾脏和头肾相比,肝胰脏中IgM 基因表715第6期 叶仕根,等:黄颡鱼免疫球蛋白M 基因的克隆与组织表达分析达在整个取样过程中变化不太明显,最高表达(免疫后第21天)和最低表达(第14天)分别为初始水平的1.06㊁0.81倍;3个组织中,头肾和脾脏中IgM 基因表达变化规律较为相似,均为短暂图2　Real-time PCR 扩增产物的熔解曲线分析Fig.2　Melting curves of the PCR products of IgM-F1/R1and ACTIN-F /R注:M 为DL 2000DNA Marker;1~8分别为8对不同简并引物扩增的PCR 产物,其中第6道(引物为IgM-F /R)有特异性扩增产物Note:M,DL 2000DNA Marker;Lane 1-8,PCR products by dif⁃ferent degenerate primers,nes 6,PCR of IgM se⁃quence in yellow catfish products by degenerate primers IgM-F /R图3　用简并引物扩增黄颡鱼IgM 序列的PCR 产物电泳Fig.3　Agarose gel electrophoresis of PCR products ofIgM sequence by degenerate primers in yellowcatfish图4　黄颡鱼IgM 部分序列编码氨基酸的分析Fig.4　Deduced amino acid sequences by IgM from yellow catfish Peltebagrusfulvidraco图5　黄颡鱼IgM 部分序列与其他鱼类IgM 重链氨基酸序列的比较分析Fig.5　Comparison of multiple amino acid sequences of IgM in yellow catfish Peltebagrus fulvidraco ,with other fishes815大连海洋大学学报 第28卷注:1)Hp 肝胰脏;Sp 脾脏;Hk 头肾;Ki 肾脏;Gi 鳃;Sk 皮肤;In 肠;Mu 肌肉㊂2)使用β-actin 为内参,数据分析采用2 △△CT 法处理,图中相对表达倍数指各组织以最低表达量组织为基准的表达倍数,下同Note:1)Hp,hepatopancreas;Sp,spleen;Hk,head kidney;Ki,kidney;Gi,gill;Sk,skin;In,intestine;Mu,muscle.2)Expression levels were assessed using the β-actin as endogenous con⁃trol,and measured on the basis of the minimum in tissues by2△△CTmethod,et sequentia图6　Real-time PCR 检测黄颡鱼IgM 基因的组织分布Fig.6　Expression levels of IgM gene in various tissuesdetected by Real-timePCR图7　OMPs 免疫对黄颡鱼各组织IgM 表达的影响Fig.7　Expression levels of IgM gene in various tissuesof yellow catfish challenged by OMPs detected by Real-time PCR下降(第8天)后再升高,最高表达分别出现在第14天和21天;脾脏IgM 基因表达的最高值和最低值分别为初始水平的2.90和0.71倍,头肾IgM 基因表达的最高值和最低值分别为初始水平的2.21和0.82倍;脾脏和头肾组织中IgM 基因表达自免疫后第14天到第28天试验结束时均维持了较高的表达水平,分别为初始表达水平的1.84~2.90和1.92~2.21倍㊂3　讨论本研究中,通过RT -PCR 方法首次成功克隆了黄颡鱼IgM 重链基因恒定区部分cDNA 序列㊂该序列编码氨基酸中包含4个保守的半胱氨酸残基,可分别形成两个二硫键,组成两个IgC (Ig 恒定区)结构域㊂同源性分析发现,其编码氨基酸与瓦式黄颡鱼㊁大鳍鳠㊁斑点叉尾鮰㊁草鱼和斑马鱼等鱼类的IgM 重链基因编码氨基酸序列的相似度为56%~88%,具有较高的同源性㊂在获得黄颡鱼IgM 重链基因部分序列的基础上,采用Real -time PCR 方法检测了IgM 重链基因在黄颡鱼不同组织中的表达情况以及经OMPs 免疫后其在各组织中表达的变化规律,为黄颡鱼乃至其他鱼类的抗感染机制和疾病防治研究等提供了理论依据㊂在新基因的克隆中,根据近缘物种相关蛋白氨基酸序列的保守区域设计简并引物是常用的方法之一㊂但由于密码子的简并性和摇摆性,常常使得设计出来的引物简并度非常高,不利于PCR 扩增反应的进行㊂张晓峰等[18]研究发现,鱼类密码子与其他生物一样具有明显的偏好性,即编码同一种氨基酸的密码子在翻译成蛋白质时并非均一使用,通常是某些密码子被优先使用,某一物种或某一基因通常会倾向于使用一种或几种特定的同义密码子,即所谓的最优密码子,此现象被称为密码子偏性㊂本研究中,参照鱼类密码子偏好性对引物进行了针对性修改,使两条引物的简并度分别由1536㊁4096下降至2,提高了引物的扩增效率,成功克隆到黄颡鱼IgM 基因重链恒定区的部分cDNA 序列㊂Real-time PCR 分析表明,黄颡鱼头肾和脾脏是IgM 基因mRNA 的主要分布组织,其表达量显著高于其他组织㊂本研究结果与李春涛等[22]对大鳍鳠以及王欣欣等[23]对草鱼IgM 基因mRNA 分布情况的研究结果类似㊂胡瑜兰[24]和彭博[25]研究发现,斑马鱼和鲫的头肾中免疫球蛋白具有较高表达水平㊂头肾和脾脏中较高的IgM 基因mRNA 或免疫球蛋白表达水平与其抗体产生细胞发生的主要器官的功能是相适应的,这从侧面证实了头肾和脾脏是鱼类免疫的主要场所[26-27]㊂值得注意是,作为鱼类造血器官之一,黄颡鱼肾脏中也有较高的IgM 基因mRNA 表达水平,为头肾和脾脏的50%左右㊂但这与欧洲鳗鲡[16]和大鳍鳠[22]肾脏中IgM 基因mRNA 表达水平高于脾脏中的研究结果有一定差异㊂这可能与物种差异㊁动物机体状况㊁环境等因915第6期 叶仕根,等:黄颡鱼免疫球蛋白M 基因的克隆与组织表达分析素有关,如季节变化会影响鱼体内免疫球蛋白的表达水平,夏季高于冬季;运输会影响斑点叉尾鮰对抗原刺激的应答反应[28-29]㊂上述结果表明,头肾㊁脾脏和肾脏是鱼类IgM基因mRNA和免疫球蛋白表达的主要场所㊂此外,本研究中还发现,黄颡鱼肌肉组织中IgM基因mRNA的表达水平是所有被检测组织中最低的,这与对欧洲鳗鲡的研究结果一致㊂肌肉组织中IgM低表达可能与肌肉组织中MHC表达量较低有关[30]㊂因为MHC在免疫应答的启动和免疫调节中发挥重要作用,肌肉组织中较低的MHC表达量使得其自身难以具备产生免疫反应的分子基础[31]㊂由于样本保存的原因,本研究中免疫前样品未能用于IgM基因表达的定量分析㊂研究表明,鳜和大鳍鳠经嗜水气单胞菌灭活疫苗免疫后以及剑尾鱼经溶藻弧菌灭活疫苗免疫后,其IgM基因表达水平在第6~10天后显著升高,但在前期如第3天(剑尾鱼)㊁第4天(鳜)和第5天(大鳍鳠)变化并不十分明显[22,32-33]㊂因此,本研究中免疫后(第4㊁8㊁14㊁21天)样品中的IgM基因表达情况仍能反映出其变化规律㊂经OMPs免疫后,黄颡鱼脾脏和头肾中IgM基因表达变化趋势相似,均为先小幅降低(第8天)后升高,且自第14天起维持在较高表达水平㊂这与嗜水气单胞菌灭活疫苗免疫后,大鳍鳠脾脏和头肾中IgM基因表达持续升高并维持在较高的表达水平的结果一致[22]㊂但与鳜经嗜水气单胞菌灭活疫苗免疫后,头肾和脾脏中转录水平峰值出现在第7天,至第28天时鳜头肾中转录水平下降到免疫前水平而脾脏仍维持在较高表达水平的研究结果有一定差异[33]㊂这可能与物种和取样时间长短有一定关系㊂Zilberg等[34]研究发现,对斑点叉尾鮰注射鮰爱德华菌后,其血细胞㊁脾脏和头肾中IgM基因表达量在13d内均显著增加㊂Raida等[35]研究发现,对虹鳟注射鲁氏耶尔森氏菌Yersinia ruckeri后,其脾脏和头肾中IgM基因表达量在21d内都有增加㊂这些结果表明,鱼类IgM 分子在3周内可以识别细菌抗原[6]㊂尽管在未经免疫刺激的情况下,肝胰脏中IgM基因表达水平较低(仅为头肾或脾脏的1/4左右),作为鱼类重要的代谢器官,肝脏组成细胞参与肝脏免疫调节[36],其仍然可能在免疫防御中发挥重要作用㊂然而,本研究结果表明,经OMPs免疫后黄颡鱼肝胰脏中IgM基因表达量无明显变化㊂作者的另一研究发现,经鮰爱德华菌OMPs免疫可显著提升黄颡鱼肝胰脏中CAT㊁AKP和SOD活性(另文发表)㊂这些结果表明,鱼类特异性免疫防御更多的是与脾脏㊁头肾等组织相关,肝胰脏更多的是通过增加一些免疫酶的活性来发挥免疫防御作用,主要参与机体的非特异性免疫防御,但其详细机制仍有待于进一步探讨㊂综上所述,本研究中成功克隆到了黄颡鱼IgM 基因的部分序列,并检测了其组织分布模式和经OMPs免疫后的变化规律,研究结果将有助于对黄颡鱼IgM结构与功能的理解,为鱼类的免疫防治技术和分子免疫应答机制研究提供了基础资料㊂参考文献:[1]　Pilstrom L,Bengten E.Immunoglobulin in fish-genes,expressionand structure[J].Fish&Shellfish Immunol,1996,6:243-262.[2]　Saha N R,Suetake H,Kikuchi K,et al.Fugu immunoglobulin D:ahighly unusual gene with unprecedented duplications in its constant region[J].Immunogenetics,2004,56:438-447.[3]　Danilova N,Bussmann J,Jekosch K,et al.The immunoglobulinheavy-chain locus in zebrafish:identification and expression of a previously unknown isotype,immunoglobulin Z[J].Nat Immnol, 2005,6(3):295-302.[4]　Hansen J D,Landis E D,Phillips R B.Discovery of a unique Igheavy-chain isotype(IgT)in rainbow trout:implications for a distinctive B cell developmental pathway in teleost fish[J].Proc Natl Acad Sci USA,2005,102:6919-6924.[5]　Savan R,Aman A,Nakao M,et al.Discovery of a novel immuno⁃globulin heavy chain gene chimera from common carp(Cyprinus carpio L.)[J].Immunogenetics,2005,57:458-463. [6]　Tian J Y,Sun B J,Luo Y P,et al.Distribution of IgM,IgD and IgZin mandarin fish,Siniperca chuatsi lymphoid tissues and their tran⁃scriptional changes after Flavobacterium columnare stimulation [J].Aquaculture,2009,288:14-21.[7]　Wilson M R,Warr G W.Fish immunoglobulins and the genes thatencode them[J].Ann Rev Fish Dis,1992,2:201-221. [8]　Reddy P S,Corley R B.The contribution of ER quality control tothe biologic functions of secretory IgM[J].Immunol Today,1999, 20:582-588.[9]　Klimovich V B,Samoilovich M P,Klimovich B V.Problem of J-chain of immunoglobulins[J].Journal of Evolutionary Biochemistry and Physiology,2008,44(2):151-166.[10]　Danilova N,Hohman V S,Kim E H,et al.Immunoglobulin varia⁃ble-region diversity in the zebra fish[J].Immunogenetics,2000,52:81-91.[11]　Ghaffari S H,Lobb C J.Cloning and sequence analysis of channelcatfish heavy chain cDNA indicate phylogenetic diversity withinthe IgM immunoglobulin family[J].J Immunol,1989,142:1356-1365.[12]　Hansen J,Leong J A,Kaattari plete nucleotide sequence ofa rainbow trout cDNA encoding a membrane-bound form of im⁃munoglobulin heavy chain[J].Mol Immnol,1994,31(6):499-501.025大连海洋大学学报 第28卷[13]　Nakao M,Moritomo T,Tomana M.Isolation of cDNA encoding theconstant region of the immunoglobulin heavy-chain from common carp (Cyprinus carpio L.)[J].Fish &Shellfish Immunol,1998,8:425-434.[14]　Zhang Y A,Nie P,Wang Y P.cDNA sequence encoding immuno⁃globulin M heavy chain of the mandarin fish Siniperca chuatsi [J].Fish &Shellfish Immunol,2003,14:477-480.[15]　Cheng C A,John J A C,Wu M S,et al.Characterization of serumimmunoglobulin M of grouper and cDNA cloning of its heavy chain[J].Veterinary Immunology and Immunopathology,2006,109:255-265.[16]　Feng J J,Guan R Z,Lin P,et al.Molecular cloning and character⁃ization analysis of immumoglobulin M heavy chain gene in Euro⁃pean eel(Anguilla anguilla )[J].Veterinary Immunology and Im⁃munopathology,2009,127:144-147.[17]　冯汉如.南方鲇IgM 重链基因cDNA 克隆及该基因在小瓜虫感染下的表达特征分析[D].重庆:西南大学,2010.[18]　张晓峰,孙效文.鲤鱼和斑马鱼同义密码子使用偏性分析[J].水产学杂志,2010,23(4):23-29.[19]　李颖,周一兵,万良,等.双齿围沙蚕Hsp70cDNA 基因的克隆及序列分析[J].大连海洋大学学报,2012,27(6):502-507.[20]　刘俊,赵金良,张敏,等.鳜胰岛素样生长因子-ⅡcDNA 基因的克隆与表达特征[J].大连海洋大学学报,2012,27(6):11-17.[21]　李强,刘海燕,黄华,等.鮰爱德华菌黄颡鱼分离株外膜蛋白的抗原性分析[J].广东海洋大学学报,2011,31(3):85-89.[22]　李春涛,张其中,杨莹莹,等.大鳍鳠免疫球蛋白M 重链基因的克隆及表达分析[J].水产学报,2011,35(11):1684-1693.[23]　王欣欣,孙宝剑,昌鸣先,等.草鱼免疫球蛋白M 重链基因的克隆及表达[J].水产学报,2008,32(1):13-20.[24]　胡瑜兰.硬骨鱼类补体关键因子C1q 及免疫球蛋白分子克隆㊁进化和功能的初步研究[D].杭州:浙江大学,2009.[25]　彭博.鲫鱼免疫球蛋白基因的鉴定㊁应答和功能研究[D].杭州:浙江大学,2008.[26]　孟庆闻,苏锦祥,李婉端.鱼类比较解剖[M].北京:科学出版社,1987:361-363.[27]　马燕梅,林树根,王全溪,等.花鲈头肾的显微结构和超显微结构[J].福建农林大学学报:自然科学版,2008,37(2):190-193.[28]　Ellsaesser C F,Clem L W.Hematological and immunologicalchanges in channel catfish stressed by handling and transport [J].J Fish Biol,1986,28:511-521.[29]　Nakanishi T.Seasonal changes in the immune responses andlymphoid tissue on the marine teleost,Sebastiscus marmoratus[J].Vet Immunol Immunopathol,1986,12:213-222.[30]　Koppang E O,Hordvik I,Bjerekas I,et al.Production of rabbitantisera against recombinant MHC classⅡ:chain and identifica⁃tion of immunoreactive cells in Atlantic salmon (Salmo salar )[J].Fish &Shellfish Immunol,2003,14(2):115-132.[31]　冯建军,关瑞章,林鹏,等.欧洲鳗鲡免疫球蛋白M 重链基因的原核表达与不同组织中表达变化的定量分析[J].华中农业大学学报,2009,28(6):719-725.[32]　韩进刚,付小哲,石存斌,等.剑尾鱼IgM 基因的克隆及免疫对其组织表达的影晌[J].广东海洋大学学报,2007,27(6):1-6.[33]　刘雨果,潘厚军,陈偿,等.嗜水气单胞菌灭活疫苗浸泡后鳜IgM 基因表达量和抗体效价的变化[J].淡水渔业,2009,36(6):41-46.[34]　Zilberg D,Klesius P H.Quantification of immunoglobulin in theserum and mucus of channel catfish at different ages and following infection with Edwardsiella ictaluri [J].Veterinary Immunologyand Immunopathology,1997,58:171-180.[35]　Raida M K,Buchmann K.Temperature-dependent expression ofimmune-relevant genes in rainbow trout following Yersinia ruckeri vaccination[J].Diseases of Aquatic Organisms,2007,77:41-52.[36]　张云霞,刘杞.肝窦内皮细胞与免疫耐受[J].世界华人消化杂志,2006,14(28):2776-2779.Molecular cloning and expression of partial IgM cDNA sequence indifferent tissues of yellow catfish Peltebagrus fulvidracoYE Shi-gen,FEI Yang-chun,LI Qiang,LI Hua(Key Laboratory of Mariculture &Stock Enhancement in North China’s Sea,Ministry of Agriculture,Dalian Ocean University,Dalian 116023,China)Abstract :Partial cDNA sequence (675base pairs)was cloned in total DNA of spleen in yellow catfish Peltebagrus fulvidraco by degenerated primers designed based on fish IgM heavy chain gene in GenBank and by RT-PCR.The expression of IgM gene in different tissues of the yellow catfish was studied by real time PCR with internal control β-actin.The results showed that the IgM gene was constitutively expressed in all detected tissues including hepato⁃pancreas,spleen,head kidney,kidney,gill,skin,intestine and muscle,the maximal expression levels in spleenand head kidney,the minimal expression level in muscle,and the mid-level in the other tissues.There was a dif⁃ferent expression pattern of the IgM gene in head kidney,spleen,and hepatopancreas in the yellow catfish chal⁃lenged with ecto-products of Edwardsiella ictaluri .Key words :Peltebagrus fulvidraco ;immunoglobulin M;clone;tissue distribution125第6期 叶仕根,等:黄颡鱼免疫球蛋白M 基因的克隆与组织表达分析。

收稿日期：2021-10-15基金项目：国家自然科学基金项目（3190210143）作者简介：王家琪（1996-），男，河南长垣人，硕士，主要从事鱼类性控传育种研究，（电话）135****6869（电子信箱）********************；通信作者，梅洁（1981-），男，湖北黄梅人，教授，博士，主要从事鱼类遗传育种研究，（电话）************（电子信箱）近年来，转录组学技术广泛应用于水产动物繁育、营养、发育和免疫等各研究［1］。

目前，转录组测序应用最广的是二代测序技术（RNA-Seq ），二代转录组测序具有测序通量高、成本低的优势。

尽管二代测序技术读取准确率高，但读长相对较短，给后续序列组装、拼接以及注释等带来困难［2］。

而基于基于PacBio 平台的黄颡鱼全长转录组测序及分析王家琪，熊阳，韩庆庆，皇培培，梅洁（华中农业大学水产学院，武汉430070）摘要：为进一步丰富黄颡鱼（Pelteobagrus fulvidraco ）信息数据库，采用PacBio 测序平台对黄颡鱼的肝脏、肾、背部肌肉、脑、脾、心脏、皮肤、血液、鳃、性腺等组织进行全长转录组混样测序。

共获得685574个全长非嵌合（Full-length non-chimeric read ，FLNC ）序列，通过与黄颡鱼基因组比对分析，共获得72509个非冗余isoforms ，平均长度为2918bp 。

共鉴定到3169个LncRNA 、45872个可变剪接事件、4881个基因存在可变多聚腺苷酸化位点和304个融合基因。

将12492个新基因与NR 、GO 、KEGG 、KOG 和Swwassprot 5个公共数据库进行比对，成功注释了7233个isoforms 。

GO 分析发现，新基因主要集中于细胞过程（Cellular process ）、细胞组分（Cell ）和结合功能（Binding ）。

KEGG pathway 分析显示，新基因主要在信号转导（Signal transduction ）和免疫系统（Immune system ）信号通路中得到富集，此外，涉及黄颡鱼生殖与繁殖相关的内分泌系统代谢途径包括催产素信号通路、雌二醇信号通路、孕酮介导的卵母细胞成熟、促性腺激素释放激素信号通路和卵巢类固醇合成。

(19)中华人民共和国国家知识产权局(12)发明专利申请(10)申请公布号 (43)申请公布日 (21)申请号 201910105345.6(22)申请日 2019.02.01(71)申请人 中国水产科学研究院珠江水产研究所地址 510000 广东省广州市荔湾区芳村西塱兴渔路1号(72)发明人 林强　李宁求　付小哲　梁红茹　刘礼辉　牛银杰　(74)专利代理机构 广州三环专利商标代理有限公司 44202代理人 颜希文　宋静娜(51)Int.Cl.C12Q 1/70(2006.01)C12Q 1/686(2018.01)C12N 15/11(2006.01)(54)发明名称用于区分鳜鱼弹状病毒vRNA、cRNA和mRNA的引物组合及试剂盒(57)摘要本发明提供了用于区分鳜鱼弹状病毒vRNA、cRNA和mRNA的引物组，所述引物组包括用于反转录的引物和用于荧光定量扩增的引物，所述荧光定量扩增的引物序列如SEQ ID NO:4～9所示。

本发明通过在鳜鱼弹状病毒vRNA、cRNA和mRNA三种不同类型RNA的反转录引物的5’端和3’端分别加上标签序列，对三种不同类型的单链RNA进行反转录，然后用特异的标签序列作为荧光定量的正向引物对获得的cDNA进行扩增，可以区分鳜鱼弹状病毒vRNA、cRNA和mRNA，并且具有操作简单、灵敏度高、快速等特点，同时还可以对三种单链RNA进行定量检测。

权利要求书1页 说明书5页序列表2页 附图2页CN 109762938 A 2019.05.17C N 109762938A1.用于区分鳜鱼弹状病毒vRNA、cRNA和mRNA的引物组，其特征在于，所述引物组包括用于反转录的引物和用于荧光定量扩增的引物，所述荧光定量扩增的引物序列如SEQ ID NO:4～9所示。

2.如权利要求1所述的引物组合，其特征在于，所述用于反转录的引物，在所述每个引物的5’端和3’端分别加上标签序列；其中反转录引物的5’加上的标签序列区别vRNA，在3’加上的标签序列区别cRNA和mRNA。

核酸适配体在食品兽药残留检测中的应用研究进展李美芬，袁　月*，马　瑞（云南孚尔质量检验检测有限公司，云南昆明 650000）摘　要：食品安全问题是社会最关注的民生问题之一，而食品检测可以为食品安全保驾护航，其中动物性食品中的兽药残留检测是重要的一环。

核酸适配体作为一类具有特异识别能力的生物分子，在众多领域得到关注。

本文主要总结了核酸适配体在动物性食品兽药残留检测中的应用研究进展，介绍了纳米比色法、荧光分析法、化学发光分析法、电化学传感器检测法以及表面增强拉曼光谱效应等检测方法的机制及应用，并总结了不同方法存在的问题及未来发展趋势，以期为核酸适配体在食品安全检测领域的应用提供参考。

关键词：核酸适配体；兽药残留；动物性食品Research Progress in the Application of Nucleic Acid Aptamers in the Detection of Veterinary Drug Residues in FoodLI Meifen, YUAN Yue*, MA Rui(Yunnan Fair Quality Inspection Co., Ltd., Kunming 650000, China)Abstract: Food safety is one of the most concerned livelihood issues in society, and food testing can safeguard food safety. Among them, the detection of veterinary drug residues in animal based foods is an important part. As a class of biomolecules with specific recognition ability, nucleic acid aptamers have received attention in many fields. This paper mainly summarizes the progress of research on the application of nucleic acid aptamers in the detection of veterinary drug residues in animal food. It introduces the mechanisms and applications of detection methods such as nanocolorimetry, f l uorescence analysis, chemiluminescence analysis, electrochemical sensor detection, and surface enhanced Raman spectroscopy effect. It also summarizes the problems and future development trends of different methods, to provide reference for application of nucleic acid aptamers in the field of food safety testing.Keywords: nucleic acid aptamer; veterinary drug residues; animal derived food随着人们生活水平不断提高，动物性产品的需求量也在不断增加[1]，兽药残留问题也越来越受重视。

改进的 QuEChERS 方法用于鱼肉中孔雀石绿、隐色孔雀石绿、结晶紫和隐色结晶紫的快速检测朱程云;魏杰;董雪芳;郭志谋;刘名扬;梁鑫淼【摘要】孔雀石绿（MG）和结晶紫（CV）具有抗菌等活性，常被违法用于水产养殖业。

但 MG、CV 及其代谢产物隐色孔雀石绿（LMG）、隐色结晶紫（LCV）具有致癌性。

所以水产品中染料的残留检测是食品安全分析的重要问题。

由于水产品基质复杂，样品前处理尤为重要。

本文发展了一种基于 QuEChERS 技术与高效液相色谱联用的方法，用于鱼肉中4种染料的同时检测。

对 QuEChERS 方法中提取剂体积、提取次数以及分散固相萃取材料进行了优化。

结果表明反相/强阴离子交换材料（C18SAX）能有效提高回收率。

在最优条件下，4种染料在0.5~100mg / L 范围内线性良好，相关系数均大于0.998。

该方法在鱼肉中的回收率为73％~91％，RSD 为0.66％~5.41％。

结果表明该方法简单、高效，适合于鱼肉中染料的快速检测。

%Triphenylmethane dyes malachite green(MG)and crystal violet(CV)have been used as antimicro-bial,antiparasitic and antiseptic agents in aquaculture. However,MG and CV,as well as their metabolitesleu-comalachite green( LMG)and leucocrystal violet( LCV)are potential mutagens and carcinogens. Thus,the efficient determination of dye residues is of great concern. Considering the complexity of the aquatic products, the sample pretreatment is significant for decreasing matrix interference and improving detection sensitivity. In this study,a simple and rapid QuEChERS procedure was developed and combined with HPLC analysis for the simultaneous determination of the four dyes in fish tissue. An XCharge C18 column was applied in HPLC analy-sis to achieve goodpeak shape and selectivity. The pretreatment method involved the extraction of dyes from fish tissue and further clean-up with dispersive solid phase extraction(d-SPE)material. The extraction volume, extraction time as well as d-SPE materials were systematically optimized. The results indicated that reversed-phase / strong anion exchange(C18SAX)adsorbent in the d-SPE procedure could effectively improve the recov-ery compared with conventional C18 or C18 incorporated with primary secondary amine(PSA)material. Under optimized conditions,good linearity was achieved in the concentration range of 0. 5-100 mg / L with R 2 greater than 0. 998. The recoveries were 73% -91% and the precisions were 0. 66% -5. 41% . The results demonstrated the feasibility and efficiency of QuEChERS procedure incorporated with HPLC for dye monitoring.【期刊名称】《色谱》【年(卷),期】2014(000)004【总页数】7页(P419-425)【关键词】QuEChERS;高效液相色谱;孔雀石绿;隐色孔雀石绿;结晶紫;隐色结晶紫;鱼肉组织【作者】朱程云;魏杰;董雪芳;郭志谋;刘名扬;梁鑫淼【作者单位】大连交通大学环境与化学工程学院，辽宁大连 116028; 中国科学院大连化学物理研究所分离分析化学重点实验室，辽宁大连 116023;中国科学院大连化学物理研究所分离分析化学重点实验室，辽宁大连 116023;中国科学院大连化学物理研究所分离分析化学重点实验室，辽宁大连 116023;中国科学院大连化学物理研究所分离分析化学重点实验室，辽宁大连 116023;大连交通大学环境与化学工程学院，辽宁大连 116028; 中国科学院大连化学物理研究所分离分析化学重点实验室，辽宁大连 116023;中国科学院大连化学物理研究所分离分析化学重点实验室，辽宁大连 116023【正文语种】中文【中图分类】O658Malachite green(MG)and crystal violet(CV)，which are triphenylmethane dyes，have been widely used in aquaculture as antimicrobial，antiparasitic and antiseptic agents in the past decades［1，2］.The major metabolites of MG and CV，leucomalachite green(LMG)and leucocrystalviolet(LCV)possess similar biological characteristics［3］.Owing to their potential carcinogenicity，the addition of MG and CV in aquaculture is nowadays absolutely forbidden［1，4］.However，illegal utilization of dyes still exists because they are cheap and efficient［5］.Thus，multi-residues determination of dyes in aquatic products is of great significance. Considering the complexity of the matrix in aquatic products，sample pretreatment for decreasing or eliminating matrix interference and improving the detection sensitivity is of great concern.Numerous sample pretreatment methods have been established in previous reports［6-11］.Traditional liquid-liquid extraction method can be easily realized although it consumes large volume of solvents［6］.Enzyme-linked immune sorbent assay(ELISA)［12-14］exhibits good selectivity，but itsuffers from false positive results.Solid phase extraction(SPE)is a time-consuming method although the matrix interference can be efficiently removed ［15，16］.Molecularly imprinted solid phaseextraction(MISPE)for sample preparation reveals good specificity.However，the preparation of MISPE materials is difficult［17，18］.QuEChERS，representing the abbreviation of“quick，easy，cheap，effective，rugged and safe”，has been acknowledged as a rapid and efficient pretreatment method for the determination of hydrophobic pesticides［19-23］as well as veterinary drug residues［24，25］.The QuEChERS procedure involves two steps:(i)extraction/partitioning basedon the use of NaCl and MgSO4for salting out，and(ii)dispersive solid phase extraction(d-SPE)for clean-up.In consideration of the hydrophobicity of MG，LMG，CV and LCV，the introduction of QuEChERS for sample preparation is advantageous via extracting dye into the organic layer and cleaning-up by d-SPE procedure.By optimizing the extraction volume，extraction time and d-SPE materials，a specific pretreatment method was developed which greatly simplified the sample preparation and increased the throughput.With the combination of the proposed QuEChERS procedure and HPLC method based on XCharge C18 column，thesimultaneous determination ofMG，LMG，CV and LCV in fish tissues was successfully realized.1 Experimental1.1 Chemicals and materialsMalachite green(MG)，crystal violet(CV)and leucocrystal violet(LCV)werepurchased from Sigma-Aldrich(St.Louis，MO，USA)；leucomalachite green(LMG)was obtained from Dr Ehrenstorfer GmbH(Augsburg，Germany).Acetonitrile(ACN)of HPLC grade was purchased fromMerck(Darmstadt，Germany).Ammonium formate(NH4FA)and formicacid(FA)were purchased from J＆K Scientific(Beijing，China).Water for HPLC mobile phase was purified with a Milli-Q system(Millipore，Billerica，MA，USA).All other reagents were of analytical grade and used without further purification.Reversedphase/strong anion-exchange mixed-mode material(C18SAX)(40-75 μm，particle size)described in our previous report ［26］was selected as d-SPE adsorption material.C18，primary secondary amine(PSA)materials and XCharge C18 column(3.0 mm×100 mm i.d.，5μm particles)were from Acchrom Corp.(Beijing，China).1.2 HPLC conditionsA Hitachi Chromaster HPLC system(Tokyo，Japan)consisting of 5110 quaternary pump，5210 auto sampler，5310 column oven and 5430 diode array detector(DAD)was employed for HPLC analysis.The separation was performed on an XCharge C18 column.The column temperature was set at 40℃and flow rate was1 mL/min.The injection volume was20 μL.The mobile phase composed of ACN(A)，water(B)and 100 mmol/L ammonium formate(pH 3.0，C).The elution condition was 0-3 min，40%A-65%A；3-8 min，65%A-75%A，while mobile phase C was kept constant at 20%to obtain a buffer concentration of 20 mmol/L.Each dye was determined atthe maximum absorption wavelength:620 nm for MG，590 nm for CV，263 nm for LCV and LMG.1.3 Sample preparationCod fish was from local supermarket(Dalian，China).The skin and bone were removed.Then the fish was cut into strips and homogenized.The homogenized sample was stored at-20℃in the refrigerator until sample pretreatment.Fig.1 Structures of malachite green(MG)，crystal violet(CV)，leucomalachite green(LMG)and leucocrystal violet(LCV)The homogenized cod sample(2 g)was weighed into a 15 mL polypropylene tube.Then，2 mL of ammonium formate(100 mmol/L，pH 3.0)and 3 mL of ACN were added.The sample was shook vigorously to extract the analytes from the matrix.Then 2 g of sodium chloride was added into the solution for partitioning.The sample was mixed for about 2 min and centrifuged at 6000 r/min for 5 min.After that，1 mL of supernatant and 50 mg of C18SAX material were transferred into a 2.5 mL polypropylene tube.The polypropylene tube was shook and kept in ultrasonic bath for 1 min.The mixtu re was filtered through 0.22 μm membrane.The resulting filtrate was mixed with ammonium formate(100 mmol/L，pH 3.0)in the ratio of 4∶1(sample solution∶ammonium formate，v/v).The prepared solution was injected into the HPLC system for analysis. 1.4 Standard and reagent solutionsThe mixed stock solution with mass concentration of 100 mg/L was dissolved by ACN.It was stored at 4℃and protected against light for less than two weeks.The working solution was prepared through diluting the stock solution with the ini tial mobile phase(ACN∶water∶100 mmol/Lammonium formate=40∶40∶20，v/v/v).The concentrations of working solution were diluted at 0.5，1，5，10，25，50 μg/mL.2 R esults and discussion2.1 Establishment of chromatographic methodsThe resolution of MG，CV，LCV and LMG dyes(structures shown in Fig.1)which are basic compounds with good selectivity and peak shape is difficult on traditional C18 columns，especially for LCV and LMG ［6，27］.According to the previous studies of our group［28，29］，XCharge C18 column displayed great superiority in the separation of basic compounds.Therefore，XCharge C18 column was selected for the development of the analytical method and the mobile phase conditions was optimized.As shown in Fig.2a，the compounds could not be well resolved with 0.1%(v/v)FA/H2O in the mobile phase and LMG was not eluted within 10 min.Since the utilization of buffer solution is advantageous for improving peak shape and selectivity，100 mmol/L ammonium formate with pH 5.1 was employed as the mobile phase additive.The peak shapes were improved，but LCV and LMG were not well separated(Fig.2b).When the pH value of ammonium formate was changed to 3.0，the resolution of the four dyes was successfully achieved in 8 min with good peak shapes(Fig.2c).2.2 Establishment of pretreatment methodFig.2 Dyes separated on XCharge C18 column with different mobile phasesMobile phase conditions:a.ACN/(0.1%FA/water)；b.ACN/water/100 mmol/L NH4FA，pH 5.1；c.ACN/water/100 mmol/L NH4FA，pH 3.0.The QuEChERS procedure is flexible as it provides a template for sample preparation.The solvent and sorbent can be optimized according to the property of the target analytes.Mostly，the organic solvent used for extraction and partitioning is acetonitrile，which can easily generate phase separation with the addition of sodium chloride.In this work，the volume of extraction solvent and the kind of adsorption sorbent were systematically optimized to obtain best recovery and sensitivity.2.2.1 Optimization of extraction and partitioningThe volume of extraction solvent in QuEChERS procedure directly affects the sensitivity and recovery.The insufficient solvent would cause incomplete extraction，resulting in poor recovery，while excessive extraction solvent severely dilutes the analytes，leading to low detection sensitivity.To balance the recovery and sensitivity，the volume of acetonitrile for extraction was optimized from 2 mL to 7 mL.As shown in Fig.3，the peak areas of four dyes increased from 2 mL to 3 mL，and then slightly decreased from 3 mL to 7 mL.The results indicated that 3 mL of acetonitrile was optimal for the extraction of dyes in 2 g of homogenized fish tissue.More acetonitrile could not improve the extraction efficiency but reduce the detection sensitivity due to the dilution of analytes.Thus the volume of extraction solvent was determined to be 3 mL.Then the extraction time was verified.The homogenized fish tissue(2 g)was first extracted by 2 mL of acetonitrile，and then 1 mL of acetonitrile for re-extraction.The supernatants were merged and detected.The results showedno obvious differences(results not shown).So the fish tissue was extracted once with 3 mL of acetonitrile.Fig.3 Influence of extraction volume on the peak areas of dyesIn QuEChERS procedure，partitioning was achieved by the use of sodium chloride and magnesium sulfate.In this case，the recovery of the dyes was obviously deteriorated when magnesium sulfate was used.Therefore only sodium chloride was used for partitioning.In order to drive water from acetonitrile layer，excess sodium chloride(2 g)was added.Hardly any dyes were detected in aqueous layer.And the acetonitrile layer was transferred into a polypropylene tube for d-SPE clean-up before HPLC analysis.2.2.2 Selectivity of adsorption sorbentAfter the extraction/partitioning，dyes and some nonpolar interferences were partitioned into the acetonitrile layer.In order to remove the interferences and clean-up the sample，d-SPE with a proper adsorptive material was performed.C18 and PSA(structures shown in Fig.4)are commonly used materials for adsorption ［30，31］.C18 mainly adsorbs fat，lipid and some other non-polar interferences，while PSA adsorbs fatty acids and organic acids，etc.In this experiment，C18 and PSA were first employed as adsorbents.As shown in Table 1，the recoveries of dyes were about 70%-90%with C18 as d-SPE material.If PSA was used together with C18，the recoveries reduced to about 40%-80%.The color of the sorbents after d-SPE was shown in Fig.5.In Fig.5a(C18 material)andFig.5b(C18 and PSA materi-als)，the sorbents were obviously changed to blue or violet，further demonstrating the adsorption of MG and CV.As thefour dyes were all hydrophobic and basic compounds，the electrostatic attraction between dyes and silanol groups on C18 material together with the original hydrophobic interaction led to low recoveries.When PSA was added，the alkaline PSA enhanced the ionization of silanols both on C18 and PSA，resulting in poor recoveries.Fig.4 Structures of C18，PSA and C18SAX materialsFig.5 Color of adsorbents in lower layers after d-SPE procedurea.C18；b.C18 and PSA；c.C18SAX.Table 1 R ecoveries of the four dyes after d-SPE with different adsorbentsAdsorbent Recoveries/%MG CV LCVLMG C18(50.0 mg) 86.8 84.8 73.6 87.7 C18(50.0 mg)and PSA(50.0 mg)46.0 40.6 78.0 83.7C18SAX(50.0 mg) 101.4 102.2 83.5 87.6In a previous work［26］，we developed a reversedphase/strong anion-exchange mixed-mode stationary phase(named C18SAX，Fig.4)based on polar-copolymerized approach.The C18SAX material possesses C18 and quaternary ammonium groups，which can provide hydrophobic and electrostatic interaction simultaneously.With C18SAX as d-SPE material，the nonpolar and acidic interferences in the acetonitrile layer were adsorbed by the sorbent.Meanwhile，the quaternary ammonium group provided electrostatic repulsion interaction against the basic dyes.As shown in Table 1 and Fig.5c，the recoveries were in the range of 83.51%-102.19% for the four dyes，and the C18SAX material was still white after the adsorption.Consequently，C18SAX mixed-mode material is superior to the mechanically mixed C18 and PSA materials in cleaning-up the sampleand avoiding adsorption of dyes.Based on the above discussions，this method was easily streamlined in the determination of dye residues in aquatic products.As shown in Fig.6，Part I concerns the extraction/partitioning，while PartⅡ involves the clean-up of the sample with d-SPE material，and PartⅢis for HPLC analysis.2.3 Method validationThe performance of the optimized approach for the determination of MG，CV，LCV and LMG dyes was validated with respect to the linearity，recovery，intraday and inter-day precisions as well as limit ofdetection(LOD)and limit of quantitation(LOQ).The calibration was performed with the use of matrix-matched standards.As shown in Table 2，the four dyes expressed good linearities in the range of 0.5-100 mg/L，with correlation coefficients all above 0.998.In the spiked range from 5 to 25 mg/L，the recoveries were between88.63%and 110.62%，with intra-day and inter-day precisions of 0.7%-3.5%and 1.6%-5.4%，respectively.The LODs(calculated by signal to noise ratio of 3)were 3.2 μg/kg for MG，Fig.6 Flowchart of dye determination:extraction/partitioning(part I)，clean-up by dispersive solid phase extraction material(part II)and HPLCanalysis(part III)Table 2 Matrix matched calibrations and validation data for fish tissueAnalyte Linear range/(mg/L)Correlation coefficientSpiked/(mg/L)Recovery/%RSD/%Intra-day(n=5)Inter-day(n=15)Sensitivity LOD/(μg/kg)LOQ/(μg/kg)MG 0.5-100.0 0.999 5 88.6 1.7 2.3 3.2 9.610 97.91.72.825 103 1.5 1.6 CV 0.5-100.0 0.999 5 96 0.7 2.1 1.9 5.710 105.4 1.22.325 110.6 0.8 1.8 LCV 0.5-100.0 0.998 5 95.8 1.4 4.2 23.4 70.210 96.3 1.6 1.925 101.2 34.2 LMG 0.5-100.0 0.999 5 94.1 3.5 3.1 24.1 72.310 92.8 0.7 3.125 110.7 3.55.42.4 Sample analysisThe optimized method was applied to detect MG，CV，LCV and LMG in fish samples which were bought from local supermarket.The results of two batches of cod samples were negative.3 ConclusionsThe present study demonstrated the simplicity and high-efficiency of QuEChERS pretreatment method combined with HPLC for the fast analysis of triphenylmethane dyes in fish tissue.XCharge C18 was applied in the separation of MG，LMG，CV and LCV with good selectivity and peak shape.The extraction/partitioning and d-SPE procedure in QuEChERS were systematically investigated.A C18SAX mixed-mode adsorbent was employed in the d-SPE procedure，exhibiting better recovery than conventional adsorbents.Method validation data showed satisfactory recoveries and precisions.On the other hand，the limitation of the present method also exists.The detection sensitivity cannot meet the demand ofthe minimum required performance limit(MRPL)for the dyes.In future work，MS detector will be applied for the improvement of the sensitivity. References:［1］Arroyo D，Ortiz M C，Sarabia L A，et al.J Chromatogr A，2009，1216(29):5472［2］Zhang Z，Zhou K，Bu Y Q，et al.Anal Methods，2012，4(2):429 ［3］Ascari J，Dracz S，Santos F A，et al.Food Addit Contam，2012，29(4):602［4］Andersen W C，Turnipseed S B，Karbiwnyk C M，et al.Anal Chim Acta，2009，637(1/2):279［5］Lee J B，Kim H Y，Jang Y M，et al.Food Addit Contam，2010，27(7):953［6］Chen G Y，Miao S.J Agric Food Chem，2010，58(12):7109［7］Tarbin J A，Chan D，Stubbings G，et al.Anal Chim Acta，2008，625(2):188［8］An L，Deng J，Zhou L，et al.J Hazard Mater，2010，175(1):883 ［9］Tsai C H，Lin J D，Lin C H.Talanta，2007，72(2):368［10］Hurtaud-Pessel D，Couedor P，Verdon E.J Chromatogr A，2011，1218(12):1632［11］Fux E，Rode D，Bire R，et al.Food Addit Contam，2008，25(8):1024［12］Shen Y D，Deng X F，Xu Z L，et al.Anal Chim Acta，2011，707(1/2):148［13］Xing W W，He L，Yang H，et al.J Sci Food Agric，2009，89(13):2165［14］Durnez L，Bortel W V，Denis L，et al.Malar J，2011，10:195 ［15］Deng J C，Li L H，Yang X Q，et al.Food Science，2012，33(14):150 ［16］Stubbings G，Tarbin J，Cooper A，et al.Anal Chim Acta，2005，547(2):262［17］Lian Z R，Wang J T.Mar Pollut Bull，2012，64(12):2656［18］Li Y H，Yang T，Qi X L，et al.Anal Chim Acta，2008，624(2):317 ［19］Chen W，Ren Y D，Liu H，et al.Journal of Henan Agricultural Sciences，2011，40(2):111［20］Paya P，Anastassiades M，Mack D，et al.Anal Bioanal Chem，2007，389(6):1697［21］Zhang Z Y，Gong Y，Shan W L，et al.Chinese Journal of Chromatography，2012，30(1):91［22］Huang Y C，Ding W W，Zhang Z M，et al.Chinese Journal of Chromatography，2013，31(7):613［23］Chen X S，Bian Z Y，Yang F，et al.Chinese Journal of Chromatography，2013，31(11):1116［24］Aguilera-Luiz M M，Vidal J L M，Romero-Gonzalez R，et al.J Chromatogr A，2008，1205(1/2):10［25］Stubbings G，Bigwood T.Anal Chim Acta，2009，637(1/2):68 ［26］Wei J，Guo Z M，Zhang P J，et al.J Chromatogr A，2012，1246:129［27］Stella C，Rudaz S，Veuthey J L，et al.Chromatographia，2001，53:S-113［28］Wang C R，Guo Z M，Long Z，et al.J Chromatogr A，2013，1281:60［29］Zhang J C，Wei J，Zhong H M，et al.Chinese Journal of Chromatography，2013，31(1):79［30］Lehotay S J，Son K A，Kwon H，et al.J Chromatogr A，2010，1217(16):2548［31］Walorczyk S.J Chromatogr A，2008，1208(1/2):202。

高效液相色谱法检测鱼肉组织中金霉素、多西环素、米诺环素的残留作者：谈暠媛来源：《湖北畜牧兽医》2013年第06期摘要：建立了鱼肉组织中金霉素、多西环素、米诺环素的多残留检测的高效液相色谱法（HPLC）。

样品经0.1 mol/LEDTA-McIlvaine缓冲液（pH 4.0）提取离心后，上清液用Oasis HLB固相萃取小柱净化，选用安捷伦Eclipse plus C18色谱柱（250 mm×4.6 mm，粒径5μm），以乙腈：甲醇：0.01 mol/L草酸溶液（2﹕1﹕7）为流动相，流速0.5 mL/min，350 nm 波长处测定，外标法定量。

其3种药物浓度在25～200 ng/mL时均与峰面积呈良好的线性关系。

结果表明，3种目标物质在鱼肉组织中的检测限为25 μg/kg，定量限为50 μg/kg。

在不同浓度添加水平下测定，平均回收率在76.5%～89.1%，批内、批间变异系数均在10.00%以内。

关键词：金霉素；多西环素；米诺环素；多残留；高效液相色谱法（HPLC）中图分类号：TS254.7 文献标识码：A 文章编号：1007-273X（2013）06-0017-05四环素类（Tetracyclines，TCs）药物是由放线菌产生的一类广谱抗生素，对革兰氏阳性和阴性细菌、立克次氏体等均有很好的抑菌作用，常被用于畜、禽和水产养殖中[1]。

目前国内四环素类抗生素主要包括六种：金霉素、土霉素、四环素、强力霉素（多西环素）、美他环素和米诺环素。

本文针对3种四环素类抗生素药物的特性，研究其在鱼肉中的多残留检测方法。

方法采用高效液相色谱法，其具有选择性高、分离效果好、检测灵敏度高等优点，其准确度和回收率均能满足国家规定的对四环素类药物残留限量的要求，具有较好的通用性和推广性。

1 材料与方法1.1 仪器与设备Waters2695高效液相色谱仪（配2487型紫外检测器）；分析天平；回旋振荡器；旋涡混合器；数显恒温水浴锅；高速冷冻离心机；氮吹仪；Waters Oasis HLB固相萃取小柱（60 mg，3 mL）1.2 药品与试剂金霉素（CTC）、多西环素（DC）、米诺环素（MINO）标准品含量均大于80.0%。

·2827·收稿日期：2020-02-18基金项目：上海市自然科学基金项目（20ZR1423600）；上海市扬帆人才计划项目（19YF1419400）作者简介：*为通讯作者，陈再忠（1972-），博士，教授，主要从事观赏鱼繁殖遗传育种生物学研究工作，E-mail ：***************.cn 。

刘怡南（1994-），研究方向为观赏鱼繁殖生物学，E-mail ：**********************七彩神仙鱼脑组织转录组mRNAs 差异表达分析刘怡南1，2，3，温彬1，2，3，陈再忠1，2，3*（1水产科学国家级实验教学示范中心（上海海洋大学），上海201306；2上海水产养殖工程技术研究中心（上海海洋大学），上海201306；3农业农村部淡水水产种质资源重点实验室（上海海洋大学），上海201306）摘要：【目的】挖掘七彩神仙鱼（Symphysodon haraldi ）脑组织性别差异基因，为揭示脑组织性别相关基因调控繁殖生理机制打下基础。

【方法】利用Illumina HiSeq 6000测序平台对七彩神仙鱼雌、雄脑组织样本进行转录组测序分析，经过滤和Trinity 组装获得基因，采用DIAMOND 进行功能注释；并选取NR 、GO 、KEGG 、Pfam 、Swiss-Prot 和egg-NOG 等数据库进行比对，筛选出差异表达候选基因；随机选取6个差异表达基因进行实时荧光定量PCR 验证。

【结果】从构建的七彩神仙鱼脑组织cDNA 文库测序获得337190200条原始数据（Raw reads ），经质量筛选后获得34109个基因（平均长度1007.00bp ）和67488个转录本（平均长度694.00bp ）。

经生物信息学分析方法筛选，最终获得85个差异表达基因（61个在雄鱼脑组织中高表达，24个在雌鱼脑组织中高表达），包括黑色素浓集激素（MCH ）、催乳素释放激素（Prlh ）、垂体同源结构域转录因子2（pitx2）、免疫球蛋白家族成员（DSCAM 和IGDCC3）、溶质载体（UNC93B1）及醛糖还原酶（AKR1B1）等功能基因。

微波萃取气相色谱法测定鱼肉中有机氯残留第23卷第1期2010年2月四川理工学院(自然科学版)JournalofSichuanUniversityofScience&Engineering(NaturalScienceEdition)V oL23No.1Feb.2010文章编号:1673—1549(2010)01-0062-03微波萃取气相色谱法测定鱼肉中有机氯残留郑林,施泽明,李佳宣,林清梅,倪师军(1.成都理工大学地球化学系,成都610051;2.福建省核工业294大队,福州350013) 摘要:探索测定鱼肉中有机氯的快速,准确和价廉的方法.采用微波萃取技术提取鱼肉中的六六六,滴滴涕,再利用毛细管气相色谱分离,微电子捕获检测器检测.8种有机氯农药回收率在89.3%一104.2%之间,相对标准偏差均小于10%.说明准确度与精密度较好,符合农药残留量分析的基本要求.探索了各成分在不同食性鱼肉中的含量特征,结果显示:肉食性鱼类>杂食性鱼类>草食性鱼类.可见,有机氯农药随着食物链在生物体内富集.关键词:微波萃取;气相色谱;鱼;有机氯中图分类号:0656.31文献标识码:A有机氯农药(OCPs)是一类全球性环境污染物,是历史上最早大规模使用过的高残毒农药,使用时间长,用量大.在环境中降解缓慢,滞留时间长,有半挥发性和较强的亲脂憎水性,可沿食物链逐级放大并可在环境中远距离迁移,使存在于大气,水,土壤内的低浓度OCPs物质通过食物链对处于高营养级的生物或人类健康造成损害,并可通过"蒸馏效应"或"蚱蜢跳效应"转移到地球的绝大多数地区,导致全球范围的污染.OCPs物质对人体产生的主要危害可能是对肝,肾等脏器和神经系统,内分泌系统,生殖系统等有急性和慢性毒性作用,可表现出对试验动物有致癌性,生殖毒性,神经毒性,内分泌干扰毒性等.在生态环境中,鱼类是食物链中的一个重要环节,因而准确,快速测定鱼组织中有机氯农药残留具有重要意义.目前,测定鱼组织样品中OCPs的处理方法较多,主要有溶剂萃取法,固相萃取法,基质固相扩散法(MSPD)及超临界液相萃取法等,但这些方法存在消耗溶剂量大或分析时间长的缺点.本文利用微波萃取,层析柱净化,大大缩短了样品处理时问,提高了方法的灵敏度和准确度,适用于大批量食品和生物样品的分析测定.1材料与方法1.1仪器与试剂微波萃取仪(MARS)(美国CEM公司);Agilent7890A系列气相色谱仪带电子捕获检测器(一ECD, "Ni)配Agilent.B03.02工作站(Agilent,USA),所用色谱柱为HP一5—19091J～413石英毛细管柱(0.32mm×0.25umX30m,甲基硅氧烷).无水硫酸钠(分析纯,650~C灼烧4h,贮于密封瓶内备用,用时先用正己烷淋洗);浓硫酸(优级纯);丙酮(分析纯,天津化学试剂厂);石油醚(色谱纯,天津化学试剂厂);正己烷(色谱纯,天津化学试剂厂,用KMnO一HSO水溶液和KMnO一NaOH水溶液各洗3次,水洗干燥后精馏备用).有机氯农药标准:Ot—HCH,13一HCH,一HCH,6一HCH,P,P一DDE,O,P一DDT,P,P一DDD,P,P一DDT8种物质混合标准(国家标准物质中心),各化合物质量浓度均为100.OOmg/L,石油醚为溶剂.溶剂处理:由于在环境样品中被测组分的浓度一般都较低,因此分析中必须十分重视溶剂空白问题.根据我们的实验结果,普通的分析纯试剂即使经过全玻璃系统重蒸馏后也难以得到满意的结果.在溶剂处理方面主要采取氧化洗涤和精馏方法以除去干扰物质.1.2样品分析1.2.1样品处理用分析天平准确称取1.000g鱼肉样,装入萃取罐.加入10.0mL萃取液(正己烷:丙酮=1:1),100℃萃取30min,后将上清液倒人离心管.残渣再萃取一次,以确收稿日期:2009-12-02基金项目:中国地质调查局"成都经济区城市生态地球化学调查与评价"和国家环保部"汶川特大地震造成矿山破坏对农田土壤污染评估与应对措施"联合资助(200314200015)作者简介:郑林(1983一),男,湖南常德人,硕士生,主要从事环境地球化学方面的研究.第23卷第1期郑林等:微波萃取气相色谱法lJ定鱼堕有扭望63保农残完全被萃取出,最后将20.0mL萃取后的溶液用离心机(4500r/rain)进行离心5min.经无水硫酸钠脱水,于旋转蒸发器中浓缩.将浓缩液用正己烷定量转移至10mL具有刻度试管中定容至5.0mL,小心加入1.0mL10%H2SO的硫酸,振摇1min,以3000r/min离心10rain,取上清液至层析柱(层析液:正己烷:丙酮:9:1),收集洗脱液(正己烷:丙酮=4:1),浓缩,定容至1.0mL于样品瓶中,待GC测试.1.2.2仪器条件GC操作条件:进样口温度260℃,检钡4器温度320~C.采用程序升温,初始温度80~C,保持1rain后以10℃/min升至200℃,保持2min;4℃/rain升至250cI二, 保持2min;10℃/rain升至280℃,保持10rain.气体流速:氮气60.0mlMmin,尾吹气20.0mL/min,脉冲不分流进样,柱流速3.8mL/min,柱压:8.586Pa.2结果与讨论2.1方法性能及指标考察2.1.18种有机氯农药的标准色谱图8种有机氯农药(1000~g/L)的标准色谱图如图1所示,从图1可以看出各化合物实现了有效分离,图2为实际样品中有机氯农药残留的色谱图.图1有机氯农药混合标样色谱图图2鱼样中有机氯农药残留的色谱图2.1.2标准曲线及线性范围将有机氯混合标样用正己烷溶解配成10.0mg/L标准储备液,保存于3℃冰箱中,取配好的标准储备液,用正己烷分别稀释成5g/L,50~g/L,100g/,L,1000~g/L,进样2L.根据浓度与峰面积的关系作标准曲线(见表1).表1八种有机氯农药的分析灵敏度2.1.3微波萃取回收率实验微波萃取可以达到常压下使用同样溶剂所达不到的萃取温度.用某一鱼肉样做萃取回收率实验,结果见表2. 表2微波萃取回收率2.1.4加标回收率和精密度在鱼肉样品中添3H8种有机氯农药的标准溶液,每种农药进行5次回收试验,其回收率和相对标准偏差(RSD) 见表l.8种有机氯农药回收率在89.3%一104.2%之间,相对标准偏差均小于10%.符合农残检测的要求.说明准确度与精密度较好,符合农药残留量分析的基本要求. 2.1.5检出限在加标鱼肉样品中,以各目标组分的3倍信噪比为检出限(LOD),获得该方法的LOD(见表1).2.1.6定性分析根据各组分的保留时间进行定性,各组分的出峰顺序分另U为:一HCH,p—HCH,一HCH,8一HCH,P,P (一DDE,O,P(一DDT,P,P(一DDD,P,P(一DDT.2.1.7定量分析用峰面积外标法进行定量分析,计算方法为:Ri=Ai×Wi×V/Ai×V,×G式中:R;一样品中i组分农药的含量,mg/kg;A.一样品中i组分农药的峰面积;W～样品中i组分农药的量,ng;V一样品定容体积,mL;Ai.一样品中i组分农药的峰面积;V;一样品的进样量,IxL;G一样品的重量,g.2.2测试结果与分析不同种类鱼的鱼肉中8种有机氯含量,结果见表3.64四川理工学院(自然科学版)2010年2月由表3可知,从市场上购买的各种鱼中有机氯农药残留量都未超过国家标准0.1mg/kg,这说明成都市东郊市场上此类食品是安全的.不同种类鱼体内8种有机氯农药含量有一定的差异,由高到低依次为:鲶鱼>鲤鱼>鲫鱼>草鱼.由于鲶鱼为底层肉食性鱼类,污染源排入水中的有机氯主要沉积于水底,加之鲶鱼的食物链长,取食含农药量多的动物性饵料,其体内脂肪成分含量较高,体内容易累积较多的有机氯.鲫鱼和鲤鱼为底层杂食性鱼类,食物链较短,鲤,鲫鱼属中下层鱼类,以浮游生物,底栖动物及水草为食,还可从底泥沉积物中吸收有机物,故体内累积的有机氯较鲶鱼少.以水草碎屑及浮游生物为食的草食性鱼类,如草鱼体内积累的有机氯最少.可以看出,不同鱼类总有机氯含量水平由高到低依次为:肉食性鱼类>杂食性鱼类>草食性鱼类, 有机氯在食物链中有逐级富集的趋势.这种比较结果,符合水生生态系统中占据较高生态位的肉食性鱼类更容易富集持久性有机物的规律,和国内外很多学者研究结果一致.窦薇对白洋淀几种不同食性鱼类对六六六,滴滴涕的富集研究显示,草食性鱼体内农药残留量最少,其次是杂食性鱼类,肉食性鱼体内农药残留量最高.鲫鱼(杂食)比草鱼(草食)富集系数高与其食性有关.董军¨¨对珠江三角洲淡水养殖沉积物及鱼体中DDT和PAH的残留分析同样显示,肉食性鱼鳜鱼和鳙鱼有机物质量分数高于杂食性和草食鱼类.此外,施治u一对天津地区鱼塘水,悬浮物,沉积物和鱼体中的DDT研究显示,所测鱼体各器官中XDDT含量均高于鱼肉.3结束语(1)建立了用微波萃取与GC—ECD联用分析测定鱼肉中六六六和滴滴涕的系统分析方法.结果表明利用微波萃取来提取鱼肉中微量六六六,滴滴涕是快速,有效的.如果很好的控制萃取操作条件,消除在分析过程中本底干扰,正确操作GC,本法具有很好的准确性及精密度.(2)对鱼体内有机氯含量进行测试,结果表明,各成分都有检出,说明鱼已受到有机氯农药的污染,但其含量远低于食用卫生标准.鱼体内∑HCH和∑DDT残留量均未超过我国鱼肉食用卫生标准,但其潜在危险不容忽视.(3)几种不同食性鱼类中,草食性鱼体内农药残留量最少,其次是杂食性鱼类,肉食性鱼体内农药残留量最高.可见,有机氯农药随着食物链在生物体内富集.参考文献:[1】WaniaF,MackayD.Trackingthedistn'butionofpenis—tentorganicpollutants[J].EnvironmentalScience& Technology,1996,30:390-396.[2】ZhangGParkerA,HouseA,eta1.SedimentaryRecords ofDDTandHCHinthePearRiverDelta,SouthChina [J].EnvironSciTechnol2003,36:3671-3677.[3】BeyerA,MackayD,MatthiesM,eta1.Assessingthe Long—rangeTransportPotentmlofPersistentOrganic Pollutants[J].EnvironSciTechnol2000,34:699—703. [4】TatsumEndo,AdiraOkuyarm,Y asutakaMatsubara,et a1.RelativeConcentrationsofOrganoch~rinesinAdi- poseTissueandSerumamongReproductiveAgeWor~n[J].AnalyticaChirrficaActa2005,531(1):7-13. [5】AtsushiMatsunaga,AkioYasuhara.BirthDefectsRisk AssocmtedwithMatemalSportFishConsurrotion:Po- tentmlEffectModificationbySexofOffspring[J].Chem- osphere2005,58(7):897-904.[6]刘晨,陈家玮,杨忠芳.地球化学样品中有机农药残留分析预处理方法新进展[J].地质通报,2007,26(11): 1499-1502.[7】李攻科,何小青,熊国华,等.用微波消解气相色谱法测定鱼肉中的有机氯农药[J].分析测试,1999,18 (4):5—8.[8】GB2763-2005,食品中农药的最大残留限量[S】.[9]窦薇,赵忠宪.白洋淀几种不同食性鱼类对六六六, DDT的富集[J].环境科学进展,1996,6(4):51-56. [10]施治,潘波,何新春,等.天津地区鱼塘水,悬浮物,沉积物和鱼体中的DDT[J].农村生态环境,2004,20 (4):1—5.[11]董军,栾天罡,邹世春,等.珠江三角洲淡水养殖沉积物及鱼体中DDTs和PAHs的残留与风险分析[J].生态环境2006.15(4):693-696.(下转第68页)68四川理工学院(自然科学版)2010年2月[3]史丰华.贯叶连翘中有效成分的分离纯化工艺及其检测方法的研究[D].重庆:重庆大学2005.[4]汪茂田,谢培山,王忠东,等.天然有机化合物提取分离与结构鉴定[M】.北京:化学工业出版社20O4. [5]SuenSAcomparisonofIsothermandkineticmodels forbinarysohteadsorptbntoaffinit~rmembranes[J].J ChemTechnolBiotechnol,1996,(65)2249-257.[6】KimY,KimC,ChoiI,eta1.Arsenicremovalusingilleso—porousaluminapreparedviaatemplatingmethod[J]. EnvironSciTechnol2oo4,(38).924—931.[7]AnaGL.Sorptionofantimonyontohydmxyapatite[J]. EnvironmentScienceandTechnology,2001,35(2):3669—3675.[8]孙磊,王玉蓉,李维峰.大孔吸附树脂吸附远志总皂苷的吸附热力学和动力学研究[J】.北京中医药大学,20o6'29(11):772?775.[9】孔凡彬,徐瑞富,谢国红,等.两种大孔树脂对水溶液中克百威的吸附行为[J].山西农业科学,2007,35(5):66-69.[10]张磊,徐环昕,刘坐镇.HZ816大孔树脂对番茄红素的吸附特性研究[J].现代食品科技,2009,25(3):232—236.[11】BellJP,TsezosM.Removalofha~rdousorganicpol- htantsbybiormssadsorption[J].JWaterPolhtCon—tmlFed,1987,59:191.[12】KunioE,FushengL,Y osh~imA,eta1.Poredistnq3ution effectofactivatedcarboninadsobingorganicmi- cropollutantsfromnaturalwater[J].WatRes200135(1):167 ThermodynamicalResearchonAdsorptionofHypericinonMacroporousHZ8160Resin ZHANGLi,LIUChun—xin,FENGXi.wen.,HEY ong,HELingxing(1.SchoolofChemistryandPharmacenticalEngineering,SichuanUniversityofScience&a mp;Engineering,Zigong643000,China;2.SchoolofMaterialandChemicalEngineering,SichuanUniversityofScience&Engi neering,Zigong643000,China)Abstract:Theadsorptionthermodynamicsofhypericininsolutionwasstudied.Studiesindic atethathypericinadsorp—tionontoHZ8160resinconformstoFreundlichadsorptionisothermequation,withn>1,A H<0,AG<0andAS<0, whichshowedthattheadsorptionprocessofhypericinonmacroporousresinHZ8160wasasp ontaneous,exothermicandfa—vourableprocess,whichbelongstothephysicaladsorption.Keywords:hypeficin;macroporousresin;thermodynamics(上接第64页) AnalysisofOrganicChlorinatedPesticidesinFishbyMicrowaveExtraction/CapillaryGC ZHENGLini.SHIZe.mingl,LIJia一~fllztnl,LINQg.meil.NIShi-junl(1.DepartmentofGeochemistry,ChengduUniversityofTechnology,Chengdu610051,Chi na;2.No.294GeologicalPaayofFujianProvincialNuclearIndust~,Fuzhou350013,China) Abstract:Theaimwastoexplorethequick,exactandcheapmethodfordetectiononfish.Theor ganicchlorinatedpes—tlcides(HCHsandDDTs)infishwereextractedbymicrowaveextractionandanalyzedbycapi llaryGCwithIxECD.Theex—tractionrecoverywas89.3%一104.2%.andRSDwaslessthan10%.Accuracyandprecisionwhichwerebetteraccordedto therequirementsofanalysisofpesticideresidues.Exploredthecontentcharacteristicsoforga nochlorineinkindsoffish.Theresultsshowedthat:flesh—eatfish>omnivorousfish>anochlorinepesticidesen richedintheorganismsbodywiththefoodchain.Keywords:microwaveextraction;GC-ECD;fish;organochlorine。

480分析化学第52卷[81]QIAN S,CHEN Y,PENG C,WANG X,CHE Y,WANG T,WU J,XU J.Anal.Chim.Acta,2023,1239:340670.[82]XING G,SHANG Y,WANG X,LIN H,CHEN S,PU Q,LIN L.Biosens.Bioelectron.,2023,220:114885.[83]ZHUANG J,ZHAO Z,LIAN K,YIN L,WANG J,MAN S,LIU G,MA L.Biosens.Bioelectron.,2022,207:114167.[84]ZHOU J,CHEN P,WANG H,LIU H,LI Y,ZHANG Y,WU Y,PAEK C,SUN Z,LEI J,YIN L.Mol.Ther.,2022,30(1):244-255.Advances of CRISPR/Cas-based Biosensor in Detection ofFood-Borne PathogensZHANG Xiao-Yuan1,2,YAO Zhi-Hao2,HE Kai-Yu2,WANG Hong-Mei2,XU Xia-Hong2,WU Zu-Fang*1,WANG Liu*21(College of Food and Pharmacy,Ningbo University,Ningbo315000,China) 2(Institute of Agro-product Safety and Nutrition,Zhejiang Academy of Agricultural Sciences,Hangzhou310021,China)Abstract Rapid and accurate detection methods for food-borne pathogens are essential to ensure food safety and human health.One promising innovation in this area is the clustered regularly interspaced short palindromic repeats/CRISPR-associated systems(CRISPR/Cas)biosensor,which utilizes Cas protein and CRISPR RNA (crRNA)ribonucleo protein to specifically recognize target genes,and converts target signals into detectable physical and chemical signals.The CRISPR/Cas biosensor shows many advantages,such as high specificity, programmability,and ease of use,making it promising to pathogen detection.This paper introduced the principles and characteristics of CRISPR/Cas systems,along with the strategies for signal recognition,amplification,and output based on different CRISPR/Cas biosensors,and their respective applications in food-borne pathogen detection.Furthermore,the construction principles and challenges of multiple biosensors based on CRISPR/Cas were explored,as well as their potential for simultaneous detection of multiple pathogens.Finally,the challenges and future development trends of CRISPR/Cas-based biosensors in rapid pathogen detection were discussed, aiming to provide valuable reference and inspiration for biosensor designers and food safety practitioners. Keywords Food-borne pathogens;CRISPR/Cas;Biosensor;Review(Received2023-09-15;accepted2024-02-01) Supported by the National Natural Science Foundation of China(No.32172307)and the Basic Public Welfare Research Program of Zhejiang Province,China(No.LGN21C200007).第52卷分析化学（FENXI HUAXUE）评述与进展第4期2024年4月Chinese Journal of Analytical Chemistry481~491DOI:10.19756/j.issn.0253-3820.221448 RNA荧光适配体在生物传感与成像中的应用研究进展邱星晨#1,3范存霞#1,2白瑞1谷雨*1李长明*1郭春显*1（苏州科技大学材料科学与工程学院1，物理科学与技术学院2，环境科学与工程学院3，苏州215009）摘要RNA荧光适配体是近年来发展起来的一种简单且有效的RNA分子荧光标记工具，可与不发光或发光微弱的小分子荧光团结合，显著增强其发光性能。

rna fish原理
RNA鱼（Fish）是一种适用于特定实验室技术的方法，用于
研究RNA的结构和功能。

该方法包括多种技术和实验步骤，
可以帮助科学家深入了解RNA的性质和功能。

RNA鱼的原理是基于互补配对的作用。

在进行RNA鱼实验时，首先需要设计和合成一系列特定的探针。

这些探针可以与感兴趣的RNA序列相互配对，形成一个稳定的复合体。

探针通常
包含了标记物，例如荧光染料或辐射性同位素，这有助于观察和检测RNA的位置和分布。

一旦探针与RNA目标序列结合，实验者可以使用不同的方法
来检测标记物的存在。

例如，可以使用荧光显微镜观察成像，以确定RNA的定位。

另外，还可以使用辐射探测器来测量辐
射性同位素的放射强度，从而确定RNA的存在和分布。

通过RNA鱼的实验，科学家可以获得有关RNA的重要信息。

他们可以确定RNA的位置、分布和数量，为研究RNA功能
提供了关键的线索。

此外，RNA鱼方法还可以用于研究RNA
诱导的表观遗传修饰和RNA-RNA相互作用等特定研究领域。

总结而言，RNA鱼是一种基于互补配对的实验方法，可以帮
助科学家研究RNA的结构和功能。

通过设计和合成特定的探针，并使用荧光显微镜或辐射探测器等技术，可以确定RNA
的位置、分布和数量，从而为RNA功能研究提供有价值的信息。