Real-time PCR method for detection of zygomycetes
Real-time PCR method for detection of zygomycetes
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DOI：
10.1128/jcm.02331-07
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发表时间：
2008-07-01
影响因子：
9.4
通讯作者：
Wengenack, Nancy L.
中科院分区：
文献类型：
--
作者：
Hata, D. Jane;Buckwalter, Seanne P.;Wengenack, Nancy L.
接合菌感染对于免疫功能低下的宿主来说可能是毁灭性的。该类真菌与其他丝状真菌（例如曲霉属、镰刀菌属）在组织病理学上难以区分，可能会导致诊断和开始适当治疗的延迟，从而显着影响患者的治疗结果。开发了一种实时 PCR 检测方法来检测培养物和组织样本中的接合菌属 Absidia、Apophysomyces、Cunninghamella、Mucor、Rhizopus 和 Saksenaea 的物种。设计引物和荧光共振能量转移杂交探针来检测多拷贝接合菌细胞色素 b 基因的 167 bp 保守区域。构建含有来自总状毛霉的靶序列的质粒作为阳性对照。该测定的分析灵敏度为 10 个靶标/μl，由其他丝状真菌、酵母（念珠菌属）和细菌组成的特异性小组在测定中证明没有交叉反应性。培养分离株检测的临床敏感性和特异性分别为 100% (39/39) 和 92% (59/64)。使用有限数量的新鲜组织标本测定的敏感性和特异性均为 100% (2/2)。福尔马林固定、石蜡包埋的组织的敏感性为 56% (35/62)，特异性为 100% (19/19)。 PCR 检测的速度、灵敏度和特异性表明它可用于快速、准确地检测接合菌。
Zygomycete infections can be devastating in immunocompromised hosts. Difficulties in the histopathologic differentiation of this class from other filamentous fungi (e. g., Aspergillus spp., Fusarium spp.) may lead to delays in diagnosis and initiation of appropriate treatment, thereby significantly affecting patient outcome. A real-time PCR assay was developed to detect species of the zygomycete genera Absidia, Apophysomyces, Cunninghamella, Mucor, Rhizopus, and Saksenaea in culture and tissue samples. Primers and fluorescence resonance energy transfer hybridization probes were designed to detect a 167-bp conserved region of the multicopy zygomycete cytochrome b gene. A plasmid containing target sequence from Mucor racemosus was constructed as a positive control. The analytical sensitivity of the assay is 10 targets/mu l, and a specificity panel consisting of other filamentous fungi, yeasts (Candida spp.), and bacteria demonstrated no cross-reactivity in the assay. The clinical sensitivity and specificity of the assay from culture isolates were 100% (39/39) and 92% (59/64), respectively. Sensitivity and specificity determined using a limited number of fresh tissue specimens were both 100% (2/2). The sensitivity seen with formalin-fixed, paraffin-embedded tissues was 56% (35/62), and the specificity was 100% (19/19). The speed, sensitivity, and specificity of the PCR assay indicate that it is useful for the rapid and accurate detection of zygomycetes.