The use of combined PCR, fluorescence in situ hybridisation and immunohistochemical staining to diagnose mucormycosis from formalin-fixed paraffin-embedded tissues
The use of combined PCR, fluorescence in situ hybridisation and immunohistochemical staining to diagnose mucormycosis from formalin-fixed paraffin-embedded tissues
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使用组合 PCR、荧光原位杂交和免疫组织化学染色诊断福尔马林固定石蜡包埋组织中的毛霉菌病
DOI：
10.1111/myc.13382
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发表时间：
2021-10-23
期刊：
影响因子：
4.9
通讯作者：
Li, Ruoyu
中科院分区：
文献类型：
--
作者：
Liu, Xiao;Song, Yinggai;Li, Ruoyu
目的建立一套完整的毛霉菌病诊断系统，包括实时荧光PCR、荧光原位杂交和免疫组化染色。方法根据基因组序列设计11条引物和探针进行实时荧光定量PCR检测，并对曲霉属、镰刀菌属、丝孢霉属、长孢霉属、隐球菌属和念珠菌属进行特异性验证。收集44只小鼠真菌感染模型的福尔马林固定石蜡包埋（FFPE）组织，采用激光捕获显微切割（LCM）法和直接提取法提取DNA，进行实时荧光定量PCR检测。此外，17例组织病理学证实为毛霉菌病的临床标本被纳入新诊断系统的特异性检测。结果实时荧光PCR法可检测每种菌最低10 CFU/ml的gDNA。与gDNA在种属间无交叉反应。从小鼠模型标本中，用LCM提取的样品与直接提取法相比，实时PCR的灵敏度分别为100%和91.43%，在毛霉目水平和80%对45.71%，在物种水平。特异性为100%。从临床标本中，LCM结合实时荧光PCR可检出88.24%（15/17）的毛霉菌。荧光原位杂交（FISH）和免疫组化（IHC）的敏感性分别为70.59%和41.18%。LCM-RT-PCR、FISH和IHC联合检测在所有样本中均得到阳性结果。结论本研究建立的毛霉菌病联合诊断系统是一种不依赖于培养的、可靠的方法，可用于快速鉴定FFPE组织中的菌种，以及时诊断毛霉菌病。
Objective To develop a comprehensive diagnostic system for mucormycosis from formalin-fixed paraffin-embedded tissues, consisting of own-designed real-time polymerase chain reaction (PCR) assays, fluorescence in situ hybridisation, and immunohistochemical staining. Methods We designed 11 primers and probes for specific real-time PCR assays based on genome sequences, and validated the specificity by Aspergillus, Fusarium, Scedosporium, Lomentospora, Cryptococcus and Candida species. Formalin-fixed paraffin-embedded (FFPE) tissues from forty-four mouse model infected by above fungi were collected and extracted DNA by laser capture microdissection (LCM) and direct extraction methods for real-time PCR assays. In addition, seventeen clinical specimens histopathologically proven for mucormycosis were included for specific detection with the new diagnostic system. Results The real-time PCR assays allowed detection of a minimum of 10 CFU/ml equivalent gDNA of each species. No cross-reaction with gDNA among species was noted. From mouse model specimens, the sensitivity of real-time PCR in samples extracted with LCM versus direct extraction method was 100% versus 91.43% at Mucorales level and 80% versus 45.71% at species level, respectively. The specificity was 100%. From clinical samples, LCM combined with real-time PCR can test 88.24% (15/17) of Mucorales. Sensitivities of fluorescence in situ hybridisation (FISH) and immunohistochemical staining (IHC) were 70.59% and 41.18%, respectively. Combined LCM-RT-PCR, FISH and IHC yielded positive results in all samples. Conclusions The combination diagnostic system we developed is a culture-independent and robust method which enables rapid species identification from FFPE tissues for timely diagnosis of mucormycosis.