CN105510570A - 一种简便检测棒曲霉毒素的方法 - Google Patents
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Abstract
本发明属于分析化学领域,具体涉及一种简便检测棒曲霉毒素的方法。首先,将棒曲霉毒素适体固定在96孔板微孔中,以转化酶修饰的适体互补序列为探针,通过DNA杂交作用,将探针固定在96孔板微孔中。当有目标物棒曲霉毒素存在时,棒曲霉毒素与棒曲霉毒素适体发生识别作用,使转化酶修饰的适体互补序列探针脱落,然后取脱落的探针溶液于小离心管中并加入蔗糖,在探针上转化酶的作用下,将蔗糖转化为葡萄糖,利用血糖仪为检测仪器,实现对棒曲霉毒素的测定。
Description
技术领域
本发明属于分析化学领域,具体涉及一种简便检测棒曲霉毒素的方法。
背景技术
发展便捷、低廉的测定食源性致病菌的方法对于食品安全具有重要的意义。已经报道可以利用家庭用血糖仪测定一些非葡萄糖目标物。棒曲霉毒素是由青霉属、曲霉属、丝衣霉属产生的真菌代谢毒素,对人体有一系列急性毒性和致畸致癌等慢性毒性。研究表明棒曲霉素具有引起动物抽搐、肺、肝部出血、肾脏损害、肠道出血等急性毒性,并具有神经毒性、免疫毒性、基因毒性、致畸、致癌等慢性毒性[MoakeMM,Padilla-ZakourOI,WoroboRW.Comprehensivereviewofpatulincontrolmethodsinfoods.ComprRevFoodSciF,2005,4(1):8-21]。目前常用于检测棒曲霉素的方法主要有化学分析法如薄层层析法、高效液相色谱法、气相色谱法、色谱联用技术[ScottPM,LawrenceJW,VanWalbeekW.Detectionofmycotoxinsbythin-layerchromatography:applicationtoscreeningoffungalextracts.ApplMicrobiol,1970,20(5):839-42;ShephardGS,LeggottNL.Chromatographicdeterminationofthemycotoxinpatulininfruitandfruitjuices.JChromatogrA,2000,882(1-2):17-22;ItoR,YamazakiH,InoueK,etal.DevelopmentofLiquidChromatography-ElectrosprayMassSpectrometryfortheDeterminationofPatulininAppleJuice:InvestigationofItsContaminationLevelsinJapan.JAgricFoodChem,2004,52(25):7464-7468]等。这些方法结果可靠、灵敏度高,但是大多数仪器设备比较昂贵,还要进行复杂的前处理,多数只能在实验室中使用。如今应用比较广泛的免疫分析法,如基于荧光标记的免疫竞争法[deChampdoréM,BazzicalupoP,DeNapoliL,etal.Anewcompetitivefluorescenceassayforthedetectionofpatulintoxin.AnalChem,2007,79(2):751-757]、化学发光免疫分析法[马强,朱定波,杨杏芳等.鲁米诺-铁氰化钾流动注射化学发光体系测定饮用水中棒曲霉素.食品工业科技,2013,15:306-308]、胶体金免疫层析技术[李康,应美蓉,盛慧萍等.胶体金免疫层析技术在真菌毒素快速检测中的应用.食品安全质量检测学报,2013,1:201-206]等。这些方法各有其优点,能不同程度的满足对棒曲霉毒素的检测要求,但方法复杂,需要贵重的仪器,检测成本高。核酸适体技术具有高特异性、高灵敏度、易操作、成本低等优点。这为棒曲霉毒素的检测提供了新的思路。
血糖仪作为临床医学中常用的医疗电子仪器,主要通过测量血液中的血糖浓度进行临床诊断。最近,美国伊利诺伊大学的化学家们将功能DNA传感器和便携式血糖仪组合在一起,与蔗糖转化酶把蔗糖转化为葡萄糖的功能结合,把血糖仪用作简单、便携、价格低廉的血液、血清、水或食物中许多靶分子的测量仪。本发明以96孔板为载体固定棒曲霉毒素适体DNA,以蔗糖转化酶修饰的适体互补DNA为探针,制备棒曲霉毒素适体生物传感器,以血糖仪为检测仪器实现了对棒曲霉毒素的快速、便捷测定。
发明内容
本发明旨在发明一种方法简单、成本低、灵敏度高、选择性好的测定棒曲霉毒素的方法。
实现发明目的技术方案是:
首先,将棒曲霉毒素适体固定在96孔板微孔中,以转化酶修饰的适体互补序列为探针,通过DNA杂交作用,将探针固定在96孔板微孔中。当有目标物棒曲霉毒素存在时,棒曲霉毒素与棒曲霉毒素适体发生识别作用,使转化酶修饰的适体互补序列探针脱落,然后取脱落的探针溶液于小离心管中并加入蔗糖,在探针上转化酶的作用下,将蔗糖转化为葡萄糖,利用血糖仪为检测仪器,实现对棒曲霉毒素的测定。
测定步骤为:
(1)棒曲霉毒素适体DNA的固定
依据链霉亲和素和生物素之间的特异性结合作用将生物素修饰的棒曲霉毒素适体DNA1固定在链霉亲和素修饰的96孔板微孔上。首先,将100μL5.0×10-7mol/L的DNA1加入到链霉亲和素修饰的微孔板中,37℃条件下在摇床上振荡反应30分钟,弃去未反应的溶液,然后用100μLpH7.4的Tris-HCl缓冲液清洗三次。
(2)探针的制备
将100μL含10mgNHS的水溶液和100μL含20mgEDC的水溶液混合,然后再加入500μL的1mg/mL的蔗糖转化酶溶液和100μL5.0×10-7mol/L的DNA2溶液。将所得的溶液混合,在室温下搅拌3h,得探针(转化酶修饰的适体互补序列探针),4℃条件下保存备用。
(3)分析检测
本方法的棒曲霉毒素传感器是建立在链霉亲和素和生物素的特异性结合、核酸序列的特异性结合和棒曲霉毒素与其适体的特异性结合的基础上的。首先,将100μL的探针溶液加入到适体固定的微孔中,37℃条件下振荡反应2小时,将未反应的溶液除去,并用100μLpH7.4的Tris-HCl缓冲液清洗三次。然后,将含棒曲霉毒素的溶液100μL加入到微孔中,通过棒曲霉毒素与其适体的特异性结合把探针释放到溶液中,吸取微孔中的溶液于另外一个微孔中,并加入100μL1.0mol/L的蔗糖溶液,37℃条件下振荡反应2小时。最后,取2μL的溶液利用血糖仪进行检测(图1)。
(4)所使用的仪器与试剂
ACCU-CHEK血糖仪(罗氏诊断产品(上海)有限公司);Anke-TGL-16C飞翁牌高速离心机(上海市安亭科学仪器厂);AR224CN型电子天平(奥豪斯仪器有限公司);KQ-50E型超声波清洗器(昆山市超声仪器有限公司);THZ型恒温振荡箱(上海一恒科学仪器有限公司)。
实验所需各种致病菌的培养基以及配套试剂均由北京陆桥技术有限责任公司提供;盐酸、蔗糖、三羟甲基氨基甲烷(Tris)等购自上海化学试剂有限公司;EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)、NHS(N-羟基琥珀酰亚胺)、蔗糖转化酶购自Sigma公司;链霉亲和素修饰96孔板购自于美国Corning公司。实验中所使用的其他试剂均为分析纯,水为二次蒸馏水。
DNA由上海生工生物工程有限公司合成,序列如下:
DNA1:5′-biotin-CAGCTCAGAAGCTTGATCCCGGCCCGCCAACCCGCATCATCTACACTGATATTTTACCTTGACTATCAGTCGTGCATCTG-3′
DNA2:5′-CAGATGCACGACTGATAGTCAAGGTAAAATATCAGTGTAGATGATGCGGGTTGGCGGGCCGGGATCAAGCTTCTGAGCTG-NH2-3′
附图说明
图1棒曲霉毒素测定原理示意图。
图2适体与棒曲霉毒素反应时间对信号强度的影响。
图3棒曲霉毒素浓度与信号关系图。
图4方法测定棒曲霉毒素的选择性。
发明的优点与效果
在最佳实验条件下,随着棒曲霉毒素浓度的增加,血糖仪读数增大。依本实验方法,根据棒曲霉毒素浓度和血糖仪读数,绘制标准工作曲线,如图3所示。棒曲霉毒素浓度在0.05~7.0ng/mL范围内与血糖仪读数呈线性关系,线性回归方程为y=69.306x+13.885,R2=0.9991(y:血糖仪读数;x:棒曲霉毒素浓度,单位为ng/mL),检出限为0.03ng/mL。
具体实施方式
下面结合具体实施例进一步说明本发明,但不构成对发明的进一步限制。
实施例1适体与棒曲霉毒素反应时间对信号的影响
在这个检测方法中,棒曲霉毒素与其适体的作用时间直接影响血糖仪的检测信号强度。图2反应了不同反应时间对血糖仪读数的影响,由图可知,最大的检测信号出现在反应50分钟时,之后随着反应时间的增长,检测信号有所降低,因此,选取50分钟作为棒曲霉毒素与其适体反应的最佳反应时间。
实施例2特异性评价
按照上述操作过程,测定了浓度为1.0ng/mL的棒曲霉毒素(a)和浓度为100.0ng/mL赭曲霉毒素(b)、金黄色葡萄球菌肠毒素C1(c)、杂色曲霉毒素(d)和单端孢霉烯族毒素(e)对传感器的响应,血糖仪读数结果如图4所示,结果显示100倍的赭曲霉毒素、金黄色葡萄球菌肠毒素C1、杂色曲霉毒素和单端孢霉烯族毒素对浓度为1.0ng/mL的棒曲霉毒素测定没有干扰,表明所建立的方法对棒曲霉毒素具有良好的选择性。
实施例3牛奶和水样中棒曲霉毒素的测定
为了验证方法的可行性,将本方法用于复杂样品中棒曲霉毒素含量的测定,将不同浓度的棒曲霉毒素加入牛奶和水样中测得的回收率如表1所示。回收率在98.1~101.3%之间,表明方法测定实际样品的可行性。
表1回收试验
aAverageoffivedeterminations.
Claims (3)
1.一种简便检测棒曲霉毒素的方法,包括以下步骤:
(1)将100μL5.0×10-7mol/L的DNA1加入到链霉亲和素修饰的微孔板中,37℃条件下在摇床上振荡反应30分钟,弃去未反应的溶液,然后用100μLpH7.4的Tris-HCl缓冲液清洗三次;
(2)将100μL含10mgNHS的水溶液和100μL含20mgEDC的水溶液混合,然后再加入500μL的1mg/mL的蔗糖转化酶溶液和100μL5.0×10-7mol/L的DNA2溶液;将所得的溶液混合,在室温下搅拌3h,得转化酶修饰的适体互补序列探针,4℃条件下保存备用;
(3)将100μL的探针溶液加入到适体固定的微孔中,37℃条件下振荡反应2小时,将未反应的溶液除去,并用100μLpH7.4的Tris-HCl缓冲液清洗三次;然后,将含棒曲霉毒素的溶液100μL加入到微孔中,通过棒曲霉毒素与其适体的特异性结合把探针释放到溶液中,吸取微孔中的溶液于另外一个微孔中,并加入100μL1.0mol/L的蔗糖溶液,37℃条件下振荡反应2小时;最后,取2μL的溶液利用血糖仪进行检测;
优选的,所述的DNA1的部分序列为:5′-biotin-CAGCTCAGAAGCTTGATCCCGGCCCGCCAACCCGCATCATCTACACTGATATTTTACCTTGACTATCAGTCGTGCATCTG-3′;
优选的,所述的DNA2的部分序列为:5′-CAGATGCACGACTGATAGTCAAGGTAAAATATCAGTGTAGATGATGCGGGTTGGCGGGCCGGGATCAAGCTTCTGAGCTG-NH2-3′。
2.根据权利要求1所述的一种简便检测棒曲霉毒素的方法,其特征在于所述的ACCU-CHEK血糖仪为罗氏诊断产品上海有限公司的血糖仪;高速离心机为上海市安亭科学仪器厂的Anke-TGL-16C飞翁牌高速离心机;天平为奥豪斯仪器有限公司的AR224CN型电子天平;超声波清洗器为昆山市超声仪器有限公司的KQ-50E型超声波清洗器;恒温振荡箱为上海一恒科学仪器有限公司的THZ型恒温振荡箱。
3.根据权利要求1所述的一种简便检测棒曲霉毒素的方法,其特征在于所述的Tris-HCl溶液的配制方法为:称量60.55gTris置于1L烧杯中,加入约400mL的去离子水,充分搅拌溶解,加入浓盐酸调节所需要的pH为7.4,即得。
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