【作者】 郑桂丽；

【导师】 唐学玺； 杨瑞馥； 徐怀恕；

【作者基本信息】 中国海洋大学 ， 生态学， 2004， 博士

【摘要】 目的：利用生物芯片进行细菌的高通量检测和鉴定已成为很多人的共识，但至今为之还未真正实现。为建立细菌性生物恐怖剂和重要医学细菌的筛查方法，本研究旨在建立一种基于16S rDNA寡核苷酸芯片的检测和鉴定常见致病菌的技术。 方法：以16S rDNA为靶标，针对待检细菌设计合成一系列寡核苷酸探针，制备寡核苷酸芯片。由于16S rDNA的保守性，针对每个细菌的四个可变区设计出4条探针。以本室保存的包括近缘和远缘细菌在内的135种共计300多株细菌对探针进行了验证和筛选。根据一种含26条种特异性探针的试验芯片，对芯片的杂交和洗涤条件等进行了摸索，并确立了新的数据分析方法。以筛选得到的探针，重新制备新的寡核苷酸芯片。待检细菌DNA经通用引物扩增标记后，与芯片杂交，对杂交图谱分析归纳，得到一套种／属特异的检测模式。待检样本与芯片杂交后，结果与种／属特异的检测模式相比，根据相似性大小判断样本的种类。以本室保存的部分样品对其进行了特异性验证。最后进行了混合样品检测试验及灵敏度试验。 结果：针对79种靶标细菌分别设计的4条探针，共316种，以本室保存的135种共计300多株细菌进行杂交验证，结果剔除了12个“交叉”反应严重的探针，60个始终没有信号的探针，以及63个最初设计时即相互重复的探针后，最终确定有效探针181个。 以含有针对7种细菌的26条探针的芯片为模型，初步探讨了利用16S rDNA寡核苷酸芯片鉴定致病菌的可行性。在已有条件基础上，优化了杂交和洗涤条件，缩短了杂交时间。在此基础上，分析比较了几种常见的数据分析方法，确定了以相对信号强度为依据的信号阴阳性判断思路，以及以特异性寡核苷酸谱（杂交图谱）为基础的细菌检测方式。结果表明，利用寡核苷酸芯片实现致病菌高通量鉴定是完全可行的，提出的信号判断方法保证了鉴定结果的稳定性，使鉴定结果受样品浓度及杂交过程差异等的影响程度减小，使用实验室保存的部分菌株进行初步考核，证实其具有较强的特异性和重复性。此外，为给后续的实验选择合适的阳性和阴性等参照，在此芯片中引入四种参照探针进行试验，结果证实EUB338ACTCCTACGGGAGGCAGC基本可以胜任阳性参照的作用，来源于λ噬菌体的AGAATATGGCGGCGATGCT还和部分菌种有交叉现象，不能作为阴性参照，而poly（T）₄9和50％DMSO位点的信号强度均很小，可以胜任阴性和空白参照的作用，以作为芯片杂交和洗涤质量控制的标准之一。 以经过验证的EUB338 ACTCCTACGGGAGGCAGC以及在探针验证过程中证实交叉反应最严重的其中两个探针:Acl’netobacte:haem口如ticus（溶血不动杆菌）的3号探针TCCTACGGGAGAAAGCAGGGGATC（ID%=100%）和4号探针AeAoAG以AoeTTG以AeTGATeT（xDo;o=1 000;0）为阳性参照，poly（T）;9和50%DMso为阴性和空白参照，加上确定的181个有效探针，重新设计并制备新的检测芯片。利用通用引物sua和gO7b对涉及31个属126种细菌（包括可获得的靶标细菌，及其同属内的其他近缘细菌，和某些不作为靶标细菌，但可供分析的菌株数较多的细菌）进行扩增和标记后，与芯片杂交，对杂交图谱分析归纳，得到一套种（属）特异的检测模式，共计62种，分别针对不同的种、属，或为几个种或属的混合模式。确立检测模式后，从已提取的模板库中随机选取了包括远缘和近缘细菌在内的 228份样品（104种菌）进行特异性验证。结果表明，大部分样品可准确地鉴定到种或属的水平，没有对应模式存在者，则没有对应的可信结果，即最大的相似性指数一般都小于75%，鉴定准确率为93.42%（213株正确，巧株错误）。该芯片检测系统不能区分李斯特氏菌（属）和除炭疽芽抱杆菌外的大部分芽抱杆菌，这两类细菌对应的阳性信号都较少，在混合样品中也容易被其他细菌的阳性信号所掩盖，因此针对这两类细菌可能需要进一步改变杂交条件，或样品制备方法，以增加它们的阳性信号数量，从而增加相互之间的分辨能力，同时在混合样品中又不致被其他细菌的杂交信号所掩盖。个别菌种检测结果出现偏差，但由于没有对应模式存在，需要进一步验证，包括硝酸盐阴性不动杆菌、摩氏摩根氏菌、沃氏葡萄球菌和和泰拉维沙门氏菌等。 以金黄色葡萄球菌、炭疽芽抱杆菌和鼠疫耶尔森氏菌三种比较典型的致病菌为例，研究了该芯片检测系统的灵敏度问题。炭疽芽抱杆菌和鼠疫耶尔森氏菌灵敏度较好，30因体系中最低可检测到的细菌数分别为5.2 X 103cfu和7.1 X 102cfu，金黄色葡萄球菌显然较差些，达7.4 xl护cfu时才被检测到，但此灵敏度较低并不意味者该芯片检测系统灵敏度就很差，因为其实际上取决于样品的提取和扩增效率。染色体稀释灵敏度试验证实了样品提取对芯片检测灵敏度的影响，三种菌（金黄色葡萄球菌、炭疽芽抱杆菌和鼠疫耶尔森氏菌）的最低检测浓度分别为4.sng/ml、12.45ng/ml和63lPg/ml，金黄色葡萄球菌灵敏度为中间水平。 混合样品的盲测试验和不同浓度比例混合样品的检测结果说明，样品中存在多个菌种时也可检测出来，但一般只能检测出占主导地位的几种细菌，如所占比例大于10%的某些细菌（如乙酸钙不动杆菌），针对不同的细菌，该比例底限会有所变化，更多还原

【Abstract】 Objective: The potential of DNA microarray technology in high-throughput detection of bacteria is widely acknowledged but has not been fully realized yet. For the rapid and efficient detection of biological agents and clinical infectious bacteria, a 16S rDNA oligonucleotides microarray based detection and identification system was developed.Methods: Oligonucleotides probes complementary to species-specific 16S rDNA regions of targeted bacteria were designed and evaluated by hybridization of different remotely related as well as closely related reference strains. The hybridization and washing conditions were optimized according to a microarray containing 26 species-specific probes. A newly formatted microarray was constructed containing the selected probes and controls. The chromosome DNA of some bacteria was amplified and labeled using a pair of universal primers targeted to 16S rDNA, and then the PCR products were applied to the oligonucleotides microarray. A series of specific hybridization patterns corresponding to each species or genus were obtained. Samples that were unknown were hybridized to the microarray, then the results were compared to the species- or genus-specific patterns and the identities were concluded according to the value of the correlation coefficient. Specificity of the oligonucleotides microarray detection system was validated by hybridization to a set of reference nucleic acids from pure cultures. Sensitivity test was performed and the capability of the microarray system in mixed samples was checked by hybridization to a few artificial samples that consist of two or three kinds of chromosome DNA.Results: 4 probes targeted to each bacteria were designed respectively, i.e. 316 probesfor 79 bacteria all together. Probes were validated against more than 300 strains covering 135 species. 12 most heavily cross-hybridized probes, 60 meaningless probes, i.e. never score positive during the former experiments, and 63 probes that is repetitive to each other at the beginning of the probes’ designation were discarded as the result. 181 effective probes were confirmed in the end.The feasibility of the 16S rDNA oligonucleotides microarray system was checked according to a microarray containing 26 species-specific probes. The hybridization and washing conditions were optimized, the hybridization time was shortened. A new method was confirmed for the judgment of positive or negative probes based on the relative signal intensity by comparing several common methods used frequently now. The identities of samples were deduced according to the oligonucleotides profiles, not the so-called specific probes. The result suggests that it is possible to realize the high-throughput detection and identification of pathogenic bacteria by the oligonucleotides microarray. The judgment method of signals guaranteed the stability of the identification result, making it less influenced by the difference of the samples and the hybridization processes. The specificity, reproducibility and reliability were then confirmed by hybridization to a set of pure cultures of targeted species. To find out suitable controls that can be used later, 4 probes were introduced in the microarray to be validated, and the result indicated that EUB338 ACTCCTACGGGAGGCAGC could be a perfect positive control, AGAATATGGCGGCGATGCT rooted in A phage was unfitted for the negative control, but poly(T)49 and 50% DMSO were suited for the negative control and the blank control, respectively. All the signal intensities on their positions were as low as enough comparing with the local background.A newly formatted microarray was constructed containing EUB338, poly(T)49, 50% DMSO and the 181 specific probes. Chromosome DNA of 126 species of bacteria was amplified and labeled using a pair of universal primers targeted to 16S rDNA, and then PCR products were applied to the oligonucleotides microarray. 62 specific hybridization patterns corresponding to each species or genus were obtained. Specificity of the microarray system was verified by hybridization t更多还原