Panfungal Polymerase Chain Reaction for Identification of Fungal Pathogens in Formalin-Fixed Animal Tissues
Panfungal Polymerase Chain Reaction for Identification of Fungal Pathogens in Formalin-Fixed Animal Tissues
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DOI：
10.1177/0300985817698207
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发表时间：
2017-07-01
影响因子：
2.4
通讯作者：
Hoffmann, Aline Rodrigues
中科院分区：
文献类型：
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作者：
Meason-Smith, Courtney;Edwards, Erin E.;Hoffmann, Aline Rodrigues
真菌生物的鉴定常常给病理学家带来问题，因为一些真菌生物的组织形态不特异，可能无法获得新鲜组织，并且培养物中的分离和鉴定可能需要很长时间。本研究的目的是验证使用全真菌聚合酶链反应 (PCR) 来识别福尔马林固定石蜡包埋 (FFPE) 组织中的真菌。对 128 个包含犬、猫、马和牛组织的福尔马林固定石蜡包埋的卷发进行了测试，这些组织有皮肤、鼻腔、肺部和全身真菌感染，通过组织学切片中真菌的存在进行鉴定。组织学切片的定量评分鉴定出罕见（11.9%）、偶尔（17.5%）、中等（17.5%）或丰富（53.1%）的真菌生物体。从 FFPE 组织中分离出 DNA，并针对内转录间隔区 2 (ITS-2) 区域（所有真核生物中发现的一段非编码 DNA）进行 PCR。使用基本局部比对搜索工具和 NCBI ITS 序列数据库对聚合酶链式反应产物进行测序并以 97% 的同一性匹配进行鉴定。在 128 个区块中，117 个（91.4%）产生了 PCR 产物，其中 89 个（69.5%）获得了高质量序列。 79 个区块 (61.7%) 的序列和组织学鉴定相匹配。当组织病理学无法做到这一点时，该测定能够提供属和种水平的鉴定，因此，是诊断真菌疾病的有益补充工具。
Identification of fungal organisms often poses a problem for pathologists because the histomorphology of some fungal organisms is not specific, fresh tissues may not be available, and isolation and identification in culture may take a long time. The purpose of this study was to validate the use of panfungal polymerase chain reaction (PCR) to identify fungal organisms from formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed paraffin-embedded curls were tested from 128 blocks containing canine, feline, equine, and bovine tissues with cutaneous, nasal, pulmonary, and systemic fungal infections, identified by the presence of fungi in histologic sections. Quantitative scoring of histologic sections identified rare (11.9%), occasional (17.5%), moderate (17.5%), or abundant (53.1%) fungal organisms. DNA was isolated from FFPE tissues and PCR was performed targeting the internal transcribed spacer 2 (ITS-2) region, a segment of noncoding DNA found in all eukaryotes. Polymerase chain reaction products were sequenced and identified at 97% identity match using the Basic Local Alignment Search Tool and the NCBI database of ITS sequences. Of the 128 blocks, 117 (91.4%) yielded PCR products and high-quality sequences were derived from 89 (69.5%). Sequence and histologic identifications matched in 79 blocks (61.7%). This assay was capable of providing genus- and species-level identification when histopathology could not and, thus, is a beneficial complementary tool for diagnosis of fungal diseases.