【目的】基质辅助激光解析电离飞行时间质谱（matrix-assisted laser  desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS）是近年来新兴的微生物检测技术。该技术在鉴定少见及罕见丝状真菌方面的研究仍较为缺乏。本研究的目的是评价MALDI-TOF MS在鉴定赛多孢霉属、枝顶孢霉样、帚霉属和小囊菌属真菌中的应用效果。

【方法】1. 试验菌株为北京大学真菌和真菌病研究中心保藏的赛多孢霉属真菌28株、枝顶孢霉样真菌26株及帚霉属和小囊菌属真菌27株。所有菌株均采用液体培养基培养，甲酸乙腈法提取蛋白。分别选取8株（4种）赛多孢霉属真菌、10株（7种）枝顶孢霉样真菌及11株（10种）帚霉属和小囊菌属真菌作为参考菌株，用MALDI Biotyper OC 4.0软件构建BMU数据库。

2. 使用布鲁克丝状真菌库v1.0分别鉴定20株赛多孢霉属真菌、16株枝顶孢霉样真菌及16株帚霉属和小囊菌属真菌的测试菌株，与DNA测序结果进行比较；联合布鲁克丝状真菌库v1.0与BMU数据库再次鉴定上述菌株。

3. 使用MALDI Biotyper OC 4.0软件分别构建28株赛多孢霉属真菌、26株枝顶孢霉样真菌及27株帚霉属和小囊菌属真菌的质谱图（mass spectrum profile, MSP）聚类分型树状图，与使用MEGA7软件构建的多位点联合系统进化树进行比较。

【结果】1. 使用MALDI-TOF MS及布鲁克丝状真菌库v1.0，20株赛多孢霉属真菌中95.0%（19/20）正确鉴定到属水平，但所有菌株均不能准确鉴定到种；16株枝顶孢霉样真菌全部无法鉴定；16株帚霉属和小囊菌属真菌中，62.5%（10/16）和68.8%（11/16）分别正确鉴定到种和属水平，正确鉴定的菌株均为S. brevicaulis。

2. 补充BMU数据库后，所有赛多孢霉属菌株均正确鉴定到复合体（species complex）水平，75.0%（15/20）正确鉴定到种水平；所有枝顶孢霉样菌株、帚霉属和小囊菌属菌株均正确鉴定到种水平。

3. 赛多孢霉属真菌的MSP聚类分型树状图不能区分尖端赛多孢霉复合体内的菌种。枝顶孢霉样真菌的MSP聚类分型树状图能够区分7个不同的菌种，与多基因系统进化树高度一致。帚霉属和小囊菌属真菌的MSP聚类分型树状图显示，各菌株聚于各自菌种或相近菌种的分支中，但小囊菌属中不同分支的位置与多基因系统进化树中的位置有所不同。

【结论】1. 仅使用布鲁克丝状真菌库v1.0，MALDI-TOF MS对赛多孢霉属真菌鉴定性能较好，对枝顶孢霉样、帚霉属和小囊菌属真菌的鉴定性能较差。

2. 完善数据库后，MALDI-TOF MS可以将赛多孢霉属真菌鉴定到复合体水平，但区分复合体内菌种的能力有限；MALDI-TOF MS可以将枝顶孢霉样真菌、帚霉属和小囊菌属真菌准确鉴定到种水平。

3. MSP聚类分型树状图能够可靠地揭示枝顶孢霉样真菌种间分群，并可以作为区分帚霉属和小囊菌属不同菌种的辅助手段，但不能区分尖端赛多孢霉复合体内的菌种。

【Objective】 Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a powerful microorganism identification tool. Research on MALDI-TOF MS identification of rare filamentous fungi is still lacking. This study aimed to evaluate the performance of MALDI-TOF MS on the identification of Scedosporium, Acremonium-like, Scopulariopsis and Microascus species.

【Methods】 1. A total of 28 Scedosporium isolates, 26 Acremonium-like isolates, 27 Scopulariopsis and Microascus isolates preserved at the Research Center for Medical Mycology of Peking University were included in this study. Liquid mold cultures and formic acid/acetonitrile protein extraction method were utilized for MALDI-TOF MS identification. An in-house BMU library containing isolates of Scedosporium spp. (7 isolates of 4 species), Acremonium-like spp. (10 isolates of 7 species), Scopulariopsis spp. and Microascus spp. (11 isolates of 10 species) was developed by MALDI Biotyper OC 4.0 software.

2. A total of 20 Scedosporium isolates, 16 Acremonium-like isolates, 16 Scopulariopsis and Microascus isolates were identified by MALDI-TOF MS with the Bruker Filamentous Fungi Library v1.0 (FFL v1.0) alone, and the FFL v1.0 plus the BMU library, respectively. The performance was evaluated by comparing the results with DNA sequencing.

3. The mass spectrum profile (MSP) dendrograms of the 28 Scedosporium isolates, 26 Acremonium-like isolates and 27 Scopulariopsis and Microascus isolates were constructed by MALDI Biotyper OC 4.0 software, respectively, and compared with the phylogenetic trees constructed by MEGA7 software based on multi-locus sequencing.

【Results】 1. Using the FFL v1.0 alone, 95.0% (19/20) and 0% (0/20) of the Scedosporium isolates were correctly identified to the genus and species levels, respectively; none of the Acremonium-like isolates had reliable identification; 68.8% (11/16) and 62.5% (10/16) of the Scopulariopsis and Microascus isolates were correctly identified to the genus and species levels, respectively, with S. brevicaulis being the only species correctly identified.

2. Using a combination of the FFL v1.0 and the BMU library, 100% (20/20) and 75.0% (15/20) of the Scedosporium isolates were correctly identified to the species complex and species levels, respectively; all of the Acremonium-like, Scopulariopsis and Microascus isolates were correctly identified to the species level.

3. The MSP dendrogram of the Scedosporium isolates cannot differentiate species in the Scedosporium apiospermum complex. The MSP dendrogram of the Acremonium-like isolates sepatated different species clearly, with a similar topology to that of the multi-locus phylogenetic tree. The MSP dendrogram of the Scopulariopsis and Microascus isolates clustered isolates of the same species or closely related species into a single clade. However, the topology of the MSP dendrogram and the multi-locus phylogenetic tree of the Microascus isolates was different.

【Conclusions】 1. MALDI-TOF MS showed unsatisfied performance on identifying Scedosporium, Acremonium-like, Scopulariopsis and Microascus isolates when using FFL v1.0 alone.

2. With a supplementary BMU library, MALDI-TOF MS was effective to identify Scedosporium isolates to the species complex level, while limited in the species-level identification; MALDI-TOF MS was reliable to identify Acremonium-like, Scopulariopsis and Microascus isolates to the species level.

3. The MSP dendrograms could be used as a method to discriminate different Acremonium-like species, and as an auxiliary method to discriminate species within genera Scopulariopsis and Microascus, but was unable to differentate species in the S. apiospermum complex.

第一章 文献综述 1
摘要 1
介绍 1
1. MALDI-TOF MS鉴定丝状真菌的样品前处理方法 2
1.1 培养方式 2
1.2 样本制备方式 3
2. MALDI-TOF MS鉴定丝状真菌的商业系统 4
3. MALDI-TOF MS鉴定丝状真菌的自建数据库 5
4. MALDI-TOF MS鉴定丝状真菌的Cutoff值 6
5. MALDI-TOF MS鉴定丝状真菌的其他影响因素 7
6. MALDI-TOF MS鉴定丝状真菌的其他进展 8
6.1 代谢产物低分子量质谱检测 8
6.2 阳性血培养直接检测 8
总结 8
第二章 基质辅助激光解析电离飞行时间质谱鉴定赛多孢霉属真菌 9
2.1 引言 9
2.2 实验材料和方法 10
2.2.1 试验菌株 10
2.2.2 主要试验仪器 10
2.2.3 主要实验试剂 11
2.2.4 主要实验耗材 12
2.2.5 主要试剂的配制及储存 12
2.2.6 实验方法 13
2.2.7 统计学处理 18
2.3 结果 19
2.3.1 赛多孢霉属菌株BT2序列测序结果及镜下结构 19
2.3.2 赛多孢属菌株MALDI-TOF MS 蛋白指纹图谱 20
2.3.3 赛多孢霉菌株MALDI-TOF MS 鉴定结果 21
2.3.4 赛多孢霉属真菌MSP聚类分型树状图 22
2.4 讨论 22
第三章 基质辅助激光解析电离飞行时间质谱鉴定枝顶孢霉样真菌 25
3.1 引言 25
3.2 实验材料和方法 26
3.2.1 试验菌株 26
3.2.2 主要实验仪器 26
3.2.3 主要实验试剂 26
3.2.4 主要实验耗材 26
3.2.5 主要试剂的配制及储存 26
3.2.6 实验方法 27
3.2.7 统计学处理 28
3.3 结果 28
3.3.1 枝顶孢霉样真菌镜下结构 28
3.3.2 枝顶孢霉样真菌MALDI-TOF MS蛋白指纹图谱 31
3.3.3 枝顶孢霉样真菌MALDI-TOF MS鉴定结果 31
3.3.4 枝顶孢霉样真菌MSP聚类分型树状图 32
3.4 讨论 33
第四章 基质辅助激光解析电离飞行时间质谱鉴定帚霉属和小囊菌属真菌 35
4.1 引言 35
4.2 实验材料和方法 36
4.2.1 试验菌株 36
4.2.2 主要实验仪器 36
4.2.3 主要实验试剂 36
4.2.4 主要实验耗材 36
4.2.5 主要试剂的配制及储存 36
4.2.6 实验方法 37
4.2.7 统计学处理 38
4.3 结果 38
4.3.1 帚霉属和小囊菌属真菌镜下结构 38
4.3.1 帚霉属和小囊菌属真菌MALDI-TOF MS蛋白指纹图谱 42
4.3.2 帚霉属和小囊菌属真菌MALDI-TOF MS鉴定结果 42
4.3.3 帚霉属和小囊菌属真菌MSP聚类分型树状图 43
4.4 讨论 44
第五章 结论及展望 47
5.1 结论 47
5.2 展望 47
参考文献 48
附录A 所有试验菌株的分离部位及DNA测序结果 56
附录B 所有测试菌株的MALDI-TOF MS鉴定结果 61
致谢 64
北京大学学位论文原创性声明和使用授权说明 65
个人简历 66
学位论文答辩委员会名单 68

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