红色毛癣菌的生物学特性研究

Study on the biological characteristics of Trichophyton rubrum

目的 观察红色毛癣菌在不同温度、不同培养基上的生长和产孢情况,并对其进行分子生物学鉴定.方法 ①大培养:采用沙堡葡萄糖琼脂(SDA)和马铃薯葡萄糖琼脂(PDA)平皿,27℃、35℃黑暗培养,测量菌落直径,绘成生长曲线.②小培养(钢圈法):采用SDA、PDA、溴甲酚紫乳固体葡萄糖琼脂(BCP-MSG)、乳蜜琼脂(M)和复合维生素B(VitB)培养基,27℃、30℃黑暗培养,观察镜下菌丝生长、孢子产生情况.③进行rDNA 18S和ITS序列测定.结果 在SDA,PDA上,27℃条件下菌落生长速度较35℃快;在5种培养基上,SDA、PDA产孢较快较多,复合维生素B培养基产孢较慢,但产生大分生孢子较多.30℃产孢更丰富.对部分菌株rDNA ITS、18S PCR扩增产物纯化后直接测序,结果在GenBank中比对、分析,相似度为98%～100%,均鉴定为红色毛癣菌.结论 SDA、PDA均为鉴定和分离红色毛癣菌的合适培养基.5种培养基均可用来刺激红色毛癣菌产孢,其中SDA、PDA产孢较早、较丰富.红色毛癣菌rDNA 18S和ITS序列测定是一种快速准确的红色毛癣菌分子生物学鉴定方法.

Objective To observe the growth situation and sporulation of Trichophyton rubrum,and to understand the rDNA sequence of Trichophyton rubrum and find a rapid identification method.Methods ①Colony observation:The strains were cultured on the Petri Dish of SDA and PDA at 27℃ and 35℃ for 4 weeks.②Slide culture: The strains were cultured on SDA,PDA,BCP-MSG, Milk Honey Agar and Vitamin B Complex Agars at 27℃and 30℃ for 3 weeks.③Molecular Biological Study:The ITS and 18S regions of the selected strains were amplified.Results The suitable temperature for growth was 27℃. 30℃ was better for sporulation.The strains on SDA and PDA sporulated more fast and abundantly,while the strains on Vitamin B complex agar produce more macroconidia. After comparison of the sequence of the PCR products, the identities of the sequence were 98%~100%.Conclusions ①SDA and PDA are the suitable culture media for isolating Trichophyton rubrum and motivating its sporulation.②Vitamin B complex agar is suitable for macroconidia production.③18S and ITS rDNA sequence can be used in clinical PCR quick identification of Trichophyton rubrum.