(Trichophyton rubrum) 90% rdna 18S ITS ATCC-28188 1 (BMU00294)
5 (BMU02464 02465 01672 01643 01645) BMU00294 02464 02465 BMU00294 01672 01643 01645 1. SDA PDA 27 35 4 2. SDA PDA BCP MSG M B VitB 27 30 3. rdna ITS 18S PCR GenBank SDA PDA 2 3 SPSS t-test 3 4 p 0.05 27 35 SDA PDA B BCP MSG
10-14 rdna ITS 18S PCR GenBank 98% 100% 1.SDA PDA 27 2 3 2.SDA PDA BCP MSG SDA PDA B 30 10-14 3. ITS1 ITS4 18Sr 18Sl DNA DNA GenBank 98% 100% rdna
Studies on the biological characteristics of Trichophyton rubrum and its clinical significance ABSTRACT Postgraduate: Bao Yingqiu Advisor: Prof. Li Ruo yu Trichophyton rubrum is the common pathogen that can cause dermatophytosis. However, the knowledge of its biological characteristics is very limited, such as the feature of sporulation, proper medium for sporulation, etc. Therefore, it is necessary to understand its biological characteristics. The purpose of the present study was: To observe the growth situation of Trichophyton rubrum and draw growth curve. To find the suitable medium for sporulation. To understand the rdna sequence of Trichophyton rubrum and find a rapid identification method. Materials and Methods: Six strains collected in the Research Center for Medical Mycology of Peking University were used: ATCC-28188(BMU 00294)and five clinical isolates : BMU 02464, 02465, 01672, 01643 and 01645.BMU 00294,02464 and 02465 were used for morphological
experiment, BMU 00294, 01672, 01643 and 01645 were used for molecular biological experiments. 1.Colony observation : The strains were cultured on the Petri Dish of SDA and PDA at 27 and 35 in dark for 4 weeks respectively. The diameter of the colony was recorded everyday. A growth curve was draw based on growth time and average diameter of the colony.. 2.Slide culture: The strains were cultured on Slide cultures with SDA,PDA,BCP MSG, Milk Honey Agar, Vitamin B Complex Agar at 27 and 30 for three weeks respectively. Next, the growth of hyphae and the situation of sporulation were observed and recorded under microscope everyday. 3. Molecular Biological Study: Fungal general primers of rdna internal transcribed spacer ( ITS ) and rdna 18S were selected to amplify the ITS and 18S regions of the selected strains. After purification, the PCR products were sequenced directly and the sequences were analyzed and compared with the data from GenBank. Results: According to the growth curve, on SDA and PDA, Trichophyton rubrum grew faster in the second and third week. The growth rate and
time of Trichophyton rubrum was in direct proportion during this period. After statistic analyzed by SPSS, there was statistically significant difference of the colony diameter between cultured on 27 and 35 (p<0.05). In different culture media and under different temperatures, the strains on SDA and PDA sporulated faster and abundant, the strains on Vitamin B complex agar sporulated slower but produce more macroconidia. The strains on BCP-MSG and Milk Honey Agar also sporulated. We can see abundant spores on day 10 14 in different media. The PCR products were sequenced directly and the sequences were analyzed and compared. After searching and comparing, the identities of the sequence were 98% 100%. All the tested strains were identified as Trichophyton rubrum. Conclusions: 1. SDA and PDA are the suitable culture media for isolating and identifying Trichophyton rubrum. The suitable temperature for growth is 27.The strains grow well in the second and third week after cultivation. 2. SDA, PDA, BCP-MSG and MilK Honey Agar can be used to motivate Trichophyton rubrum to sporulate, Vitamin B complex agar can motivate Trichophyton rubrum to produce macroconidia. 30 is the suitable temperature for sporulation.
3. All strains were demonstrated to be Trichophyton rubrum. This rdna sequence can be used in clinical PCR quick identification of Trichophyton rubrum. Key words: Trichophyton rubrum growth curve characteristics of sporulation rdna sequence.
[2] 40 [3] [2] 30 - - - [4] [1] 90% 67% [5]. 1. 25-28 a. b.
[32] 2 2-3um,, [32] Trichophyton raubitschekii [32] 3 25 35 [33] [38] ph [38] 4 [8]
Norris [9] Summerbell RC [10] Fischer Kane [11] 1971 BCP-MSG [10] 4-5. Colin J J 1% 4-7 7 30-40% 7 84% [9] 15% B1 [1] 28 92% [12] 10 14 Kaminski GW MBLA 5
[15] 1 4 BCP-MSG [32] 7 ph 6. MIC 50 50% MIC 90 90% MIC MFC E-test [2] NCCLS 1998
M-38P [41] C J Jessup [3] NCCLS M-38P Fluconazole, (Griseofulvin), (Itraconazole), Terbinafine 132 MIC MIC 50 0.001 g/ml MIC 90 0.002 g/ml. DNA RAPD 8-20bp 10bp PCR DNA DNA DNA RAPD Liu [16] Zhong [17] DNA
[18] DNA rdna ribosomal ribonucleic acid rdna 18S rdna ITSI 5.8S rdna ITSII 28S rdna 100-200 18S 5.8S 28S rdna internal transcribed spacer ITS, [40] ITS ITS [39] ITS 1 ITSI ITSII ITS 4 18S rdna 5.8S rdna 28S rdna
ITS [19] ITS [20] Fari M.E1 ITS2 TR20S LRI SR6R ITS TR20S DNA [21] Colin J J ITS1 ITS4 3 17 ITS PCR M ITS 692bp [22] DNA DNA DNA. 1.
[13] 1990 Sekiguchi K ph [14] ph [33] [34] PMN [35] [13] 2 [5] [36 37]
Majocchi s granuloma 2000 Bulajic 1997 1998 2447 591 20% 41.47% 22.06% 10.88% [6] 1992 Lugo Somolinos A [24] Trichophyton raubitschekii 1981 Tietz HJ 2002 Trichophyton raubitschekii [25] 80% 35 [4] 1997 Ploysangam T 7 1988 1993 7 [26] 1994 Chang P 26 8 88.05% 18 69.2% [27]