双特异分子探针技术的建立及对十二种常见赤潮藻的检测
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摘要
赤潮的发生、发展和消亡等是当前海洋生态学研究的热点之一,而对赤潮藻种类和数量的分析是其中的基础内容。除了传统的显微镜观察外,越来越多的新兴技术正被引入这一领域并发挥着越来越重要的作用。
本研究利用S1酶能够保护完全匹配的双链核酸的性质,将分子生物学中的酶保护分析技术和夹心杂交技术融合在一起,创立了双特异分子探针技术以实现对赤潮藻的检测。该技术将酶保护分析探针(NPA探针)与细胞裂解液中的rRNA进行杂交,再用S1酶处理(降解)不匹配的双链杂合体和单链分子,然后采用微孔板夹心杂交技术测定保留下来的与rRNA分子等量匹配的NPA探针及其数量,从而实现对微藻种类和数量的鉴定。本研究还从细胞破碎程度、酶保护分析和夹心杂交三个方面对诸多影响因素进行条件优化,最终建立了针对裸甲藻、红色赤潮藻、海洋原甲藻、微小原甲藻、东海原甲藻、链状亚历山大藻、锥状斯氏藻、旋链角毛藻、中肋骨条藻、尖刺拟菱形藻、圆海链藻和球形棕囊藻等7种甲藻、4种硅藻和1种定鞭藻的相关检测曲线和方法,获得了良好的特异性和灵敏度。将双特异分子探针技术应用于模拟样品和现场样品分析,取得了理想的检测结果。
双特异分子探针技术避免了核酸定量检测技术一般需要的核酸纯化步骤,将目标生物的rRNA转化为等量的互补DNA探针,既解决了RNA不稳定易于降解的难题,又利用S1酶能够降解单链DNA、RNA或不完全匹配的双链核酸分子的特性,将探针的专一性从DNA探针与藻类的RNA杂交一个层次,提高到对杂交真实性进行校验的第二个层次(也就是说特异结合的探针被保留,其它探针被分解),从根本上解决了探针种属特异性的难题。本技术方法简单,条件易于控制,不但满足了微藻分析技术鉴定到种并且定量的要求,而且降低了关键探针的设计难度,使研发针对多种微藻并行分析的技术成为可能,为海洋微藻、特别是赤潮藻的快速、准确、连续大面积观测研究开辟了一条新路。
It is one of the research focuses for marine ecology that the emergence, development and vanishment of harmful algae blooms (HABs) at present. Among them, qualitative and quantitative detection for HAB species is the basic task. Besides traditional microscopy technology, more and more new technologies are introduced into this field. In this study, combining traditional S1 enzyme analysis with sandwich hybridization, sandwich hybridization integrated with nuclease protection assay (NPA-SH) was developed. The assay consists of three steps: firstly, cell-derived rRNA sample was hybridized with the nuclease-protection-assay probe (NPA probe) and subjected to digestion with S1 nuclease to clear all mismatched probes and preserve perfectly matched probes stoichiometrically; secondly, the remaining NPA probes were sandwich hybridized with the corresponding capture DNA probes immobilized on a microplate and then the corresponding signal probes were hybridized with the NPA probes; finally, the probe‘sandwich’was quantified with enzyme-mediated color reaction. The signal intensity correlated to the abundance of the target species in the original samples. On the basis of feasibility, this method was optimized from cells splitting, nuclease protection assay and sandwich hybridization. Finally, twelve species-specific NPA probes were respectively designed, the standard curves for each alga, Gymnodinium sp., Akashiwo sanguinea, Prorocentrum micans, Prorocentrum minimum, Prorocentrum donghaiense, Alexandrium catenella, Scrippsiella trochoidea, Chaetoceros curvisetus, Skeletonema costatum, Pseudo-nitzschia pungens, Thalassiosira rotula and Phaeocystis globosa, were established and the equations were deducted. Good specificity and sensitivity had achieved. Moreover, there were good results when cultured samples and field ones were analyzed with this method.
NPA-SH avoided complicated pre-treatments of purification of nucleic acid, transformed the targeted rRNA into the complemental DNA probes equally. This method solved the problem of RNA easy to degrade and upgraded the probe specificity from DNA probes hybridizing with RNA of microalgae to check reality of hybridization, using the ability of S1 nuclease, which degrades single-stranded nucleic acids to yield 5' phosphoryl mono or oligonucleotides to leave double stranded DNA, RNA and DNA/RNA hybrids intact (That is to say, the specific probes were kept and the others
本研究利用S1酶能够保护完全匹配的双链核酸的性质,将分子生物学中的酶保护分析技术和夹心杂交技术融合在一起,创立了双特异分子探针技术以实现对赤潮藻的检测。该技术将酶保护分析探针(NPA探针)与细胞裂解液中的rRNA进行杂交,再用S1酶处理(降解)不匹配的双链杂合体和单链分子,然后采用微孔板夹心杂交技术测定保留下来的与rRNA分子等量匹配的NPA探针及其数量,从而实现对微藻种类和数量的鉴定。本研究还从细胞破碎程度、酶保护分析和夹心杂交三个方面对诸多影响因素进行条件优化,最终建立了针对裸甲藻、红色赤潮藻、海洋原甲藻、微小原甲藻、东海原甲藻、链状亚历山大藻、锥状斯氏藻、旋链角毛藻、中肋骨条藻、尖刺拟菱形藻、圆海链藻和球形棕囊藻等7种甲藻、4种硅藻和1种定鞭藻的相关检测曲线和方法,获得了良好的特异性和灵敏度。将双特异分子探针技术应用于模拟样品和现场样品分析,取得了理想的检测结果。
双特异分子探针技术避免了核酸定量检测技术一般需要的核酸纯化步骤,将目标生物的rRNA转化为等量的互补DNA探针,既解决了RNA不稳定易于降解的难题,又利用S1酶能够降解单链DNA、RNA或不完全匹配的双链核酸分子的特性,将探针的专一性从DNA探针与藻类的RNA杂交一个层次,提高到对杂交真实性进行校验的第二个层次(也就是说特异结合的探针被保留,其它探针被分解),从根本上解决了探针种属特异性的难题。本技术方法简单,条件易于控制,不但满足了微藻分析技术鉴定到种并且定量的要求,而且降低了关键探针的设计难度,使研发针对多种微藻并行分析的技术成为可能,为海洋微藻、特别是赤潮藻的快速、准确、连续大面积观测研究开辟了一条新路。
It is one of the research focuses for marine ecology that the emergence, development and vanishment of harmful algae blooms (HABs) at present. Among them, qualitative and quantitative detection for HAB species is the basic task. Besides traditional microscopy technology, more and more new technologies are introduced into this field. In this study, combining traditional S1 enzyme analysis with sandwich hybridization, sandwich hybridization integrated with nuclease protection assay (NPA-SH) was developed. The assay consists of three steps: firstly, cell-derived rRNA sample was hybridized with the nuclease-protection-assay probe (NPA probe) and subjected to digestion with S1 nuclease to clear all mismatched probes and preserve perfectly matched probes stoichiometrically; secondly, the remaining NPA probes were sandwich hybridized with the corresponding capture DNA probes immobilized on a microplate and then the corresponding signal probes were hybridized with the NPA probes; finally, the probe‘sandwich’was quantified with enzyme-mediated color reaction. The signal intensity correlated to the abundance of the target species in the original samples. On the basis of feasibility, this method was optimized from cells splitting, nuclease protection assay and sandwich hybridization. Finally, twelve species-specific NPA probes were respectively designed, the standard curves for each alga, Gymnodinium sp., Akashiwo sanguinea, Prorocentrum micans, Prorocentrum minimum, Prorocentrum donghaiense, Alexandrium catenella, Scrippsiella trochoidea, Chaetoceros curvisetus, Skeletonema costatum, Pseudo-nitzschia pungens, Thalassiosira rotula and Phaeocystis globosa, were established and the equations were deducted. Good specificity and sensitivity had achieved. Moreover, there were good results when cultured samples and field ones were analyzed with this method.
NPA-SH avoided complicated pre-treatments of purification of nucleic acid, transformed the targeted rRNA into the complemental DNA probes equally. This method solved the problem of RNA easy to degrade and upgraded the probe specificity from DNA probes hybridizing with RNA of microalgae to check reality of hybridization, using the ability of S1 nuclease, which degrades single-stranded nucleic acids to yield 5' phosphoryl mono or oligonucleotides to leave double stranded DNA, RNA and DNA/RNA hybrids intact (That is to say, the specific probes were kept and the others
引文
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