CN104145025A - 用于检测微囊藻素生成性毒性蓝细菌的方法和引物 - Google Patents
用于检测微囊藻素生成性毒性蓝细菌的方法和引物 Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
本发明提供了用于检测样品中微囊藻素生成性毒性蓝细菌的存在的方法,其包括使自环境样品获得的核酸与引物接触的步骤,所述引物与存在于铜绿微囊藻、鱼腥藻和阿氏浮丝藻中的mcyB基因的核酸序列特异性杂交。本发明进一步提供了设计用于本发明方法的引物、引物对和探针。
Description
发明领域
本发明涉及检测来自环境样品的微囊藻素生成性毒性(microcystin-producing toxic)蓝细菌的领域。具体而言,本发明提供了用于检测所述毒性蓝细菌的基于聚合酶链式反应(PCR)的测定方法。本发明进一步提供了设计用于本发明方法的材料,诸如引物、引物对和探针。
发明背景
在近几年,毒性蓝细菌水华在全世界已经越来越引起科学团体、官方和一般公众的关注。由蓝细菌块发生产生的毒素已经引起野生生物和家畜的死亡(关于综述,参见Stewart等,2008),并且已经与人生病和死亡关联起来(Turner等,1990;Pilotto等,1997;Jochimsen等,1998)。微囊藻素,形成具有超过90种已知结构变体的主要蓝毒素(cyanotoxin)组的环状七肽肝毒素(Welker和von2006),是由鱼腥藻属(Anabaena)、微囊藻属(Microcystis)、浮丝藻属(Planktothrix)和念珠藻属(Nostoc)品系生成的(Sivonen和Jones,1999),尽管已经描述了微囊藻素生成性软管藻属(Hapalosiphon)(Prinsep等,1992)、席藻属(Phormidium)(Izaguirre等,2007)和费氏藻属(Fischerella)的孤立发生(Fiore等,2009)。这8种属中,通常,鱼腥藻属、微囊藻属和浮丝藻属引起淡水肝毒性水华。为了更好了解蓝细菌水华的动力学和毒素生成的机制,以及预测与这些现象有关的风险,需要用于不同微囊藻素变体和毒素生成性蓝细菌的容易使用的检测方法。已经将对微囊藻素连续低水平的暴露与升高的肝癌风险联系起来(Yu,1995;Ueno等,1996;Svircev等,2009),这进一步加剧这这些方法的需要。
微囊藻素和非微囊藻素生成性蓝细菌的可靠鉴定可以是挑战性的。同一物种的形态学难于辨认的毒性和无毒性品系的混合水华是常见的(Vezie等,1998)。mcyS基因簇的发现揭示了一组用于基于PCR的微囊藻素生产者检测的潜在靶物。与毒素结构中的非蛋白质生成性的罕见氨基酸残基联系的基因特别好地适合于此目的(Mbedi等,2005)。许多研究描述了检测微囊藻素生产性蓝细菌的基于核酸的方法,及其尝试评估与毒性水华有关的风险的用途(关于综述,参见Sivonen,2008)。例如,WO2011003184披露了设计用于检测微囊藻素生成性蓝细菌的引物。公开的引物与铜绿微囊藻株UTCC299的mcyD基因的ss-酮脂酰合酶(KS)域的保守区,或与铜绿微囊藻UTCC300的mcyD基因的第一个脱水酶域的保守区互补。在WO2006128230中,披露了另一种用于检测肝毒性蓝细菌的基于PCR的方法。扩增的靶序列位于源自微囊藻素合成酶基因复合物的mcyE基因和节球藻素(nodularin)合成酶基因复合物的ndaF基因的肝毒素关联氨基转移酶域序列中。
然而,已经描述了能够检测超过一种微囊藻素生成性属的少数实时定量PCR(Al-Tebrineh等,2011;Vaitomaa等,2003;Briand等,2008),并且目前大多数发表的qPCR法仅集中于一种属,最常是微囊藻属(Foulds等,2002;Kurmayer and Kutzenberger,2003;Rinta-Kanto等,2005;Furukawa等,2006;Fortin等,2010)。
样品制备和标记物技术选择在qPCR中是重要的。与由PCR抑制剂的不完全除去引起的低效扩增组合的在多步骤DNA提取方法期间的样品损失可以导致重大的量化误差(Wilson,1997)。解决这些问题会改善PCR可靠性。升高的特异性继而可以用经标记的序列特异性探针(例如TaqMan)实现,并且与更常用的瞬发荧光团(prompt fluorophore)不同,镧系螯合物标记物容许时间分辨荧光测定法检测,由于较低的背景信号而导致改善的灵敏性,因为任何自身荧光在激发和测量之间的时间窗期间衰变(Soini and1987)。
本研究的目的是开发用于检测潜在微囊藻素生成性鱼腥藻属、微囊藻属和浮丝藻属的定量实时PCR方法。为了实现可靠的量化,就qPCR性能和模板产率而言比较DNA提取和简单的细胞裂解。开发的qPCR测定法适用于环境样品分析,并且检查基因拷贝数目和微囊藻素浓度之间的关联。
附图简述
图1:mcyB qPCR测定法的标准曲线。将基数10log mcyB拷贝数相对于a)鱼腥藻属,b)微囊藻属和c)浮丝藻属的其相应阈值循环(Ct)绘图。可以对每个目标属检出范围为101–107个拷贝的mcyB靶序列。扩增效率对于所有靶物是相似的(92.7–94.2%)。4个重复反应内的Ct变化是非常低的,标准差以通常在符号内部隐藏的误差棒显示。
图2:两种qPCR样品制备方法的比较。比较显微镜测定的细胞量(有图案的柱)以检测提取的基因组DNA(白色柱)和过滤且受到破坏的细胞(灰色柱)中的mcyB基因拷贝。对微囊藻素生成性铜绿微囊藻NIVA-CYA140、水华鱼腥藻(Anabaena cf.flos-aquae)NIVA-CYA267/4和阿氏浮丝藻NIVA-CYA299进行比较。与自相同量的细胞提取的DNA相比,过滤和细胞裂解产生高得一致的拷贝数。误差棒标示样品间和样品内拷贝数变化二者。
图3:在2008年4月-6月期间在Hauninen水库(reservoir)(Raisio,Finland)中浮丝藻mcyB基因拷贝数(■,实线)、浮丝藻细胞数目(▲,点线)、和通过LC-MS测定的总微囊藻素浓度(□,虚线)的形成。细胞数目基于100μm细丝单元的显微镜计数和每一个此类单元30个细胞的估值。观察到mcyB拷贝数目和总微囊藻素浓度(dmMC-RR和dmMC-LR)之间的明显正相关联。没有检测到潜在毒性的微囊藻或鱼腥藻。
图4:鱼腥藻属(Anabaena sp.)、铜绿微囊藻和阿氏浮丝藻的mcyB基因中的靶序列,和实施例中使用的引物和探针。
图5:mcyB基因的总基因拷贝数与通过ELISA测量的环境水生样品中微囊藻素浓度[MC]的关联。
图6:mcyB基因的总基因拷贝数与通过HPLC测量的环境水生样品中微囊藻素浓度[MC]的关联。
图7:mcyB基因的总基因拷贝数与通过LC-MS测量的环境水生样品中微囊藻素浓度[MC]的关联。
发明详述
本发明涉及用于检测样品中微囊藻素生成性毒性蓝细菌的存在的方法,其包括下列步骤:
a)对所述样品中的细胞实施裂解;
b)在聚合酶链式反应混合物中使从裂解细胞获得的核酸与引物接触,所述引物与铜绿微囊藻、阿氏浮丝藻和鱼腥藻属中存在的mcyB基因的核酸序列特异性杂交,其中所述引物扩增mcyB基因中如SEQ ID NO:1,SEQ IDNO:2,和/或SEQ ID NO:3中列出的靶序列的至少一部分;
c)用自步骤b)获得的反应混合物实施聚合酶链式反应,从而所述mcyB基因的序列在所述序列存在于样品时得到特异性扩增;并
d)检测扩增核酸序列的存在,其中扩增mcyB基因序列的存在指示所述样品中存在微囊藻素生成性毒性蓝细菌。
优选地,包含蓝细菌材料的制备用于本发明方法的样品是环境样品,诸如水样品,其可以是海水样品或淡水样品或其它合适的环境样品。
虽然方法主要涉及检测毒性蓝细菌,诸如铜绿微囊藻、阿氏浮丝藻和鱼腥藻属,但是其也可以用于量化所述样品中的微囊藻素生成性毒性蓝细菌。这可以通过使用本发明的引物对提取的DNA实施定量-PCR(qPCR),并测定mcyB基因的基因拷贝数来完成。此外,也可以通过将通过qPCR获得的所述基因拷贝数与相应的微囊藻素浓度关联来监测样品中的毒性微囊藻素浓度。
在步骤a)中,可以通过常规方法将在步骤b)的PCR中用作模板的核酸从裂解细胞提取并纯化。然而,优选地,直接使用从裂解细胞获得的悬浮液作为PCR模板。因而,在本发明的一个实施方案中,首先可以过滤怀疑包含毒性蓝细菌的未稀释的水样品。然后,将过滤的细胞在无菌去离子水中悬浮,并通过热处理实施细胞裂解。因而,获得的悬浮液和其中的核酸可以用于步骤b)的反应混合物。
在步骤b)中,所述引物扩增mcyB基因中靶序列的至少一部分,所述靶序列如SEQ ID NO:1中所列且对应于图4中描述的铜绿微囊藻基因组的位置6147-6251,或如SEQ ID NO:2中所列对应于图4中描述的铜绿微囊藻基因组的位置6203-6305,或如SEQ ID NO:3中所列对应于图4中描述的鱼腥藻属藻的位置6170-6272。
在本发明的实施方案中,步骤b)中扩增的扩增子(即靶序列)包含如由SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3限定的靶序列的至少20,优选至少50,更优选至少80,且最优选103或105个连续核苷酸。
优选地,用于方法的引物包含下列一种引物或由下列一种引物组成:
5’-GCTTTAATCCACAAGAAGCTTTATTAGC-3’(SEQ ID NO:4)
5’-AGATTTTAATCCACAAGAAGCTTTATTAGC-3’(SEQ ID NO:5)
5’-GGTTTAATCAACAAGAGGCTTTATTAGC-3’(SEQ ID NO:6)
5’-CTGTTGCCTCCTAGTTCAAAAAATGACT-3’(SEQ ID NO:7)。
在本发明的一个最优选的实施方案中,所述方法以可用几种商业试剂盒的实时聚合酶链式反应实施。
对于在实时聚合酶链式反应中使用,本发明还提供了与铜绿微囊藻、阿氏浮丝藻和鱼腥藻属中存在的mcyB基因的核酸序列特异性杂交的寡核苷酸探针,其中所述探针具有序列:
5’-ACTGAATTATTGGAGGTAGAGGTGAGTGATAC-3’(SEQ ID NO:8)
5’-CCTCTACCTCCAATAATTCA-3’(SEQ ID NO:9)
5’-GGGTGAGTTATTAGAAGCAGAAGTTAGTAACAG-3’(SEQ IDNO:10)
5’-TTCTGCTTCTAATAACTCACC-3’(SEQ ID NO:11)
5’-GGGGTGAATTATTAGAAATAGAAGTAAGTGACAA-3’(SEQ IDNO:12)
5’-TTACTTCTATTTCTAATAATTCACC-3’(SEQ ID NO:13)。
本发明还涉及引物,其包含如SEQ ID NO:4-7中所列的任一种序列或由如SEQ ID NO:4-7中所列的任一种序列组成。优选地,引物具有28-40个核苷酸。更优选地,引物包含小于30、35或40个核苷酸。
此外,本发明提供了探针,其包含如SEQ ID NO:8-13中所列的任一种序列或由如SEQ ID NO:8-13中所列的任一种序列组成。优选地,探针具有小于40个核苷酸。
本发明的寡核苷酸引物和探针是短的核苷酸(诸如RNA或DNA,优选DNA)序列,通常具有25至30或更小的碱基。然而,自动合成仪容许合成多达160至200个碱基的寡核苷酸,并且目前的寡核苷酸可以延长以将例如限制酶切割位点添加至寡核苷酸。优选地,引物的典型长度是26-32,更优选28-30个核苷酸。
本发明还提供了用于检测微囊藻素生成性毒性蓝细菌存在的试剂盒。试剂盒可以包含如上文定义的引物和探针。
上文和下文实施例中描述此类引物和探针。
优选地,所述试剂盒包含用于实时聚合酶链式反应的手段,诸如标记的探针、酶聚合酶、缓冲液和核苷酸。
本领域中公知的是,同一基因的序列在微生物物种的菌株中略有变化,如此,铜绿微囊藻、阿氏浮丝藻和鱼腥藻属中存在的mcyB基因的序列在物种的所有菌株中不是100%相同的。然而,从本文中的描述,特别是从下文实施例看清楚的是基于这些基因序列的相似性和同源性,本领域技术人员会识别出任何相关菌株中的mcyB基因序列。
在本文中,术语“特异性杂交”意味着寡核苷酸和靶序列之间的互补杂交。术语“同源性”指由容许寡核苷酸与序列之间的微小错配的互补杂交显示的特异性,所述微小错配不能危及用于检测杂交信号的退火。
本文中用于说明本发明背景,且特别是用于提供关于其实施的更多详情的出版物和其它材料通过提及并入本文。本发明在以下实施例中进一步描述,所述实施例不意图限制本发明的范围。
实施例
材料和方法
1.1.蓝细菌菌株和环境样品
表1中列出用于此研究的蓝细菌菌株。菌株购自巴斯德培养物保藏中心(Pasteur Culture Collection)(PCC,Paris,France)和挪威水质研究所(NorwegianInstitute for Water Research)蓝细菌培养物保藏中心(NIVA,Oslo,Norway),并且根据提供者的推荐维持。将所有NIVA株在Z8中培养(Staub,1961,修饰的NIVA1972,1976)。使用由PCC描述的配方,将来自PCC的菌株在BG11(Sigma)、省去硝酸盐的BG110、或添加NaNO3和NaHCO3的BG110中培养。还维持来自其它资源的三种额外的菌株。将铜绿微囊藻NIES-107(NationalInstitute of Environmental Studies,Tsukuba,Japan)在Z8中培养。将拉普鱼腥藻(Anabaena lapponica)菌株966(Finnish Environment Institute,Dr.Jarkko Rapala)及鱼腥藻90(University of Helsinki,Prof.Kaarina Sivonen)在不添加氮的改良Z8中培养。使用PowerGlo20W水族馆灯泡(Hagen,Japan)作为光源将所有培养物维持于23℃。用显微镜从NIVA-CYA267/4、NIVA-CYA140和NIVA-CYA299的2.5周龄培养物计算蓝细菌细胞。将样品在去离子水中以1:100–1:200稀释,并且在鲁戈(Lugol)氏碘中保存。在10ml样品等分试样的16小时沉积后,在具有10x和40x物镜的Nikon TE-200倒置显微镜(Nikon,Japan)上实施计数。收获来自计数的培养基的细胞以进行mcyB量化。在2008年4月-6月期间从Hauninen水库,Raisio,Finland(11份样品)及在2008年7月期间从岛的淡水湖(13份样品)收集环境样品。所有样品(包括提取的DNA和其它PCR模板)于-20℃保持冷冻,直至进一步分析。将培养物和环境样品快速冷冻或冷冻干燥。
1.2.微囊藻素的提取
用1.2ml75%甲醇(HPLC级;Rathburn,Walkerburn,UK)提取8-10mg冷冻干燥的蓝细菌材料或含有过滤的蓝细菌材料的冷冻干燥的GF/C纤维玻璃滤纸(25mm;Whatman,Maidstone,UK)的样品。将提取物在水浴超声发生器(Bandelin Sonorex RK156,Berlin,Germany)中处理15分钟,且另外用探头超声波仪(probe sonicator)(具有3mm微尖端(microtip)的Bandelin Sonopuls HD2070,30%脉冲,30%能量)处理1分钟。在以10,000xg离心10分钟后,将上清液分成等分试样,并于40℃用氩气蒸发至干燥。将意图通过HPLC-DAD和LC-ESI-MS-MS进行微囊藻素分析的提取物在75%甲醇中重悬,而那些以ELISA为目的的提取物在水中重悬。
1.3.HPLC及二极管阵列UV检测(HPLC-DAD)
使用由脱气器、四元泵(quaternary pump)、40℃的恒温柱室(thermostattedcolumn compartment)和以200–300nm运行的二极管阵列检测仪(以238nm量化)组成的Agilent(Waldbronn,Germany)1100系列HPLC系统分析样品。固定相是来自Merck(Darmstadt,Germany)的Purospher STAR柱,3μm颗粒,55mmx4mm或来自Supelco(Bellefonte,PA,USA)的Ascentis RP-酰胺柱,3μm颗粒,100mm x4.6mm I.D.。流动相由乙腈(HPLC S级,Rathburn,Walkerburn,UK)(溶剂B)-Milli-Q超纯水(Millipore,Molsheim,France)(溶剂A)组成,两者都含有0.05%三氟乙酸(TFA;Fluka,Buchs,Switzerland),线性梯度程序如下:0分钟25%B,7分钟70%B,10分钟70%B,10.1分钟25%B;停止时间15分钟;流速1ml/分钟。注射体积是10μl。另外,在具有3μm颗粒的Merck PurospherSTAR RP-18e柱,55mm x4mm I.D.上分析样品(Spoof and Meriluoto,2005)。各个实验室纯化的微囊藻素和以下参照样品用于鉴定微囊藻素:a)铜绿微囊藻PCC7820的提取物和b)如(Spoof等,2003)中描述的铜绿微囊藻NIES-107的提取物。
1.4.LC-MS和LC-MS-MS
在Agilent1100系列HPLC系统上实施LC-MS实验,所述Agilent1100系列HPLC系统与装备有电喷雾接口的Waters Micromass(Manchester,UK)QuattroMicro三级四极质谱仪偶联。在Merck Purospher STAR RP-18封端柱(30mmx4mm,3μm颗粒)上量化毒素。流动相由0.1%含水甲酸(溶剂A)和乙腈(溶剂B)的梯度组成,线性梯度程序如下:在10分钟里25%B至70%B,然后在2分钟里至90%B,其中其保持1分钟。注射时间间隔是16分钟,注射体积是10μl,流速是0.5ml/分钟,并且柱炉温度是40℃。毛细管电压设置为3.8kV,且锥电压(cone voltage)设置为40V(dmMC-RR和MC-RR)或75V(微囊藻素和节球藻素的剩余部分)。去溶剂化气体(氮气)温度和流速分别设置为300℃和650L/h。离子源温度设置为150℃。以正电喷雾离子化模式检测离子。选择离子记录(SIR)模式的监测信号是m/z[dmMC-RR+2H]2+512.8、[MC-RR+2H]2+519.8、[MC-LF+H]+986.5和[MC-LF+Na]+1008.5、[dmMC-LR+H]+986.5、[MC-LR+H]+995.5、[MC-LY+H]+1002.5、[MC-LW+H]+1025.5和[MC-LW+Na]+1047.5、[MC-YR+H]+1045.5、[dmNod+H]+811.5和[Nod+H]+825.5。用Masslynx v.4.0软件(Micromass)完成数据采集。在Agilent1200快速解析(RR)LC上实施LC-MS-MS实验,所述Agilent1200快速解析(RR)LC与具有电喷雾离子(ESI)源的Bruker DaltonicsHCT超离子阱质谱仪(Bremen,Germany)偶联。1200RR LC系统包括二元泵(binary pump)、真空除气器、SL自动采样器、和恒温柱室。以正电喷雾离子模式操作离子阱。离子源参数如下设置:干燥温度350℃,雾化器压力40psi,干气流动10.0L/min,毛细管电压4.0kV。采用具有Smart Parameter Setting(SPS)功能的500-1200m/z的MS扫描范围。ICC靶标设置于300000,最大积累时间是100ms。通过SmartFrag设置辅助丰富的MS-MS片段化。在具有3μm颗粒的Ascentis C18,50mm x3mm I.D.柱(Supelco)上于40℃实现毒素的分离。注射体积是5μl。流动相由水-乙腈-甲酸(99:1:0.1;溶剂A)和乙腈-甲酸(100:0.1;溶剂B)组成,线性梯度程序如下:0分钟25%B,5分钟70%B,6分钟70%B,6.1分钟25%B;停止时间10分钟;流速0.5ml/min。主要监测信号与用Quattro Micro仪相同,但是用HCT Ultra仪获得的质谱容许基于片段化样式鉴定别的毒素。用Bruker Compass1.3软件完成数据采集。
1.5.ELISA
用Quantiplate微囊藻素试剂盒(Envirologix,Portland,ME,USA)使用由制造商提供的方案分析样品。依照HPLC结果用水稀释提取物以调节微囊藻素浓度至测定法的工作范围。对一些样品生成两个不同稀释物。
1.6.PCR样品制备
使用两种方法制备样品以进行PCR分析。使用NucleoSpin Plant II DNA提取试剂盒(Macherey-Nagel,Düren,Germany)依照制造商的说明书从培养菌株的冷冻干燥细胞或冷冻干燥的岛环境样品提取基因组DNA。用分光光度法(ND-1000,NanoDrop Technologies,Wilmington,DE,USA)测定DNA浓度和质量。如下实施过滤和细胞裂解:将10ml收获的细胞培养物在90ml无菌去离子水中悬浮,并且在玻璃纤维滤纸(47mm GF/C,Whatman)上真空过滤。在Hauninen水库样品的情况中,将50ml未稀释的水过滤。将每个滤纸切出段,并在100μl无菌去离子水中悬浮。于80℃将细胞裂解实施5分钟。此后,直接使用悬浮液作为PCR模板。为了量化目的,记录滤纸段尺寸。在有几处修改的情况下使用上文描述的两种方法处理显微镜计数的培养物样品:将细胞以5ml和10ml二者的等分试样收获,并且经受过滤或DNA提取。对新鲜收获的细胞实施样品制备。通过离心(4℃,3220g,20min,Eppendorf5810R,Hamburg,Germany)收集细胞以提取DNA。
1.7.引物和探针
从GenBank核苷酸数据库检索mcyB序列(Nishizawa等,1999;Tillett等,2000;Christiansen等,2003;Rouhiainen等,2004;Kaneko等,2007),并且基于序列比对设计引物和探针。寡核苷酸由Thermo Scientific(Ulm,Germany)和biomers.net(Ulm,Germany)制备(表2)。合成具有5’-C6-氨基接头和3’-磷酸根基团的探针。在5’端氨基接头用有机Tb3+螯合物(2,2’,2”,2”’-{{6,6’-{4”-[2-(4-异硫氰基苯)乙基]吡唑-1”,3”-二基}二(吡啶)-2,2’-二基}二(亚甲基次氮基)}四(乙酰)}铽(III))(2,2’,2”,2”’-{{6,6’-{4”-[2-(4-Isothiocyanatophenyl)ethyl]pyratzole-1”,3”-diyl}bis(pyridine)-2,2’-diyl}bis(methylenenitrilo)}tetrakis(acetato)}terbium(III))标记所有检测探针,如先前描述的(Nurmi等,2002)。所有淬灭剂探针具有在其3’端由寡核苷酸制造商缀合的BHQ1(Black hole淬灭剂1)分子。对表1中列出的蓝细菌菌株测试引物特异性。各个PCR反应含有5nmol dNTP(Finnzymes,Espoo,Finland)、2.67pmol每种正向引物和8pmol反向引物、0.5个单位的DyNAzyme II HotStart DNA聚合酶(Finnzymes)、1X DyNAzyme II HotStart缓冲液和1μl模板,即1ng基因组DNA或1μl含有裂解细胞的悬浮液。用无菌Milli-Q水充满反应体积20μl。使用PTC-200热循环仪(MJ Research,Watertown,MA,USA)实施热循环,以于95℃10分钟开始,接着是于95℃30秒,于58℃30秒和于72℃1分钟的35个循环,且以于72℃10分钟结束。在用溴化乙啶(0.5ng L-1)染色的2%琼脂糖凝胶上分析PCR产物。
1.8.qPCR
实时定量PCR技术的原则已经详细记载于别处(Nurmi等,2002)。简言之,通过测量荧光半衰期检测扩增产物积累,所述荧光通过由DNA聚合酶的5’→3’外切核酸酶活性从检测探针释放的螯合物模块获得。借助于淬灭剂探针,将背景荧光保持于最小。使用qPCR和来自所有培养菌株的纯化的基因组DNA确认检测探针特异性。每个qPCR反应含有4nmol dNTP、2pmol每个正向引物和6pmol反向引物、0.2个单位的DyNAzyme II HotStart DNA聚合酶、1X DyNAzyme II HotStart缓冲液、0.25pmol检测探针(mcyB-mP、mcyB-pP或mcyB-aP)、2.5pmol相应的淬灭剂探针(mcyB-mQ或mcyB-pQ,对于mcyB-aQ,使用3.0pmol的量)和1ng模板DNA。用无菌Milli-Q水将反应充满至20μl。在具有Applied Biosystems光学帽(Optical Caps)(Foster City,CA,USA)的ThermoFast96Robotic PCR板(Abgene,Surrey,UK)上运行所有反应。qPCR运行以于95℃5分钟开始,接着是于95℃30秒和于62℃1分钟的8个循环。在循环8后,将温度降低到35℃达15秒,用于荧光测量。每隔一个循环重复测量,直到循环40后,并且用Victor21420多标记物计数器(Multilabel Counter)(PerkinElmer Life Sciences Wallac,Turku,Finland)使用制造商的标准方案实施以进行Tb荧光的时间解析测量。
1.9.标准曲线形成
从鱼腥藻90、PCC7806(微囊藻)和NIVA-CYA299(浮丝藻)中通过PCR生成双链DNA标准品,如上文描述的。使用Qiaquick PCR纯化试剂盒(Qiagen,Hilden,Germany)实施mcyB扩增产物的纯化,随后使用QuantIt PicoGreen试剂盒(Invitrogen,Eugene,OR,USA)纯化,两者均依照制造商的说明书进行。计算纯化的扩增产物的摩尔浓度,并且制备每种属特异性标准品的一系列10倍稀释(n=7)。将标准品保持于-20℃,直至使用。如上文描述的那样运行qPCR。将阈值循环(Ct)相对于对数mcyB拷贝数绘图,并从线性回归的斜率计算扩增效率(E)(E=10-1/斜率-1,其中数值1.00对应于100%的效率)。
1.10.通过qPCR分析环境和培养物样品
制备从显微镜计算的培养物样品制备的模板的一系列10倍稀释物(n=4)。目的是比较qPCR量化和细胞密度并且评估扩增效率和可能的PCR抑制。除了增加的模板量(4ng DNA或4μl热处理的样品)外,如上文描述的那样实施所有qPCR。在四个重复反应中分析培养物样品,一式三份分析未稀释的环境样品。通过将1ng鱼腥藻90基因组DNA添加至每个反应,并且在溴化乙啶染色的琼脂糖凝胶上显现PCR产物对产物mcyB阴性结果的环境样品测试PCR抑制,如上文描述的。
结果
2.1.引物和探针特异性
对30种蓝细菌菌株的提取DNA和热处理的细胞测试mcyB引物和检测探针的特异性。表1中列出了探针特异性、微囊藻素和由菌株生成的其它毒素。没有观察到假阴性。除了节球藻素生成性夏威夷节球藻(Nodularia harveyana)PCC7804外,没有从非微囊藻素生成性菌株观察到扩增产物。然而,通过qPCR中的任何检测探针没有检测到节球藻(Nodularia)扩增产物。如此,所有属特异性检测探针以100%特异性和灵敏性运行。可以使用三种可能的引物组合中的每种从不依赖于属的所有微囊藻素生成性菌株扩增靶mcyB序列,但是扩增效率在菌株间有所变化(数据未显示)。在三种正向引物一起使用时,实现有效的扩增。
2.2.标准曲线和扩增效率
使用从纯化PCR产物制备的标准品测定灵敏性和扩增效率。对于所有三种靶标属,分析灵敏性是每个反应10个mcyB拷贝和对数线性范围10至107个拷贝的mcyB(图1)。对于鱼腥藻、微囊藻和浮丝藻,从标准曲线得到的扩增效率和相关系数分别是93.8%(r2=0.997)、91.7%(r2=0.999)和95.3%(r2=0.998)。
2.3.样品制备方法的比较
为了比较两种样品制备方法,从已知细胞密度的培养物样品制备PCR模板。从通过过滤和热处理制备的模板获得的扩增效率是较高的或者等于相应纯化的DNA模板的那些扩增效率(分别为89–100%,平均值r2=0,9993±0,0008和83–97%,平均值r2=0,9976±0,0018)。提取DNA的质量有所变化,A260/A280比率范围为1.7至2.0。经热处理的样品太稀以致不能用分光光度法检查。样品的过滤和热处理产生相应提取DNA的mcyB拷贝数的4–330倍(图2)。在NIVA-CYA299(一种阿氏浮丝藻菌株)观察到最大的差异,其中过滤样品的检出的拷贝数仅是细胞计数的1,5倍,但是是相应DNA提取样品的拷贝数的330倍。对于鱼腥藻,DNA的拷贝数和细胞裂解物与细胞计数具有相同的量级。对于微囊藻,其适合于细胞计数和来自提取DNA的拷贝数,而细胞的过滤和热破坏产生细胞量大致30倍的拷贝数。
2.4.对环境样品的qPCR分析
分析总共24份环境样品。大多数岛湖样品(9/13)含有源自仅微囊藻,或微囊藻以及鱼腥藻或浮丝藻的mcyB基因拷贝(表3)。微囊藻素存在于所有样品中,其在qPCR中对mcyB产生阳性结果。然而,不能建立总拷贝数和毒素浓度之间的线性关系。4份样品不含靶标属mcyB拷贝,也没有显示可以已经引起假阴性结果的PCR抑制的证据。ELISA结果对于所有mcyB阴性岛样品呈阳性,而相同样品的HPLC-DAD结果均呈阴性,并且LC-MS-MS分析产生范围为每g样品(干重)0至0.67μg微囊藻素的浓度。
11份Hauninen水库样品中的5份在mcyB方面呈阳性。与显微镜检查一致,群体以浮丝藻占优势,并且没有检出潜在毒性的微囊藻或鱼腥藻。自从掺杂样品获得的扩增产物的视觉检查后没有观察到PCR抑制。发现浮丝藻mcyB拷贝数和总微囊藻素浓度之间的明显正相关(图3)。细丝量从4月3日起逐渐增加,并且在5月29日达到峰值,之后观察到快速的下降。总微囊藻素浓度和浮丝藻特异性mcyB拷贝数同时达到峰值与观察到的最高的浮丝藻群体密度不一致。两种主要的微囊藻素变体存在于含有毒素的样品中,即dmMC-RR和dmMC-LR。
讨论
3.1.mcyB qPCR和遗传背景
适合于检测几种毒性属的靶物的鉴定是至关重要的。在本文描述的qPCR测定法中,靶序列位于肽基载体蛋白域编码序列(用来将生长的多肽链从McyB转移至McyC以进一步延长的域)中mcyB的第二硫醇化基序内(Nishizawa等,1999;Tillett等,2000)。据我们所知,其用作qPCR检测的靶物是第一次。与许多研究中靶定的mcyB第一腺苷酸化域(例如(Mbedi等,2005;Kurmayer and Kutzenberger,2003;Nonnemann and Zimba,2002;Mikalsen等,2003)不同,第二硫醇化基序不可能受到微囊藻素结构变化影响。因此,不依赖于毒素谱,几种潜在毒性的属的同时检测是有可能的。通过从节球藻素生成性夏威夷节球藻PCC7804扩增与mcyB同源的ndA证明一大批检测的潜在肝毒素生产者。mcyB的第二缩合(condensation)、腺苷酸化和硫醇化基序在ndaA基因中具有其对应物(Moffitt and Neilan,2004),并且节球藻中的扩增子与鱼腥藻、微囊藻和浮丝藻中的相应扩增子分别共享80%、75%和76%序列同一性。然而,由于显示检测探针仅与其意图的靶物杂交,mcyB qPCR特异性不受ndaA的扩增危害。
大多数现有的定量mcyB qPCR测定法基于几种探针(若使用TaqMan检测化学)和引物对(Kurmayer and Kutzenberger,2003;Nonnemann and Zimba,2002;Kaebernick等,2002),其具有单一靶标属。凭借设计为检测同一基因的不同部分的新引物和属特异性探针,定量mcyB检测扩展为还包括鱼腥藻和浮丝藻,并且对设计为检测超过一种微囊藻素生成性属的全集测定法(repertoirassay)进行新靶物的添加。虽然通用引物提供关于所有毒性物种的丰度的信息(Al-Tebrineh等,2011),属特异性检测具有群体动力学监测的添加益处。使用的灵敏性标记物技术(Nurmi等,2002)给测定法提供良好的灵敏性和较宽的量化范围。凭借检测限每个反应10个mcyB拷贝和7个数量级的范围,目前的测定法非常类似于先前发表的方法(Al-Tebrineh等,2011;Vaitomaa等,2003;Foulds等,2002;Kurmayer and Kutzenberger,2003;Rinta-Kanto等,2005;Furukawa等,2006)。
3.2.样品制备
qPCR标准品和样品的扩增效率之间的差异对量化结果可以具有实质性影响(Wilson,1997)。通过不同方法制备的样品间也存在相同的误差来源,并且需要注意进行任何比较。在此研究中,由观察到的扩增效率的变化引起的样品类型间的理论拷贝数差异仍然在3-5倍的范围中。这不能解释检出的拷贝数的1-2个数量级的差异(对于微囊藻和浮丝藻),显示了蓝细菌细胞的简单裂解和基因组DNA的提取和纯化产生的每个细胞的可用模板量间存在有实际的显著差异。许多原因可以引起此类结果,可能是DNA提取期间的样品材料损失和低效的细胞裂解。也可能的是,基因拷贝数和细胞计数间的差异在一定程度上是由显微镜量化的误差引起的,尽管专业人员实施的计数会使其变得可能性相当小。
基于我们的观察,若假设每个细胞1个基因拷贝,在热处理样品中微囊藻mcyB数目会导致细胞浓度的估计过高。然而,在相同的假设下,从纯化的基因组DNA量化mcyB会导致细胞浓度的估计不足,特别是在阿氏浮丝藻的情况下。对于鱼腥藻和微囊藻,先前已经观察到导致细胞数目估计过高的定量PCR结果(Vaitomaa等,2003),并且研究支持含有多个基因组的蓝细菌细胞(Labarre等,1989;Becker等,2002)。若情况如此,则它会解释估计过高,但是同时使估计不足变得更严重。因此,我们选择在只要可能时在环境样品的分析中使用过滤和热处理,并且直接比较拷贝数与毒素浓度,避免成问题的基因拷贝转化成细胞数。
先前对于定性分析已经报告了本文进行比较的类似结果,备选的样品制备方法已经产生与纯化的DNA相当的模板(Hisbergues等,2003;Ouahid等,2005)。不同样品制备方法的定量性能评估已经发现DNA的酚-氯仿提取比DNA提取的商业试剂盒产生更高的拷贝数,以及因此产生较高的细胞浓度评估(Schober and Kurmayer,2006)。Rasmussen等,2008发现了在去污剂溶液中的超声处理和微波处理与由商业试剂盒进行的DNA提取一样好地运行。我们的观察结果与先前发表的结果一致,所述先前发表的结果指示在不纯化的情况下的细胞裂解和DNA释放是mcy基因靶物的检测和量化前用于样品制备的有效方法。
3.3.环境样品
通过在2008年秋季和初夏量化mcyB拷贝数和总微囊藻素浓度追踪Hauninen水库中的蓝细菌群体。虽然在理论上微囊藻素合成酶基因的存在仅揭示毒素生成的潜力,阿氏浮丝藻mcyB拷贝和毒素浓度之间的正相关是明显的。由于两种其它靶定毒素生产者都没有被检出,假设阿氏浮丝藻是水库的唯一微囊藻素生产属。此结论得到样品中找到的微囊藻素变体MC-dmLR和MC-dmRR(其对于阿氏浮丝藻是典型的)支持(Fastner等,1999)。细丝计数与mcyB拷贝数或毒素浓度没有明显联系,这指示Hauninen水库中的阿氏浮丝藻群体是毒性和无毒性菌株的混合物,其相对比例在监测期期间广泛变化。已经报告了阿氏浮丝藻中的无活性mcy基因型,并且它们似乎相当常见(Kurmayer等,2004;Ostermaier and Kurmayer,2009)。基于观察到的毒素浓度和基因拷贝数,不能推论Hauninen水库中的此类基因型的存在,并且因此假设忽略不计。
与Hauninen水库形成对比,从岛上的不同淡水湖收集的单一样品没有显示微囊藻素浓度和mcyB拷贝数之间的明显关联,尽管没有观察到假阳性定性mcyB结果(即检出基因拷贝,但是样品中不存在微囊藻素)。环境因素可以将变化引入mcy基因表达样式和毒素生成中(Kaebernick等,2000;Sevilla等,2008),因此湖环境的差异最可能促成此结果。另一种可能的影响因素是样品制备。考虑关于由不同方法生成的模板的qPCR性能的结果,可能的是降低的DNA提取产率将方法依赖性误差引入量化结果。
4份样品在用HPLC-DAD和LC-MS-MS方法分析时一起显示非常低的微囊藻素浓度或无毒素,并且在qPCR中不产生信号。然而,ELISA测定法指示这些样品中不同微囊藻素量的存在。由于微囊藻素免疫测定法可以不受样品基质影响(Metcalf等,2000),采用的其它两种方法没有指示高毒性浓度,评估过高的或假阳性的ELISA结果不能作为可能性排除。微囊藻素是相对稳定的,并且可以在水中持续几周(Lahti等,1997),如此样品中可以存在痕量,即使在不再有生成性生物体后。
结论
随着毒性蓝细菌团出现的健康风险的认识增长,监测方法的需要增加。至今为止与目前的科学知识一起提出了关于蓝细菌风险评估的指导和立法。与有效的样品制备结合的检测和量化三种主要水华形成属的所有潜在微囊藻素生成性成员的qPCR方法提供了快速的预警监测的手段。响应环境中细胞密度的重大变化,可以以良好的灵敏性和较宽的量化范围检测潜在微囊藻素生成性蓝细菌。虽然与层析数据的定性一致性较好,岛基因量化结果显示小的分开样品收集点不足以为我们提供一般性的基因拷贝数准则。此类准则仅在分析较大的样品数目后才能建立。Hauninen水库阿氏浮丝藻群体展示显微镜量化在毒素浓度评估中的不可靠性,并且强调qPCR检测的益处。所有结果共同鼓励在微囊藻素生成性蓝细菌的环境监测中使用分子方法。
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Claims (19)
1.用于检测样品中微囊藻素生成性毒性蓝细菌的存在的方法,其包括下列步骤:
a)对所述样品中的细胞实施裂解;
b)在聚合酶链式反应混合物中使自裂解细胞获得的核酸与引物接触,所述引物与存在于铜绿微囊藻(Microcystis aeruginosa)、阿氏浮丝藻(Planktothrix agardhii)和鱼腥藻属的藻(Anabaena sp.)中的mcyB基因的核酸序列特异性杂交,其中所述引物扩增所述mcyB基因中如SEQ ID NO:1、SEQ IDNO:2、或SEQ ID NO:3列出的靶序列的至少一部分;
c)用自步骤b)获得的反应混合物实施聚合酶链式反应,从而使得在所述序列存在于样品时所述mcyB基因的序列得到特异性扩增;并
d)检测扩增核酸序列的存在,其中扩增mcyB基因序列的存在指示所述样品中存在微囊藻素生成性毒性蓝细菌。
2.依照权利要求1的方法,其中至少一种所述引物包含一种下述序列:
5’-GCTTTAATCCACAAGAAGCTTTATTAGC-3’(SEQ ID NO:4),
5’-AGATTTTAATCCACAAGAAGCTTTATTAGC-3’(SEQ ID NO:5),
5’-GGTTTAATCAACAAGAGGCTTTATTAGC-3’(SEQ ID NO:6),
5’-CTGTTGCCTCCTAGTTCAAAAAATGACT-3’(SEQ ID NO:7)。
3.依照权利要求1或2的方法,其中所述聚合酶链式反应混合物用于实时聚合酶链式反应。
4.依照权利要求3的方法,其中在所述反应中使用至少一种下述探针:
5’-ACTGAATTATTGGAGGTAGAGGTGAGTGATAC-3’(SEQ ID NO:8),
5’-CCTCTACCTCCAATAATTCA-3’(SEQ ID NO:9),
5’-GGGTGAGTTATTAGAAGCAGAAGTTAGTAACAG-3’(SEQ IDNO:10),
5’-TTCTGCTTCTAATAACTCACC-3’(SEQ ID NO:11),
5’-GGGGTGAATTATTAGAAATAGAAGTAAGTGACAA-3’(SEQ IDNO:12),
5’-TTACTTCTATTTCTAATAATTCACC-3’(SEQ ID NO:13)。
5.依照权利要求3或4的方法,其中所述实时聚合酶链式反应是定量PCR反应,且所述方法进一步包括测定所述样品中所述mcyB基因的基因拷贝数的步骤。
6.依照权利要求5的方法,其进一步包括监测所述样品中毒性微囊藻素浓度的步骤,其通过将由定量PCR获得的所述基因拷贝数与相应的微囊藻素浓度关联进行。
7.一种核苷酸引物,其包含如SEQ ID NO:4-7中列出的任一种序列。
8.依照权利要求7的引物,其由如SEQ ID NO:4-7中列出的任一种序列组成。
9.依照权利要求7的引物,其中所述引物具有28-40个核苷酸。
10.一种核苷酸探针,其包含如SEQ ID NO:8-13中列出的任一种序列。
11.依照权利要求10的探针,其由如SEQ ID NO:8-13中列出的任一种序列组成。
12.依照权利要求10的探针,其中所述探针具有小于40个核苷酸。
13.选自由SED ID NO:4-13组成的组的核苷酸引物或探针用于检测样品中微囊藻素生成性毒性蓝细菌的存在的用途。
14.依照权利要求13的用途,其中所述微囊藻素生成性毒性蓝细菌选自下组:铜绿微囊藻、阿氏浮丝藻和鱼腥藻的藻。
15.用于检测样品中微囊藻素生成性毒性蓝细菌的存在的试剂盒,其包含依照权利要求7-9的至少一种引物。
16.依照权利要求15的试剂盒,其进一步包含依照权利要求10-12中任一项的探针。
17.依照权利要求16的试剂盒,其用于实时聚合酶链式反应。
18.依照权利要求15的试剂盒,其中所述微囊藻素生成性毒性蓝细菌选自下组:铜绿微囊藻、阿氏浮丝藻和鱼腥藻的藻。
19.依照权利要求15-18中任一项的试剂盒用于检测样品中微囊藻素生成性毒性蓝细菌的存在的用途。
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CN110741090A (zh) * | 2017-05-09 | 2020-01-31 | 赛安诺生物技术有限责任公司 | 用于修饰微囊藻毒素和节球藻毒素的方法 |
WO2020232734A1 (zh) * | 2019-05-21 | 2020-11-26 | 武汉藻优生物科技有限公司 | 使不产毒微藻产生微囊藻毒素的方法及得到的产毒微藻 |
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CN104278081A (zh) * | 2013-07-03 | 2015-01-14 | 宁波大学 | 一种用lamp-lfd芯片高通量检测微囊藻毒素的方法 |
CN103484394B (zh) * | 2013-07-04 | 2015-02-18 | 河南师范大学 | 有毒微囊藻菌株及其毒素纯化方法 |
US11130784B2 (en) * | 2016-10-17 | 2021-09-28 | Newsouth Innovations Pty Ltd | Recombinant microcystin production |
CN111518936A (zh) * | 2019-09-02 | 2020-08-11 | 广州微芯生物科技有限公司 | 用于检测产毒铜绿微囊藻的荧光定量pcr方法及相应的试剂盒 |
KR102421942B1 (ko) * | 2020-10-20 | 2022-07-19 | 한국수자원공사 | 마이크로시스티스, 독성 마이크로시스티스 검출용 프라이머 세트 및 이를 이용한 독성 마이크로시스티스 검출방법 |
CN113652472B (zh) * | 2021-07-27 | 2022-07-26 | 壹健生物科技(苏州)有限公司 | 一种检测产毒微囊藻菌型的探针组合、芯片、试剂盒及方法 |
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US20070026430A1 (en) * | 2005-06-30 | 2007-02-01 | Applera Corporation | Proximity probing of target proteins comprising restriction and/or extension |
WO2011003184A1 (en) | 2009-07-08 | 2011-01-13 | National Research Council Of Canada | Detection of microcystin-producing cyanobacteria |
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WO2020232734A1 (zh) * | 2019-05-21 | 2020-11-26 | 武汉藻优生物科技有限公司 | 使不产毒微藻产生微囊藻毒素的方法及得到的产毒微藻 |
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