Detection of microbial genes in a single leukocyte by polymerase chain reaction following laser capture microdissection
Detection of microbial genes in a single leukocyte by polymerase chain reaction following laser capture microdissection
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激光捕获显微切割后通过聚合酶链反应检测单个白细胞中的微生物基因
DOI：
10.1016/j.mimet.2018.11.005
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发表时间：
2018
影响因子：
2.2
通讯作者：
Okayama Akihiko
中科院分区：
文献类型：
--
作者：
Yamada Akiteru;Umeki Kazumi;Saeki Yuji;Hashikura Yuuki;Nomura Hajime;Yamamoto Ikuo;Umekita Kunihiko;Takajo Ichiro;Koshimoto Chihiro;Okayama Akihiko
虽然临床样品中细菌的分离和鉴定构成了细菌感染诊断的基本步骤，但尽管通过革兰氏染色观察细菌，但并不总是获得细菌培养的阳性结果。白细胞吞噬的细菌被认为是感染性疾病的病原体，本研究采用激光捕获显微切割（LCM）技术，在动物模型中采集中性粒细胞吞噬的细菌，然后用聚合酶链反应（PCR）进行DNA鉴定。我们将代表性细菌（大肠杆菌和金黄色葡萄球菌）接种到无特定病原体的C57 BL/6 J小鼠腹腔中。接种6 h后，从小鼠腹腔收集液体样品，通过中性粒细胞增加证明腹膜炎。然后将中性粒细胞涂片于细胞膜上，用LCM法收集单细胞吞噬菌。将细胞裂解物的上清液提供用于PCR反应以扩增16 S rRNA基因，并且我们验证了接种细菌的特异性DNA序列。此外，还建立了E. coli和革兰氏阳性菌S.金黄色葡萄球菌鉴定了每种细菌。本研究提示，LCM与PCR相结合可作为一种新的检测感染性疾病病原菌的方法。然而，需要进一步研究以检测各种额外的细菌分类群，以证明该方法对临床样本的普遍适用性。
Although isolation and identification of bacteria in a clinical specimen constitute essential steps for the diagnosis of bacterial infection, positive results of the bacterial culture are not always attained, despite observing the bacteria by Gram staining. As bacteria phagocytosed by the leukocytes are considered as the causative agents of infectious diseases, this study aims to introduce a new approach for the collection of only bacteria phagocytosed by the neutrophils in an animal model using laser capture microdissection (LCM) followed by the DNA identification using polymerase chain reaction (PCR). We inoculated representative bacteria (Escherichia coli and Staphylococcus aureus) into the abdominal cavities of specific pathogen-free C57BL/6 J mice. After 6 h inoculation, we collected the fluid samples from the peritoneal cavities of mice and demonstrated peritonitis by the increase of neutrophils. Then, we smeared the neutrophils on the membrane slides and collected single-cell phagocytosing bacteria by LCM. The supernatant of the cell lysate was supplied for the PCR reaction to amplify the 16S rRNA gene, and we validated the DNA sequences specific for the inoculated bacteria. In addition, PCR using specific primers for E. coli and S. aureus identified each species of bacteria. Hence, this study suggests that the combination of LCM and PCR could be a novel approach to determine bacteria in infectious diseases. Nevertheless, further investigation is warranted to test various additional bacterial taxa to demonstrate the general applicability of this method to clinical samples.