Gade Lalitha, Hurst Steven, Balajee S Arunmozhi, Lockhart Shawn R, Litvintseva Anastasia P
Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA.
Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA.
Med Mycol. 2017 Jun 1;55(4):385-395. doi: 10.1093/mmy/myw083.
Molecular methods of detection based on DNA-sequencing of the internal transcribed spacer 1 and 2 (ITS1 and ITS2) or 5΄ end region of 28S (D1-D2 region) of ribosomal RNA gene (rDNA) have been used extensively for molecular identification and detection of fungal infections. However, these regions are not always informative for identification of mucormycetes and other rare fungal pathogens as they often contain large introns, heterogenic regions, and/or cannot be PCR-amplified using broad range fungal PCR primers. In addition, because of the difficulties of recovering intact fungal DNA from human specimens, smaller regions of DNA are more useful for the direct detection of fungal DNA in tissues and fluids. In this study, we investigated the utility of 12F/13R PCR primers targeting a 200-230 bp region of the extended 28S region of rDNA for molecular identification of fungal DNA in formalin fixed paraffin embedded tissues and other clinical specimens. We demonstrated that this region can be successfully used for identification of all genera and some species of clinically relevant mucormycetes, as well as other medically important fungi, such as Aspergillus, Fusarium, Coccidioides, and Cryptococcus. We also demonstrated that PCR amplification and direct sequencing of the extended 28S region of rDNA was more sensitive compared to targeting the ITS2 region, as we were able to detect and identify mucormycetes and other fungal pathogens in tissues from patients with histopathological and/or culture evidence of fungal infections that were negative with PCR using ITS-specific primers.
基于核糖体RNA基因（rDNA）的内转录间隔区1和2（ITS1和ITS2）或28S的5΄端区域（D1-D2区域）的DNA测序的分子检测方法已被广泛用于真菌感染的分子鉴定和检测。然而，这些区域对于毛霉目真菌和其他罕见真菌病原体的鉴定并不总是提供有用信息，因为它们通常包含大的内含子、异质区域，和/或不能使用通用真菌PCR引物进行PCR扩增。此外，由于从人体标本中回收完整真菌DNA存在困难，较小的DNA区域对于在组织和体液中直接检测真菌DNA更有用。在本研究中，我们研究了靶向rDNA扩展28S区域的200-230 bp区域的12F/13R PCR引物在福尔马林固定石蜡包埋组织和其他临床标本中对真菌DNA进行分子鉴定的效用。我们证明该区域可成功用于鉴定所有临床相关毛霉目真菌属和一些种，以及其他医学上重要的真菌，如曲霉属、镰刀菌属、球孢子菌属和隐球菌属。我们还证明，与靶向ITS2区域相比，rDNA扩展28S区域的PCR扩增和直接测序更敏感，因为我们能够在组织病理学和/或培养有真菌感染证据但使用ITS特异性引物进行PCR呈阴性的患者组织中检测和鉴定毛霉目真菌和其他真菌病原体。
