微生物学通报 JUN 20, 2009, 36(6): 918~922 Microbiology tongbao@im.ac.cn 生物实验室 2009 by Institute of Microbiology, CAS PCR-SSCP 技术在假丝酵母属菌种鉴别中的应用 * 宋维娟程池 ( 100027) 摘要 : 选取中国工业微生物菌种保藏管理中心 (CICC) 保藏的假丝酵母属的 7 个种 30 株菌, 对其 rdna 的 ITS1 区及 ITS2 区进行了 PCR-SSCP 指纹图谱分析, 结果表明在假丝酵母属种水平的区分鉴定中, ITS1 区与 ITS2 区的 PCR-SSCP 图谱均能对本研究所选 7 个种的菌株进行显著区分, 比较两个区段的 PCR-SSCP 图谱及鉴别效果, 发现 ITS2 区的应用效果要优于 ITS1 区 关键词 : ITS1, ITS2, PCR-SSCP, 假丝酵母, 鉴别 Differentiation of Candida Species by PCR-SSCP Fingerprinting Analysis SONG Wei-Juan CHENG Chi * (China National Research Institute of Food & Fermentation Industries, China Center of Industrial Culture Collection, Beijing 100027, China) Abstract: Thirty strains of seven Candida species from CICC(China Center of Industrial Culture Collection)were studied. The strains were differentiated by ITS1 region PCR-SSCP fingerprinting analysis and ITS2 region PCR-SSCP fingerprinting analysis. Results showed that both ITS1 region and ITS2 region were able to differentiate the seven species of Candida clearly. Contrasting the maps and effects on the identification of Candida species of ITS1 region with that of ITS2 region, result indicated that on the identification of Candida species the application of ITS2 region was better than ITS1 region. Keywords: Internal transcribed spacer 1, Internal transcribed spacer 2, PCR-SSCP, Candida, Differentiation (Candida Berkhout), 163, 1/3 [1],,,, PCR-SSCP(Polymerase chain reactionsingle strand conformation polymorphism) PCR-SSCP [2,3] DNA, DNA, DNA, DNA,, [4] rdna PCR-SSCP 基金项目 : (No.2005DKA21204) * 通讯作者 :Tel: 86-10-64616613; : cheng100027@163.com 收稿日期 :2008-10-17; 接受日期 :2008-12-17
: PCR-SSCP 919 ; [5], PCR-SSCP ; rdna, ITS (ITS1-5.8S rdna-its2) 26S rdna D1/D2 [6], [3,7], 100 bp~300 bp SSCP 150 bp~300 bp ITS1 ITS2 7 30 PCR-SSCP, Candida 1 材料与方法 1.1 材料 1.1.1 供试菌株 : CICC 30, 1 1.1.2 培养基 : 1.1.3 主要试剂和仪器 : PCR, PCR, Tris, PCR (MJ Minicycler TM ), (KUBOTA 3500), (DYY-8B), (24EN), (D-77WL) Table 1 表 1 供试菌株 Test strains of Candida sp. Sample No. Species CICC No. Origins 1 Candida utilis 1767 ATCC 22023 2 1768 ATCC 9226 3 1769 ATCC 9950 4 1314 Institute of Microbiology, Chinese Academy of Sciences, Beijing 5 Candida guilliermondii 31346 The factory of Yeast, Shanghai 6 31552 Institute of Microbiology, Chinese Academy of Sciences, Beijing 7 1274 Institute of Microbiology, Chinese Academy of Sciences, Beijing 8 1945 Institute of Biobotany, Chinese Academy of Sciences, Shanghai 9 1951 ATCC 22984 10 Candida shehatae 1766 ATCC 34887 11 Candida maltosa 1962 Institute of Agriculture, Chinese Academy of Sciences, Beijing 12 1658 Institute of Industrial Microbiology, Tianjin 13 1659 Institute of Industrial Microbiology, Tianjin 14 31590 Institute of Industrial Microbiology, Tianjin 15 31591 Institute of Industrial Microbiology, Tianjin 16 Candida tropicalis 1956 Institute of Microbiology, Chinese Academy of Sciences, Beijing 17 1253 Institute of Industry, Shanghai 18 1254 Institute of Industry, Shanghai 19 1316 Institute of Microbiology, Chinese Academy of Sciences, Beijing 20 1318 Paper Research Institute of the light industry Ministry 21 1403 Paper Research Institute of the light industry Ministry 22 1407 Jiang Men Chemical Plant of Sugar and Cane, Guangdong province 23 1426 Institute of Fermentation Industry, First Ministry of light industry 24 1655 Jiang Men Chemical Plant of Sugar and Cane, Guangdong province 25 1729 Buttery of the first light industry, Guangdong province 26 Candida parapsilosis 1627 The factory of Yeast, Shanghai 27 31253 Institute of Microbiology, Chinese Academy of Sciences, Beijing 28 31271 Institute of Microbiology, Chinese Academy of Sciences, Beijing 29 31861 Institute of light industry, Hunan province 30 Candida lipolytica/y. lipolytica 1440 China Center of Industrial Culture Collection
920 微生物学通报 2009, Vol.36, No.6 1.2 基因组 DNA 的提取 DNA [8] DNA 1.3 ITS1 区与 ITS2 区的扩增 ITS1 ITS2 [9] 2 ITS1 ITS2 PCR 1%, SSCP, PCR 20 C 1.4 SSCP 分析 1.4.1 单链样品的制备 : Feng-Yan Bai [5], 3 μl PCR 2 μl ddh 2 O, 5 μl, 95 C 10 min, 15 min,, SSCP 1.4.2 非变性聚丙烯酰胺凝胶电泳 : 8% (40% / (37.5:1, V/V) 8 ml, 10 TBE 4 ml, 100% 2 ml, TEMED 40 μl, 10% 400 μl, 25.56 ml, 100 mm 130 mm 1.0 mm), 1 h 1 TBE 8 C, PCR, 2.5 μl, 200 V 10 min, 120 V 4 h~ 5 h, 8 C 1.4.3 银染 : Wallace [10] :,, 300 ml 10% 5 min;, 300 ml 0.5% 5 min;, 300 ml 0.1%, 100 r/min 15 min;, 300 ml ; 300 ml 1.5% 450 μl, 2 min~5 min,,,, 2, 300 ml 0.75%, 15 min, 2 结果 2.1 ITS1 PCR-SSCP 分析结果 1 1A 1B 7 30 Table 2 表 2 PCR 引物与扩增程序 Primers of polymerase chain reaction and reaction program Position Primer Primer Sequence(5 3 ) Direction Reaction Program ITS1 region ITS2 region ITS1 TCCGTAGGTGAACCTGCGG Forward ITS2 GCTGCGTTCTTCATCGATGC Reverse ITS3 GCATCGATGAAGAACGCAGC Forward ITS4 TCCTCCGCTTATTGATATGC Reverse 94 C 4 min, 36 Cycles 94 C 1 min, 62 C 1 min, 72 C 30 s, 72 C 10 min. 94 C 4 min, 36 Cycles 94 C 1 min, 58 C 1 min, 72 C 40 s, 72 C 10 min. 图 1 ITS1 PCR-SSCP 指纹图谱 Fig. 1 SSCP patterns of ITS1 region from type or authentic strains of seven Candida species Note: A: 1: C. maltosa (CICC 1962); 2~5: C. utilis (P. jadini) (CICC 1768, CICC 1767, CICC 1769 and CICC 1314, respectively); 6~9: C. parapsilosis (CICC 31271, CICC 31861, CICC 1627 and CICC 31253, respectively); 10~14: C. guilliermondii (CICC 1951, CICC 1945, CICC 1274, CICC 31552 and CICC 31346, respectively); 15: C. tropicalis (CICC 1956); 16: C. shehatae (CICC 1766); B: 17~21: C. maltosa (CICC 31591, CICC 31590, CICC 1659, CICC 1658 and CICC 1962, respectively); 22: C. lipolytica (Y. lipolytica) (CICC 1440); 23~32: C. tropicalis (CICC 1956, CICC 1729, CICC 1655, CICC 1426, CICC 1407, CICC 1403, CICC 1318, CICC 1316, CICC 1254 and CICC 1253, respectively); T was instead of type strain.
: PCR-SSCP 921 ITS1 PCR-SSCP 1A 6 ;, ; 1A, C. parapsilosis(6~9), 6 7, 8 9, ; C. utilis(2~5) C. guilliermondii(10~14) 1 B, C. maltosa(17~21) C. tropicalis (23~32) C. lipolytica(22) ; C. maltosa (21T) 4 (17~20 ); C. tropicalis (23T) 9 1A 1B 2 ( 1A: 8 9 15, 1B: 17 18 19 20) 1A 1B, ITS1 PCR-SSCP 7, 2, 1 2.2 ITS2 PCR-SSCP 分析结果 2 A B 7 30 ITS2 PCR-SSCP 2A 6, ; C. parapsilosis(1~4)4 ; C. guilliermondii(6~10)5, C. utilis(11~14)4 2B, C. tropicalis(17~25) C. maltosa(27~32) C. lipolytica(26)3 ; C. tropicalis (25T) 8, 8 ; C. maltosa (32T) 4 (27~31 ), 4 2A 2B, 2 ( 2A: 1~4 15, 2B: 17~25 32), ITS2 PCR-SSCP 7, 3 讨论 ITS1 ITS2 PCR-SSCP Candida, : 1) ITS1 ITS2 PCR-SSCP 7, ; 2) Candida, ITS2 PCR-SSCP ITS1, ITS2 ITS1, ITS2 ITS1, ITS1 [11] ; 3) ITS1 ITS2 图 2 ITS2 PCR-SSCP 指纹图谱 Fig. 2 SSCP patterns of ITS2 region from type or authentic strains of seven Candida species Note: A: 1~4: C. parapsilosis (CICC 1627, CICC 31861, CICC 31271 and CICC 31253, respectively); 5: C. tropicalis (CICC 1956); 6~10: C. guilliermondii (CICC 1945, CICC 1951, CICC 1274, CICC 31552 and CICC 31346 respectively); 11~14: C. utilis (P. jadinii) (CICC 1314, CICC 1769, CICC 1768 and CICC 1767, respectively); 15: C. maltosa (CICC 1962); 16: C. shehatae (CICC 1766); B: 17~25: C. tropicalis (CICC 1729, CICC 1254, CICC 1316, CICC 1407, CICC 1403, CICC 1318, CICC 1655, CICC 1956, CICC 1426, respectively); 26: C. lipolytica (Y. lipolytica) (CICC 1440); 27~32: C. maltosa (CICC 31590, CICC 31590, CICC 1659, CICC 31591, CICC 1658 and CICC 1962, respectively); T was instead of type strain.
922 微生物学通报 2009, Vol.36, No.6 ( ),, ITS1 ITS2 : 10% (>15 bp), 1% (<3 bp);, 3 bp : C. tropicalis C. maltosa,, 参考文献 [1] Kurtzman CP. The yeasts, a taxonomic study. Holland: Elsevier, 1998, pp.454 475. [2],. PCR-SSCP., 2005, 1(25): 89 93. [3] Orita M, H Iwahana, H Kanazawa, et al. Detection of polymorphisms of human DNA by gel electrophoresis as single strand conformation polymorphisms. Proc Natl Acad Sci, 1989, 86: 2766 2770. [4].., 2004. [5] Qi-Ming Wang, Juan Li, Feng-Yan Bai, et al. Rapid differentiation of phenotypically similar yaest species by single-strand conformation polymophism analysis of ribosomal DNA. Applied and Environmental Microbiology, 2008, 5: 2604 2611. [6] Scoretti G, Fell JW, Fonseca A, et al. Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rdna regions. FEMS Yeast Res, 2002, 2: 495 517. [7] Masato Orita, Youichi Suzuki, Takao Sekiya, et al. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics, 1989, 5: 874 879. [8],,. rdnad1/d2., 2002, 21(1): 27 32. [9] Manish Kumar, Shukla PK. Use of PCR targeting of internal transcribed spacer regions and single-stranded conformation polymorphism analysis of sequence variation in different regions of rrna genes in fungi for rapid diagnosis of Mycotic Keratitis. Journal of Clinical Microbiology, 2005, 43(2): 662 668. [10] Wallace AJ. SSCP/Heteroduplex analysis in PCR mutation detection protocols. Methods in molecular biology, 2000, 187: 151 163. [11] Leaw SN, Barton R, Chang SC, et al. Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions. Journal of Clinical Microbiology, 2006, 3: 693 699. 稿件书写规范 论文中计量单位的表示方法, GB3100-3102-93 ( ),, : d; h; min; s : mol/l, M ( ) N( ) : r/min, rpm : Pa kpa MPa : OD( ) : D kd, bp kb :,, : t(h) (, ) :, ( C % ) : 20 cm 0.3 cm, 20 0.3 cm; 3 C~5 C 3~5 C; 3%~6% 3~6%