Waltimo T, Kuusinen M, Järvensivu A, Nyberg P, Väänänen A, Richardson M, Salo T, Tjäderhane L
Department of Cariology, Institute of Dentistry, University of Turku, Turku, Finland.
Int Endod J. 2003 Sep;36(9):643-7. doi: 10.1046/j.1365-2591.2003.00707.x.
To examine the occurrence of Candida spp. in refractory periapical granulomas.
One hundred and three surgically removed periapical granulomas were subjected to molecular analysis for the occurrence of Candida albicans. DNA was extracted from the samples using a modified phenol/chloroform/isoamyl alcohol method and was subjected to polymerase chain reaction (PCR) with OPA-03 and repetitive sequence (GACA)4 primers. The PCR products were separated in agarose gel electrophoresis, stained with ethidium bromide, visualized using UV light and the sequences were analysed. Samples indicating possible occurrence of Candida were further investigated by histological and immunohistological methods. Periodic acid-Shiff staining (PAS) was used to detect yeast cells and hyphae, and specific monoclonal antibodies to recognize high molecular mass mannoproteins present in the C. albicans cell wall. DNA extraction was controlled by running PCR using beta-actin primers (a housekeeping gene). C. albicans CCUG19915, C. tropicalis ATCC750, C. krusei ATCC6258, C. guilliermondii ATCC6260 and C. glabrata CCUG32725 served as positive controls in PCR. A tissue preparation of chronic atrophic candidosis in oral buccal mucosa served as a positive control for histological and immunohistological examinations.
Polymerase chain reaction with beta-actin primers indicated successful DNA extraction in 68 out of 103 samples. The majority of the samples (50) were negative whereas 18 of the samples showed PCR products indicating possible occurrence of Candida spp. PAS-staining and immunohistological examination of these samples were, however, negative. Further analysis of the PCR products revealed sequences not typical for Candida spp.
Candida spp. do not seem to occur in periapical granuloma.
研究念珠菌属在难治性根尖周肉芽肿中的发生情况。
对103例手术切除的根尖周肉芽肿进行白色念珠菌发生情况的分子分析。采用改良的酚/氯仿/异戊醇法从样本中提取DNA，并使用OPA - 03和重复序列（GACA）4引物进行聚合酶链反应（PCR）。PCR产物在琼脂糖凝胶电泳中分离，用溴化乙锭染色，紫外光下观察并分析序列。对显示可能存在念珠菌的样本进一步采用组织学和免疫组织学方法进行研究。采用过碘酸希夫染色（PAS）检测酵母细胞和菌丝，并使用特异性单克隆抗体识别白色念珠菌细胞壁中存在的高分子量甘露糖蛋白。通过使用β - 肌动蛋白引物（一种管家基因）进行PCR来控制DNA提取。白色念珠菌CCUG19915、热带念珠菌ATCC750、克鲁斯念珠菌ATCC6258、季也蒙念珠菌ATCC6260和光滑念珠菌CCUG32725用作PCR的阳性对照。口腔颊黏膜慢性萎缩性念珠菌病的组织标本用作组织学和免疫组织学检查的阳性对照。
用β - 肌动蛋白引物进行的聚合酶链反应表明，103个样本中有68个成功提取了DNA。大多数样本（50个）为阴性，而18个样本显示PCR产物，表明可能存在念珠菌属。然而，对这些样本进行的PAS染色和免疫组织学检查均为阴性。对PCR产物的进一步分析显示，其序列并非念珠菌属所特有。
根尖周肉芽肿中似乎不存在念珠菌属。
