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首页> 外文期刊>Journal of Clinical Microbiology >Nucleotide Sequence-Based Analysis for Determining the Molecular Epidemiology of Penicillium marneffei
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Nucleotide Sequence-Based Analysis for Determining the Molecular Epidemiology of Penicillium marneffei

机译：基于核苷酸序列的分析法测定马尔尼菲青霉菌的分子流行病学

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The dimorphic fungus, Penicillium marneffei, is an emerging opportunistic pathogen endemic in Southeast Asia, especially for those with impaired cellular immunity such as human immunodeficiency virus-infected persons. A discriminatory and reproducible method based on the analysis of nucleotide sequences would facilitate epidemiologic investigations of this fungus. Twenty-four clinical or environmental isolates of P. marneffei obtained from China, Thailand, and Vietnam were analyzed by nucleotide sequence analysis. A total of 3,803 bp, consisting of eight nuclear gene fragments (transcription factor [AbaA], catalase [CpeA]], homodomain transcription factor [StlA], isocitrate lyase [Icl1], polyaromatic amino acid biosynthesis [PAA], NADH-dependent glutamate synthase [NGS], lovastatin nonaketide synthase [LNS], a cell wall mannoprotein [MP1], and a gene fragment of the cytochrome oxidase subunit 1 gene [COX1] of the P. marneffei mitochondrial genome) were amplified by PCR and then sequenced. No polymorphic sites within the Cox1 gene fragment were observed. Likewise, no nucleotide sequence polymorphisms were observed for three gene fragments: StlA, AbaA, and NGS. Seven single-nucleotide polymorphisms were observed for three gene fragments, Icl1, CpeA, and PAA, providing only a low degree of discriminatory power (D = 0.747). In contrast, the gene fragment for an antigenic cell wall glycoprotein, MP1, a useful immunologic marker for infection, was observed to be highly polymorphic with 12 different MP1 types (D = 0.887). Single-nucleotide polymorphisms were observed at 21 different locations in the MP1 gene fragment. Indels of 3, 21, 24, and 42 bp were observed and were in frame for protein translation. The relatively high degree of MP1 polymorphisms suggests the sequence is rapidly evolving in order to evade host immune responses. After all polymorphic gene sequences were combined, a high degree of genetic variation was observed (D = 0.949) for a total of 16 different haploid sequence types with 11 genotypes represented by single isolates. Phylogenetic analysis detected clusters composed of isolates obtained only from China or Thailand, as well as clusters with a combination of isolates from these two countries, indicating some mixing or common descent. Identical sequences were observed for isolates passed in vitro for 8 weeks, suggesting good reproducibility. The low degree of nucleotide diversity in housekeeping and regulatory genes suggests the recent emergence and spread as a species or an evolutionary bottleneck. In summary, multilocus sequence typing demonstrated a high degree of discriminatory power and reproducibility and may provide a robust and reliable adjunct method for genotyping isolates of P. marneffei and facilitating interlaboratory comparisons.
机译：二形真菌 Marneffei 是东南亚地区一种新兴的机会病原体，尤其是对于那些免疫力低下的人，例如人类免疫缺陷病毒感染者。基于核苷酸序列分析的歧视性和可重复性方法将有助于对该真菌进行流行病学研究。 P的二十四个临床或环境分离株。通过核苷酸序列分析对从中国，泰国和越南获得的marneffei 进行了分析。总共3,803 bp，由八个核基因片段组成（转录因子[AbaA]，过氧化氢酶[CpeA]，同域转录因子[StlA]，异柠檬酸裂解酶[Icl1]，多环芳族氨基酸生物合成[PAA]，NADH依赖的谷氨酸合成酶[NGS]，洛伐他汀非酰胺合成酶[LNS]，细胞壁甘露糖蛋白[MP1]和 marneffei 线粒体基因组的细胞色素氧化酶亚基1基因[COX1]的基因片段）通过PCR扩增然后测序。没有观察到Cox1基因片段内的多态性位点。同样，对于三个基因片段：StlA，AbaA和NGS，也未观察到核苷酸序列多态性。观察到三个基因片段Icl1，CpeA和PAA的七个单核苷酸多态性，仅提供了较低的辨别力（D = 0.747）。相反，观察到抗原性细胞壁糖蛋白MP1（一种有用的免疫学标记物）的基因片段具有高度多态性，具有12种不同的MP1类型（D = 0.887）。在MP1基因片段的21个不同位置观察到单核苷酸多态性。观察到3、21、24和42 bp的插入缺失，它们符合蛋白质翻译要求。相对较高的MP1多态性表明该序列正在快速进化，以逃避宿主的免疫反应。合并所有多态性基因序列后，共观察到16种不同单倍体序列类型的高度遗传变异（D = 0.949），其中11个基因型由单个分离株代表。系统发育分析检测到仅由中国或泰国获得的分离株组成的簇，以及来自这两个国家的分离株组合的簇，表明有些混合或共同下降。观察到在体外传代8周的分离株具有相同的序列，表明其可重复性良好。管家基因和调节基因中核苷酸多样性的低程度表明最近出现并作为物种或进化瓶颈传播。总而言之，多基因座序列分型显示出高度的鉴别能力和可重复性，并且可以提供强大且可靠的辅助方法用于 P的基因分型。 marneffei 并促进实验室间的比较。

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