茎环结构探针的设计及其检测致病菌的研究
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摘要
继Tyagi和Kramer于1996年提出分子信标学说之后，Bockisch等基于分子信标检测的原理提出了一种新颖、廉价的酶联构象转换分析系统并在细菌核酸检测中得到了应用，即利用亲和标记了的茎环结构寡聚核苷酸探针来检测靶核酸。但是这种方法与分子信标一样在对于存在一个核苷酸误配的时候能检测出信号出现假阳性。因此我们利用序列比对的方法在目的病原菌16s rRNA高度保守的区域寻找一段序列并设计为茎环结构探针的环序列，这段序列与其它的高度同源菌种的16s rRNA基因序列片段差异两个核苷酸以上，从而避免探针环序列与其他非目的病原菌核酸杂交时出现误配一个核苷酸的情况，克服出现假阳性的问题。设计的探针一端固定在96孔酶标板上，另一端标记上亲合大分子。当没有靶核酸的时候，亲合分子紧密地靠近在固相支持物的表面，由于探针的闭合构象使得酶连接的报告分子不能与亲合分子结合产生信号；当存在靶核酸的时候，探针的环序列与靶核酸完全互补配对使得环被打开，构象发生变化呈现线性结构，暴露在外的亲合分子与报告分子结合产生信号。这种结合了序列比对的新探针技术其灵敏度比其他常规核酸检测技术至少高出一个数量级。本论文的研究内容包括以下两个方面：
一、基于金黄色葡萄球菌16S rRNA基因序列，采用序列比对设计了一种茎环结构的寡聚核苷酸探针，根据分子信标技术和酶联免疫分析的原理，评估一个实验方法，即利用能构象转换的、固定化的茎环结构探针酶联检测靶核酸。因为探针的环序列即为金葡球菌16S rRNA基因序列的其中一个片段，同其他菌种的16S rRNA基因序列误配2个以上的核苷酸，有效的排除假阳性即不会出现误配一个核苷酸的情况
二、为了验证新实验方法能应用在所有的靶细菌核酸检测中，选取革兰氏阳性金黄色葡萄球菌和革兰氏阴性大肠杆菌为研究对象，应用此方法成功的分别检测出金黄色葡萄球菌和大肠杆菌。
本论文实验表明，在知道靶细菌基因序列前提下，通过生物信息学方法设计的专一性探针和酶联构象转换分析系统能代替传统检测技术，甚至能检测含有变异的靶细菌和应用到单核苷多态性的研究中。
After Tyagi and Kramer proposes the molecular beacon theoryin 1996, Bockisch provided a novel enzymatic conformationalswitch system based on the principle of molecular beacon forthe detection of bacterial nucleic acid. They utilized stem-loop structured oligodeoxynucleotide probes for the detectionof nucleic acid targets. This system, however, can display afalse-positive signal when the sequence of nucleic acidmismatches the loop segments of probe in a single position aswell as molecular beacon. Therefore, Using sequencecomparisons, we found out a segment of 16S rRNA gene sequencesof the bacteria, which is different from 16S rRNA gene sequencesof other highly homologous bacteria in more than two positions.As the loop of the stem-loop structured oligonucleotide probeis designed by this segment, its specificity has beenstrengthened and the system is able to successfully eliminatefalse-positive result that is mismatch in an oligonucleotide.The probes are immobilized on 96-well microtiter plates throughone terminus and carrying an affinity label at the other as theconformational switch. In the absence of a target, the labelis forced into close proximity to the surface of the solid support by the closed conformation of the probe so as toinaccessible to detector molecules. Upon target hybridization,exposure of the affinity label is able to be close to reportmolecules. This is might be due to the fact that the probe opensthe loop and turns to a linear conformation. Thus, this newprobe technology combining sequence comparisons is moresensitive than other conventional method at least one order ofmagnitude. The main researches included in this thesis arepresented as following:
1) Based on Staphylococcus aureus 16S rRNA gene sequences,sequence comparisons have been applied to design a kind ofstem-loop structured oligonucleotide probes whose loopsequence mismatched other bacterial strains 16S rRNA genesequences in more than two positions with high specificity andsensitivity. According to the principle of molecular beacontechnology and enzyme linked immunosorbent assay, a method hasbeen evaluated using immobilized stem- loop structuredoligonucleotides probe as the conformation switch which isapplied to enzymatic detection of nucleic acid targets. As itsspecificity has been strengthened, the system is able tosuccessfully eliminate false-positive result that is mismatchin an oligonucleotide.
2) In order to confirm the possibility of this novelapproach in the detection of all bacterial nucleic acid targets,the Gram-positive bacterium (Staphylococcus aureus) and Gram-negative bacterium (Escherichia coli) are studied. As a result,they can be respectively detected.
This thesis strongly implied that the novel approachcombining sequence comparisons and enzymatic conformationalswitch system could replace conventional techniques and detectmutations and single nucleotide polymorphism.
一、基于金黄色葡萄球菌16S rRNA基因序列，采用序列比对设计了一种茎环结构的寡聚核苷酸探针，根据分子信标技术和酶联免疫分析的原理，评估一个实验方法，即利用能构象转换的、固定化的茎环结构探针酶联检测靶核酸。因为探针的环序列即为金葡球菌16S rRNA基因序列的其中一个片段，同其他菌种的16S rRNA基因序列误配2个以上的核苷酸，有效的排除假阳性即不会出现误配一个核苷酸的情况
二、为了验证新实验方法能应用在所有的靶细菌核酸检测中，选取革兰氏阳性金黄色葡萄球菌和革兰氏阴性大肠杆菌为研究对象，应用此方法成功的分别检测出金黄色葡萄球菌和大肠杆菌。
本论文实验表明，在知道靶细菌基因序列前提下，通过生物信息学方法设计的专一性探针和酶联构象转换分析系统能代替传统检测技术，甚至能检测含有变异的靶细菌和应用到单核苷多态性的研究中。
After Tyagi and Kramer proposes the molecular beacon theoryin 1996, Bockisch provided a novel enzymatic conformationalswitch system based on the principle of molecular beacon forthe detection of bacterial nucleic acid. They utilized stem-loop structured oligodeoxynucleotide probes for the detectionof nucleic acid targets. This system, however, can display afalse-positive signal when the sequence of nucleic acidmismatches the loop segments of probe in a single position aswell as molecular beacon. Therefore, Using sequencecomparisons, we found out a segment of 16S rRNA gene sequencesof the bacteria, which is different from 16S rRNA gene sequencesof other highly homologous bacteria in more than two positions.As the loop of the stem-loop structured oligonucleotide probeis designed by this segment, its specificity has beenstrengthened and the system is able to successfully eliminatefalse-positive result that is mismatch in an oligonucleotide.The probes are immobilized on 96-well microtiter plates throughone terminus and carrying an affinity label at the other as theconformational switch. In the absence of a target, the labelis forced into close proximity to the surface of the solid support by the closed conformation of the probe so as toinaccessible to detector molecules. Upon target hybridization,exposure of the affinity label is able to be close to reportmolecules. This is might be due to the fact that the probe opensthe loop and turns to a linear conformation. Thus, this newprobe technology combining sequence comparisons is moresensitive than other conventional method at least one order ofmagnitude. The main researches included in this thesis arepresented as following:
1) Based on Staphylococcus aureus 16S rRNA gene sequences,sequence comparisons have been applied to design a kind ofstem-loop structured oligonucleotide probes whose loopsequence mismatched other bacterial strains 16S rRNA genesequences in more than two positions with high specificity andsensitivity. According to the principle of molecular beacontechnology and enzyme linked immunosorbent assay, a method hasbeen evaluated using immobilized stem- loop structuredoligonucleotides probe as the conformation switch which isapplied to enzymatic detection of nucleic acid targets. As itsspecificity has been strengthened, the system is able tosuccessfully eliminate false-positive result that is mismatchin an oligonucleotide.
2) In order to confirm the possibility of this novelapproach in the detection of all bacterial nucleic acid targets,the Gram-positive bacterium (Staphylococcus aureus) and Gram-negative bacterium (Escherichia coli) are studied. As a result,they can be respectively detected.
This thesis strongly implied that the novel approachcombining sequence comparisons and enzymatic conformationalswitch system could replace conventional techniques and detectmutations and single nucleotide polymorphism.
引文
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[5] 安利峰，胜利．基因芯片及其应用进展．西北民族学院学报，2002，23(6)：40-42．
[6] 翟新验，刘钧．基因技术的应用前景．当代畜牧，2003，6：41-43
[7] 白东亭，祁自柏．基因芯片应用前景．微生物学免疫学进展，2002，30(4)：100-106．
[8] 杨明星，高志贤，王升启．基因芯片及其应用．传感器技术，2002，2l(6)：54-58．
[9] Pease A C, Solas D, Sullivan E J, et al. Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc Natl Acad Sci USA, 1994, 91(11):5022-5026.
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[14] Margulies M, et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature, 2005, 437:3?6-380.
[15] Ke L D, Chen Z, Yung W K. A reliability test of standardbased quantitatire PeR: exogenous vs endogenous standards. Mol Cell Probes, 2000, 14(2):127-135.
[16] Becker K, Pan D, Whitely C B. Real-time quantitative polymerase chain reaction to assess gene transfer. Hum Gene Ther, 1999, 10:2559-2566.
[17] Higgis J A, et al. 5' nuclease PCR assay to detect Yersinia pesits. J Clin Microbiol, 1998, 36:2284-2288.
[18] Bieche I, et al. Real-time reverse transcription-PCR assay for future nagement of ERBB-based clinical apolications. Clin Chem, 1999, 45:1148-1156.
[19] Wang X, Li X, et al. Application of real-time polymerase chain reaction to quantirate induced expression of interleukin-1α beta mRNA in ischemic brain tolerance. Neurosci Res, 2000, 59(2):238-246.
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