CN116970719B - 一种间接检测水中镉污染的方法 - Google Patents
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Abstract
本发明属于重金属检测方法技术领域,具体涉及一种间接检测水中镉污染的方法。本发明通过检测样品水体的红条毛肤石鳖中基因ArWz‑2含量,发现ArWz‑2含量与水体中镉含量相关,通过二者的对应关系可以检测水中镉含量,以对水质进行监测。相对于传统的直接检测方法,本发明避免了强腐蚀性试剂对检测人员的危害,且对水中镉检测的敏感程度更高。
Description
技术领域
本发明属于重金属检测方法技术领域,具体涉及一种间接检测水中镉污染的方法。
背景技术
镉是人体非必需元素,在自然界中常以化合物状态存在,一般含量很低,正常环境状态下,不会影响人体健康。但当环境受到镉污染后,镉可在生物体内富集,通过食物链进入人体引起慢性中毒。因此对水中的镉含量进行监测十分必要。
水体中镉污染主要来自地表径流和工业废水。大气中的铅锌矿以及有色金属冶炼、燃烧、塑料制品的焚烧形成的镉颗粒都可能进入水中;用锅作原料的触媒、颜料、塑料稳定剂、合成橡胶硫化剂、杀菌剂等排放的镉也会对水体造成污染,在城市用水过程中,往往由于容器和管道的污染也可使饮用水中镉含量增加。工业废水的排放使近海海水和浮游生物体内的镉含量高于远海。
海水中的镉含量一般在0.1-0.15μg/L,对于海水水质检测中的镉含量标准,一般认可:对于第一类适用于保护海洋生物资源和人类的安全利用(包括盐场、食品加工、海水淡化、渔业和海水养殖等用水),以及海上自然保护区,镉含量不超过0.005mg/L;对于第二类适用于海水浴场及风景游览区和第三类适用于一般工业用水,港口水域和海洋开发作业区等,镉含量不超过0.01mg/L。
测试水样中的镉含量,现有技术中存在多种已公开方法,最常用的是分光光度法,如萃取—分光光度法,其对技术要求较高,且分析时间长,为了提高原子吸收分光光度法的灵敏度,一般也需先用萃取法将试样预作浓集处理。但检测方法中多涉及强腐蚀性试剂,且灵敏度不够高。
借助于水中的生物特征进行水质检测在现有技术中已经公开。如根据水体中的菌群特征判定水体污染情况,如申请号为CN202210983119.X的专利公开了一种基于微生物群落特异性响应的河流生态***健康评价方法,包括测定水体pH、水温(WT)、溶解氧(DO)、COD、TN、总有机碳、硝态氮、铵态氮、亚硝态氮、总磷、硫化物和氟化物这12项指标,计算水质等级指标WQI;提取水体微生物DNA并进行细菌16s rRNA扩增子测序;获取细菌属水平信息表,筛选有效菌属信息;基于生态位模型进行微生物敏感性划分;评价结果可靠性分析。该方法可以快速、准确、客观地反映城市河流生态***健康状况,但一方面对微生物进行测序耗费的时间和物力较大,另一方面对于水体特定物质的针对性不强,此外,灵敏度也不够高。
现有技术中还公开了通过检测特定物种中的特定生物标志物反映水体质量水平的方法,如申请号为CN202110227984.7的专利公开了一组红裸须摇蚊Hb基因及其在水质生物监测中的应用。包括:PaHb-3,PaHb-5,PaHb-13,PaHb-15、PaHb-19五个基因序列,实验结果显示:这五个Hb基因可作为监测重金属铜水体污染的生物标志物。但该方法所检测的基因数量较多,且其针对的是铜检测,对于镉污染检测效果不佳。
对于海水中镉污染生物标志物的检测,现有技术中也有公开,如申请号为CN202211658961.2的专利申请公开了一种用于水生生态***海洋污染物风险评估的生物标志物,考察了两种典型海洋污染物对海洋聚球藻PCC7002藻株的毒性机制,从毒性试验中获得的生长和光合作用参数中筛选出用于环境污染物风险评估的生物标志物,通过方差分析法进行显著差异性分析,在Pearson相关系数分析的基础上,结合IC50值筛选出新型高效的毒理学生物标志物δFIP。该方法中样本前处理方法简单,可以运用叶绿素荧光仪对藻株进行原位检测,结果稳定性好,可重复性高,可以准确评估及预测海洋有害物质的风险,提供海洋污染物风险评估的早期预测预警。但该方案针对性不强。
发明内容
本发明提供了一种间接测定水中镉污染的方法,充分考虑镉污染产生的生物富集作用或环境作用对生物体本身代谢的影响,通过检测水体中红条毛肤石鳖体内基因ArWz-2含量侧面反应水体中镉含量。
一方面,本发明提供了红条毛肤石鳖中ArWz-2在检测水镉污染中的应用。
具体地,通过检测红条毛肤石鳖中ArWz-2含量对水中镉污染进行检测。
具体地,检测样本为海水。
所述的ArWz-2为红条毛肤石鳖中存在的一段基因,具体序列如SEQ ID NO.1所示。
在NCBI中比对到与SEQ ID NO.1相似性较高的序列ID为XM_009066399.1;
另一方面,本发明提供了一种水中镉污染的检测方法。
具体地,所述的检测方法中包括测定红条毛肤石鳖中ArWz-2的含量。
进一步地,所述ArWz-2的含量为ArWz-2的表达量,所述ArWz-2的表达量检测方法可以选自现有技术已公开或未公开中的任意一种,能够体现ArWz-2表达量数据的检测方法均能实现本发明的技术方案。
优选地,所述ArWz-2的表达量可以通过Northern印迹杂交、Sorthern印迹杂交、扩增、微阵列技术、RNA测序、DNA测序或蛋白组学中的任意一种或多种进行检测,但并不局限于以上方法。
具体地,所述的微阵列技术通过分析芯片上信号强度确定ArWz-2的表达量。
具体地,所述的Northern印迹杂交、Sorthern印迹杂交为将RNA或DNA从琼脂糖凝胶中转印到硝酸纤维素膜上以确定表达量。
具体地,所述的扩增包括但不限于聚合酶链式反应扩增,作为优选,可以是实时荧光定量PCR。
具体地,所述的蛋白组学为通过检测相应蛋白的表达量测定ArWz-2的表达量。
进一步地,所述的蛋白的表达量的检测方法包括但不限于免疫印迹法、免疫组化法或质谱法。
在一些实施例中,根据不同的检测方法要求,所述的ArWz-2的含量为相对于内参基因的含量。实际上,在一些绝对定量方法中,不需要参照内参基因,这也是本领域技术人员应当理解的。
当ArWz-2的含量为相对含量时,所述的内参基因包括但不限于CYTB;若内参基因为CYTB,当ArWz-2的含量相对于内参基因的含量<0.5,可判定水中镉含量大于0.01mg/L;优选地,当ArWz-2的含量相对于内参基因的含量<0.2,可判定水中镉含量大于0.01mg/L。
本发明中,所述的红条毛肤石鳖中ArWz-2的含量可以通过现有技术已公开或未公开的方法进行检测,根据本领域技术人员能够认知的,本发明的主要目的在于发现ArWz-2的含量与镉污染存在对应关系;对ArWz-2的含量的检测方法不进行限定。
此外对于具体的检测标准,本发明不进行限定,原因在于,本领域技术人员在本发明公开的ArWz-2的含量与镉污染的关系基础上,能够通过更换内参基因或者检测方法以调整相应检测标准,且该技术手段的替换是常规的,不能根据此种替换限制本发明的保护范围。在不脱离本发明核心思想的情况下进行的技术改进、常规调整都属于本发明保护的范围。
本发明的有益效果:
本发明通过检测样品水体红条毛肤石鳖中ArWz-2含量,发现ArWz-2含量与水体中镉含量相关,通过二者的对应关系可以检测水中镉含量,以对水质进行监测。相对于传统的直接检测方法,本发明避免了强腐蚀性试剂对检测人员的危害,且对水中镉检测的敏感程度更高。
附图说明
图1为红条毛肤石鳖ArWz-2的扩增产物凝胶电泳图。
图2为红条毛肤石鳖ArWz-2的PCR定量结果。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
红条毛肤石鳖(Acanthochiton rubrolineatus)经过15天人工配制海水(不含镉)中培养后,分别在镉浓度为0 mg/L、0.01mg/L、1mg/L、7.5mg/L的人工配制海水进行培养,培养时间为3天。
随机选取镉浓度为0 mg/L、0.01mg/L、1mg/L、7.5mg/L的人工配制海水中的红条毛肤石鳖(Acanthochiton rubrolineatus),每组选择3个平行,对ArWz-2的含量进行验证,以确定ArWz的含量与水体中镉浓度的关系。
PCR为现有技术中检测基因表达量较为便捷的方法,本实施例以PCR方法为例检测红条毛肤石鳖中ArWz-2的含量。本实施例中如无特别说明,实验条件均为常规条件,RNA提取、逆转录等都可以通过常规市售试剂盒完成,具体本实施例RNA提取采用Trizol提取法,逆转录试剂盒(PrimeScript™ RT reagent Kit with gDNA Eraser(Perfect RealTime),购自宝日医生物技术有限公司,货号:RR047A)。
总RNA的提取和纯化包括以下步骤:
(1)将样品用液氮速冻,迅速转移至液氮预冷的研钵中,用研杵研磨,期间不断加入液氮,直至研磨成粉末状(无明显可见颗粒)。
(2)将研磨成粉的样品50mg转移至离心管中,加入1mL RNAisolater,静置;待样品完全融化后,再继续吹打至裂解液透明。
(3)将裂解液转移至离心管中,11200rpm 4℃离心5 min,吸取上清。
(4)向上述裂解液中加入1/5体积的氯仿。剧烈震荡15 sec,成乳浊液,4℃静置5min,11200rpm 4℃离心15 min。小心取出离心管。此时溶液分为三层:无色的水相(上层)、白色中间层以及红色的有机层(下层),小心吸取上层水相至一个新的离心管中。
(5)加入等体积预冷的异丙醇,上下颠倒混匀,4℃静置10 min。11200rpm,4℃离心10min,可见白色沉淀。
(6)小心弃去上清,加入1mL 75%乙醇(RNase-free ddH2O配制)。轻弹管底,使沉淀悬浮,并上下颠倒数次,室温静置3-5min。
(7)11200rpm 4℃离心5 min,弃去上清。
(8)在洁净的环境中室温敞口干燥沉淀2-5 min,注意不可过分干燥,否则会导致RNA难以溶解。
(9)加入适量的RNase-free ddH2O溶解沉淀,待完全溶解后,取少量检测,其余在-85℃-65℃保存。
RNA反转录成cDNA包括以下步骤:
(1)去除基因组DNA反应
表1
(2)反转录反应
表2
本实施例中ArWz-2定量引物为:
F: TCGTGCTCATCAGTTAGTT(SEQ ID NO.2);
R:GTAGTCTGGCTGTGGCTTC(SEQ ID NO.3);
使用本领域常用的内参基因CYTB为内参,定量引物:
F: GGAACAGGATTATTCTCAGCCAT(SEQ ID NO.4);
R: GGATTAGTCAGCCGTGATTTACA(SEQ ID NO.5)。
扩增体系为:反应体系20 μL,包括2 μL cDNA、上下游引物(10 μmol/L)各0.4 μL、SYBR qPCR Master Mix 10 μL、7.2 μL ddH2O。。
扩增程序为:95℃预变性30 min;95℃变性10 s,60℃延伸30 s,共40个循环;熔解曲线程序:95℃进行15 s,60℃进行60 s,95℃进行15 s。
凝胶电泳检测结果见图1,定量结果见图2,定量结果为相对内参的表达量,无单位。
根据图2可以看出,当采用本实施例中的方法进行测定时,红条毛肤石鳖中ArWz-2相对表达量<0.2时,可以判定水体中镉含量>0.01mg/L,超过第一类、第二类、第三类海水的水质检测中的镉含量标准,可以判定镉污染。
实施例2
参照实施例1中的红条毛肤石鳖培养方法,采用微阵列技术技术测定ArWz-2含量,具体为:在镉浓度为0.5mg/L的人工配制海水进行培养,培养时间为3天。
随机选取镉浓度为0.5mg/L的人工配制海水中的红条毛肤石鳖,每组选择3个平行,对ArWz-2的含量进行验证,以确定ArWz-2的含量与水体中镉浓度的关系。
结果为ArWz-2表达量对比结果与实施例1一致。
实施例3
参照实施例1中的红条毛肤石鳖培养方法,采用Northern印迹杂交测定ArWz-2含量,具体为:随机选取镉浓度为1mg/L的人工配制海水中的红条毛肤石鳖,每组选择3个平行,对ArWz-2的含量进行验证,以确定ArWz-2的含量与水体中镉浓度的关系。
结果为ArWz-2表达量对比结果与实施例1一致。
实施例4抗干扰实验/特异性
参照实施例1的方法进行实验,区别在于分别在0 mg/L、0.01mg/L镉浓度组中添加其他常见污染因素,然后检测ArWz-2相对表达量,污染物及添加量如下:
铜含量(μg/L):4.8、9.2;
铅含量(μg/L):0.6、2.2;
铬含量(μg/L):2.0、3.9;
磷含量(μg/L):0.6、2.6;
酸性pH:6;
设置空白对照(不添加干扰物,仅添加镉),共计20个组别,每个组别设置3个平行。
结果:添加含铜污染物的2组,表达量并无明显差异;添加含铅污染物的2组,表达量并无明显差异;添加含铬污染物的2组,表达量并无明显差异;添加含磷污染物的2组,表达量并无明显差异;酸化组,表达量并无明显差异。
结果表明,以上污染因素对结果判定无影响。
Claims (3)
1.一种引物对在检测水镉污染中的应用,其特征在于,所述的引物对用于扩增红条毛肤石鳖,所述引物对的核苷酸序列如SEQ ID NO.2-3所示。
2.一种水中镉污染的检测方法,其特征在于,采用权利要求1中所述的引物对对红条毛肤石鳖进行相对定量检测。
3.根据权利要求2所述的检测方法,其特征在于,所述相对定量的内参基因为CYTB,当扩增产物的表达量相对于内参基因的表达量<0.2,判定水中镉含量大于0.01mg/L。
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