方法：选取海南眼科医院检验科分离培养并镜检确定为不产孢子菌的真菌菌株19株，所有菌株均分离自感染性眼病患者眼部感染组织，接种于土豆葡萄糖琼脂培养基和沙氏培养基37℃温箱孵育7～21d后，镜检未发现生殖结构。采用玻璃研磨器研磨联合化学法提取真菌DNA，PCR扩增核糖体ITS保守序列，通过GenBank基因数据库对扩增出的ITS序列进行比对分析，确定致病菌种属。

结果：经PCR扩增后19株样本菌均得到清晰的目的条带。基因测序比对结果显示，19株不产孢子菌分属12个种属，包括可可毛色二孢子菌6株，月状弯孢霉1株，节菱孢霉菌2株，neodeightonia subglabosa 2株，红贝菌1株，肉座菌1株，多喙茎点霉1株，红色毛藓菌1株，深海曲霉菌1株，roussoella siamensis 1株，撕裂蜡孔菌1株、茄病镰刀菌1株。

结论：ITS基因测序技术可较准确地鉴定不产孢子菌的种属，是对传统真菌鉴定方法的有益补充。我国海南岛这一热带地区致病性不产孢子菌种属多样，且与多种感染性眼病的发生相关。

METHODS: Nine teen fungal strains, identified as NSM by conventional method in the laboratory department of Hainan Province Eye Hospital, were involved in this study. All of the strains were isolated from the infectious eye tissues from patients, and cultured with potato-dextrose agar and Sabourand's agar in 37℃ incubator for 7-21d. Any reproductive structure was detected by microscopy for up to 21d. After growth for 3-7d, the genomic DNA of specimens were extracted by grinding method combined with chemical method. Then, ITS sequences in the ribosome were amplified by PCR and analyzed using the National Center for Biological Information(NCBI)GenBank database. Finally, the species and genus were confirmed by sequence alignment.

RESULTS: The corresponding target bars could be observed in all 19 specimens after PCR. The results from genetic alignment classified the 19 specimens into 12 species, including Lasiodiplodia theobromae(6 isolates), Curvalaria lunata(1 isolate), Arthrinium sp.(2 isolates), neodeightonia subglabosa(2 isolates), Earliella scabrosa(1 isolate), Hypocreales sp.(1 isolate), phoma multirostrata(1 isolate), Trichophyton rubrum(1 isolate), Aspergillus westerdijkiae(1 isolate), roussoella siamensis(1 isolate), Ceriporia lacerata(1 isolate), Fusarium solqni(1 isolate).

CONCLUSION: ITS sequence alignment can identify NSM to genus and species level. The NSM in Hainan Island contains varies species, and is associated with multiple infectious diseases of the eye.