福尔马林固定石蜡包埋组织中致病真菌的分子鉴定。
Molecular identification of pathogenic fungi in formalin-fixed and paraffin-embedded tissues.
机构信息
Present address: Lecturer of Microbiology, Xavier University School of Medicine, Oranjestad, Aruba.
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
出版信息
J Med Microbiol. 2021 Feb;70(2). doi: 10.1099/jmm.0.001282.
Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus/species level Available protocols for the molecular identification of fungi from formalin-fixed and paraffin-embedded (FFPE) tissues have limitations in terms of extraction and target selection, and standardisation. Development of sequence-based fungal identification protocol after extraction of DNA from formalin-fixed and paraffin-embedded (FFPE) tissues. A total of 63 FFPE tissues from histopathology proven IFI cases were used to standardize the DNA extraction (commercial QIAamp kit-based extraction and conventional phenol-chloroform-isoamyl alcohol [PCI] method) and sequence-based fungal identification protocols. The PCR targeted different ribosomal DNA (rDNA) regions including complete internal transcribed spacer (ITS1-5.8S-ITS2), separate ITS1 and ITS2, 18S and D1/D2 of 28S regions. Semi-nested PCR targeting Mucorales-specific 18S rDNA region was performed in tissues having aseptate hyphae. The optimized ITS1-PCR protocol was evaluated in 119 FFPE tissues containing septate hyphae or yeast, and Mucorales-specific semi-nested PCR in 126 FFPE tissues containing aseptate hyphae. The DNA yield by conventional PCI method was significantly higher (<0.0001) than commercial kit, though the quality of DNA was similar by both protocols. The test accuracy was best while using ITS1 (61.9 %) as the target compared to 7.9, 29.9 and 22.2 % on targeting ITS1-5.8S-ITS2, ITS2, the D1/D2 region of 28S, respectively. The test accuracies of ITS1-PCR in tissues containing septate hyphae, aseptate hyphae and yeasts were 75.5, 18.7 and 100 %, respectively. The amplification (targeting ITS1 region) improved by increasing the thickness of tissue section (up to 50 µm) used for DNA extraction. ITS1-PCR protocol could amplify fungal DNA in 76 (63.8 %) tissues and Mucorales-specific semi-nested PCR in 86 (68.3 %) tissues. Conventional PCI-based DNA extraction from thick tissue (50 µm) may be used until optimal commercial fungal DNA extraction kit is developed. Subsequent ITS1-PCR for septate fungi and yeast, and semi-nested PCR targeting 18S rDNA for Mucorales are recommended to identify the fungus in FFPE tissues.
组织的组织病理学检查（HPE）有助于侵袭性真菌感染（IFI）的诊断，但不能将真菌鉴定到属/种水平。现有的从福尔马林固定和石蜡包埋（FFPE）组织中鉴定真菌的分子方法在提取和目标选择以及标准化方面存在局限性。从福尔马林固定和石蜡包埋（FFPE）组织中提取 DNA 后，开发基于序列的真菌鉴定方案。使用 63 份来自组织病理学证实的 IFI 病例的 FFPE 组织来标准化 DNA 提取（基于商业 QIAamp 试剂盒的提取和传统的酚-氯仿-异戊醇[PCI]方法）和基于序列的真菌鉴定方案。PCR 针对不同的核糖体 DNA（rDNA）区域，包括完整的内部转录间隔区（ITS1-5.8S-ITS2）、单独的 ITS1 和 ITS2、28S 区的 18S 和 D1/D2。在具有无隔菌丝的组织中进行针对毛霉科特异性 18S rDNA 区域的半巢式 PCR。在含有有隔菌丝或酵母的 119 份 FFPE 组织中评估优化的 ITS1-PCR 方案，在含有无隔菌丝的 126 份 FFPE 组织中评估毛霉科特异性半巢式 PCR。与商业试剂盒相比，传统 PCI 方法的 DNA 产量明显更高（<0.0001），尽管两种方法的 DNA 质量相似。当使用 ITS1（61.9%）作为目标时，测试准确性最佳，而针对 ITS1-5.8S-ITS2、ITS2、28S 区的 D1/D2 区域的目标的测试准确性分别为 7.9%、29.9%和 22.2%。在含有有隔菌丝、无隔菌丝和酵母的组织中，ITS1-PCR 的测试准确性分别为 75.5%、18.7%和 100%。通过增加用于 DNA 提取的组织切片的厚度（高达 50μm），可以提高 ITS1-PCR 的扩增（针对 ITS1 区域）。ITS1-PCR 方案可在 76（63.8%）份组织中扩增真菌 DNA，在 86（68.3%）份组织中扩增毛霉科特异性半巢式 PCR。在开发出最佳的商用真菌 DNA 提取试剂盒之前，可能会使用基于传统 PCI 的从厚组织（50μm）中提取 DNA。建议随后使用 ITS1-PCR 检测有隔真菌和酵母，使用针对 18S rDNA 的半巢式 PCR 检测毛霉科，以鉴定 FFPE 组织中的真菌。
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