多重PCR技术在检测外耳道真菌菌种中的应用研究

Application of multiplex polymerase chain reaction in the detection of fungal species of otomycosis

摘要:

目的 研究外耳道真菌病的真菌菌种分布，探讨多重PCR技术检测和鉴定外耳道真菌病常见真菌菌种的方法。方法 取2022年3月—2023年6月就诊于荆州市中心医院耳鼻咽喉科门诊的137例外耳道真菌病患者外耳道标本，采用改良的马丁固体培养基对同一标本的一部分进行了常规培养，培养后提取DNA，PCR扩增及测序鉴定真菌菌种。同时，该标本另一部分直接经DNA提取后，行两次多重PCR，分别使用通用真菌引物和特异性真菌引物，根据琼脂糖凝胶电泳得到的条带的长短可鉴定真菌菌种。结果 通过传统方法和多重PCR技术行真菌菌种鉴定，本地区137例外耳道真菌病患者曲霉菌共128株(93.4%)，其中土曲霉73株、黄曲霉24株、黑曲霉18株和烟曲霉13株；念珠菌共9株(6.6%)，均为白念珠菌。传统方法需4～7 d，而多重PCR技术6～8 h即可鉴定真菌菌种，对比两组方法结果，符合率为100%。结论 本地区外耳道真菌病患者最常见的真菌菌种是曲霉菌和念珠菌。除了常见菌种，多重PCR技术还可以鉴定高耐药和高致死率的耳念珠菌、近平滑念珠菌等罕见菌种。与传统方法相比，多重PCR技术检测和鉴定外耳道真菌病具有快速、准确等优点，值得临床推广。

关键词:外耳道真菌病;菌种鉴定;多重PCR技术;快速诊断

Abstract:

Objective To study the distribution of fungal species in otomycosis, and to explore the method of multiplex polymerase chain reaction (PCR) to detect and identify common fungal species in otomycosis.Methods Specimens were collected from external ear canal of 137 patients with otomycosis who visited the department of otolaryngology, Jingzhou central hospital from March 2022 to June 2023. Part of the specimen was routinely cultured with modified Martin solid medium. After culture, DNA was extracted, and the fungal species were identified by PCR amplification and sequencing. At the same time, the other part of the same specimen was directly extracted by DNA, and multiple PCR were performed twice, using the universal and specific fungal primers respectively. The fungal species could be identified according to the length of bands obtained by agarose gel electrophoresis.Results A total of 128 Aspergillus strains (93.4%) were identified by traditional methods and multiplex PCR in 137 otomycosis specimens, including 73 strains of Aspergillus terreus, 24 strains of Aspergillus flavus, 18 strains of Aspergillus niger, and 13 strains of Aspergillus fumigatus. There were 9 strains (6.6%) of Candida albicans. The traditional method took 4 to 7 days, while the multiplex PCR technique could identify the fungal species in 6 to 8 hours. The results of the two methods were compared, and the coincidence rate was 100%.Conclusions The most common fungal species in patients with otomycosis in this region are Aspergillus and Candida. In addition to common species, multiplex PCR can also be used to identify rare species such as Candida auris and Candida parapsilosis with high drug resistance and high mortality. Compared with traditional methods, multiplex PCR has the advantages of rapid and accurate detection and identification of otomycosis, which is worthy of clinical promotion.

Key words:Otomycosis;Species identification;Multiplex polymerase chain reaction technique;Rapid diagnosis