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1 HEREDITAS (Beijing) , 29(8): ISSN 研究报告 DOI: /yc PCR 高斌 1, 肖白 1, 邹起练 2, 黄尚志 3, 王立荣 3, 钮淑兰 4, 闫梅 1, 雷箴 1, 贾兴元 1, 王战勇袁海昕 1, 吴燕 1 1, 刘敬忠 1., ; 2., ; 3., ; 4., : 为了建立一种基于多重实时荧光相对定量 PCR 技术并应用之于唐氏综合征分子诊断, 选择 21 号染色体上唐氏综合征特异区域基因片段 (DSCR3) 为目的基因, 以 12 号染色体上的磷酸甘油醛脱氢酶基因 (GAPDH) 为参照基因, 设计合成两对引物以及分别以不同荧光标记的 TaqMan 探针, 在同一个反应管中进行扩增以相对定量指标 CT 值区分唐氏综合征患者与正常人采用 EB 病毒转化技术, 把唐氏综合征患者外周血 B 淋巴细胞转化成永生淋巴母细胞系作为标准品通过优化反应条件, 使得目的基因和参照基因的扩增效率基本一致, 接近 100%, 模板浓度在 3~300 ng/μl 范围内, CT 值的变异系数小于 15%, 浓度在 30 ng/μl 时, 变异系数最小 (<10%), 以该浓度的 DNA 作为模板进行批内和批间实验的 CT 值重复性好, 变异系数分别为 9.8% 和 13.3% 运用建立的方法检测 20 例唐氏综合征患者的血标本和 30 例正常人的血标本, 正常人 CT 值范围是 1.90~ 1.30, 患者的 CT 值范围是 2.95~ 2.15, 两组之间无交叉重叠, 有明显差异 (P<0.001) 唐氏综合征患者永生细胞系建系成功, 染色体核型和 DNA 分析表明建系前后遗传是稳定的因此, 实时荧光定量 PCR 比较 CT 值的相对定量法快速诊断唐氏综合征是可行的 : 唐氏综合征 ; 荧光定量 PCR; 相对定量 ; CT 值 ; 永生细胞系 1, Rapid diagnosis of Down s syndrome by multiplex real-time fluorescence relative quantitative PCR GAO Bin 1, XIAO Bai 1, ZOU Qi-Lian 2, HUANG Shang-Zhi 3, WANG Li-Rong 3, NIU Shu-Lan 4, YAN Mei 1, LEI Zhen 1, JIA Xing-Yuan 1, WANG Zhan-Yong 1, YUAN Hai-Xin 1, WU Yan 1, LIU Jing-Zhong 1 1. Basic Medical Research Center, Capital Medical University Affiliated Beijing Chaoyang Hospital, Beijing , China; 2. Department of Cell Biology and Genetics, Fujian Medical University, Fuzhou , China; 3. Medical Genetics Laboratory, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing , China; 4. Central Laboratory, Peking University First Hospital, Beijing , China Abstract: To establish a multiplex real-time fluorescence relative quantitative PCR method for diagnosis of Down s syndrome. The fragment from Down's syndrome critical region gene 3 (DSCR3) on chromosome 21 was used as the target gene, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene on chromosome 12 was used as the control gene. The two genes were amplified in the same tube. The relative quantitative index- CT value was used to differentiate trisomy 21 收稿日期 : ; 修回日期 : 基金项目 : ( : JS96004) [Supported by the Natural Science Foundation of Beijing (No.JS96004)] 作者简介 : (1978 ),,, : wenwugao@sina.com 通讯作者 : (1942 ),,,,, : liujz@public.bta.net.cn

, 100034 : 为了建立一种基于多重实时荧光相对定量 PCR 技术并应用之于唐氏综合征分子诊断, 选择 21 号染色体上唐氏综合征特异区域基因片段 (DSCR3) 为目的基因, 以 12 号染色体上的磷酸甘油醛脱氢酶基因 (GAPDH) 为参照基

2 8 : PCR 935 patient from normal person. The peripheral blood sample from a Down s syndrome patient was collected and the B-lymphocytes were transformed by Epstein-Barr virus to establish the immortalized cell lines as standard material. The reaction conditions were optimized to obtain an equal amplification efficiency from both the target and the control genes. The slopes of both genes were almost 3.32, indicating that the efficiencies of the two amplifications were approximately equal. Among a certain range from ng/pcr, the variation of detected CT value were less than 15%, and amplification showed the highest reproducibility when the concentration of DNA template was 30 ng/μl. Then, the variation of CT value with inter- and intra-assay were 9.8% and 13.3% at this DNA concentration of the templates. Clinical samples, including 20 blood samples from patients and 30 blood samples from normal persons, were detected using the established method. The CT value from Down s syndrome group were dramatically different from normal group (P<0.001). The trisomy 21 immortalized cell lines were established and the genetic integrity of the cell lines was stable as evaluated by karyotype and DNA analysis. The relative quantitative PCR with CT value could be used to rapidly diagnose Down s syndrome. Keywords: Down s syndrome; fluorescence quantitative PCR; CT value; immortalized cell line (Down s syndrome) 21,, 1/600 1/800,,,,,,, (FISH) (STR), [1, 2], PCR, CT, CT, ,, 1 ml 20 DNA, G, 21,,, DSCR3 (Down s syndrome critical region gene3, DSCR3), 121 bp : Forward: 5 -TAG- CAAGTCAGAGGTTTCCTT-3 ; Reverse: 5 -AACAT- TGACTGTGGCTCTTGC-3 TaqMan : 5 -FAM-CACTCTCCAGCAAGCTCAAACTGC-TA- MRA-3 12 glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 150 bp : Forward: 5 -CTGTCCAGTTAATTT- CTGACC-3 ; Reverse: 5 -CTTTGTACATGGTATTC- ACCAC-3 TaqMan : 5 -VIC- AATGCT- TGCTGCTGCCTAGCTCAG-TAMRA-3, ABI 1.3 PCR PCR ABI Prism 7000 PCR ( ABI ) 25 μl, DNA 1 μl, 200 nmol/l, TaqMan 100 nmol/l, 2 TaqMan Universal PCR Master Mix( ABI )12.5 μl : 50 2 min, min, s, 60 1 min, 40,, 0.2; 6,, CT 3 CT, CT

The reaction conditions were optimized to obtain an equal amplification efficiency from both the target and the control genes. The slopes of both genes were almost 3.

3 936 HEREDITAS (Beijing) CT 1.4 CT (mean) ; (CV) ; t 1.5 EB (EBV), B [3], DNA PCR ABI Prism 7000,, CT, 1 ABI Prism 7000, R , 0.99,, , 3.32, 100% PCR,, CT 2.2 PCR DNA, 10, 5, 300 ng/μl 30 ng/μl 3 ng/μl 0.3 ng/μl 0.03 ng/μl 2, 3, 6 CT,, CT, DNA 300 ng/μl 30 ng/μl 3 ng/μl 0.3 ng/μl,,,, DNA 图 1 目的基因参照基因的扩增曲线和标准曲线 A: DSCR3 ; B: DSCR3 ; C: GAPDH ; D: GAPDH Fig.1 Amplification and standard curves of target and control genes A: Amplification curves of the target gene DSCR3; B: Standard curve of the target gene DSCR3; C: Amplification curves of the control gene GAPDH; D: Standard curve of the control gene GAPDH.

3 ng/μl,,,, DNA 图 1 目的基因参照基因的扩增曲线和标准曲线 A: DSCR3 ; B: DSCR3 ; C: GAPDH ; D: GAPDH Fig.

4 8 : PCR ng/μl, CT 14.1% 9.9% 14.9% 17.6%, 30 ng/μl, 2.3 PCR CT DNA, 30 ng/μl 10, CT 1.58, (SD) 0.16, (CV) 9.8%,, 3, 10, CT 1.67, SD 0.22, CV 13.3%,, 2.4 DNA 5 10, PCR, CT 30 CT 1.6, SD 0.15, , 9.4%, CT 20 CT 2.55, SD 0.20, , 7.9%, CT, 2, CT, (P= ) CT, CT 3, SD Cut off, CT ,, 图 2 30 例正常人和 20 例患者的 CT 值 Fig. 2 The delta CT values from 30 normal persons and 20 Down syndrome patients ,, 10 DNA,, 3, 21q q q22, 21q22 21 [4], 21q22, (Down Syndrome Critical Region, DSCR), DSCR3, DNA,, Freeman [5], GAPDH PCR, PCR, : (1)GC 40% 60%; (2), G; (3) 5 G, G, C G; (4) 15 25, ; (5) Tm Tm 10,, 3,, PCR PCR [6],,, CT, CT CT, 21,,, CT, DNA CT DNA CT, FAM VIC,, CT [6],,

05 21,, 图 2 30 例正常人和 20 例患者的 CT 值 Fig. 2 The delta CT values from 30 normal persons and 20 Down syndrome patients 2.5 21,, 10 DNA,, 3, 21q22.1 21q22.

5 938 HEREDITAS (Beijing) [7] CT, PCR, 100% PCR, 3.32, 0.99, 3.32; 0.99,, 100%,, CT,,, CT 9.8% 13.3%, 15%, Chan [8],, CT, ng/μl, CT 15 30, CT, 30 ng/μl,,, PCR 20 30, CT , CT ,,, 3, 21,,,, EBV B, A T,,, 10 DNA, EBV A,,, PCR,,,, (References): [1] Bryndorf T, Christensen B, Xiang Y, Lind AM, Philip J. Rapid detection of numerical aberrations of chromosomes 13, 18 and 21 in chorionic mesenchymal cells. Prenat Diagn, 1993, 13(9): [2] CHEN Zhen-Bin, ZHU Jin-Ling, YAN Mei, LIANG Yan, ZHOU Yan, TAN Shu-Zhen, XIAO Bai, LIU Jing-Zhong. Genetic polymorphisms of five STR loci on chromosome 21 in Chinese Han population. Hereditas(Beijing), 2004, 26(4): ,,,,,,, STR., 2004, 26(4): [3] HUANG Xiao-Yi, LIU An, YU Yang, MA Lin-Lin,SHI Rong-Qian, LV Fu-Qu, JIANG Yan, SUN Wen-Jing, XUE Ya-Li, FU Song-Bin, LI Pu. Establishment and preservation of immortal lymphoblastoid cell lines of the 10 ethnic groups in China. Hereditas(Beijing), 2002, 24(6): ,, 旸,,,,,,,,. 10., 2002, 24(6): [4] Park J, Wurster-Hill DH, Andrews PA, Cooley WC, Graham JMJr. Free proximal trisomy 21 without the Down syndrome. Clin Genet, 1987, 32(5): [5] Freeman WM, Walker SJ, Vrana KE. Quantitative RT-PCR: pitfalls and potential. Biotechniques, 1999, 26(1): , [6] ZHENG Ming-Ming, HU Ya-Li, XU Zheng-Feng, WANG Zhi-Qun, SHI Ting-Qi, YE Xiao-Dong, CUI Heng-Mi. Application of real-time fluorescence quantitative polymerase chain reaction techniques for prenatal diagnosis of Down syndrome. Chin J Obstet Gynecol, 2004, 39(10): ,,,,,,.., 2004, 39(10): [7] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods, 2001, 25(4): [8] Chan V, Yip B, Lam YH, Tse HY, Wong HS, Chan TK. Quantitative polymerase chain reaction for the rapid prenatal diagnosis of homozygous alpha-thanlassaemia (Hb Barts hydrops fetalis). Br J Haematol, 2001, 115(2):

Rapid detection of numerical aberrations of chromosomes 13, 18 and 21 in chorionic mesenchymal cells. Prenat Diagn, 1993, 13(9): 815 823.

供试材料试验试剂和仪器基因组提取分析方法反应体系最佳配比的筛选退火温度对扩增效果的影响

供试材料试验试剂和仪器基因组提取分析方法反应体系最佳配比的筛选退火温度对扩增效果的影响生物技术通报胡立敏陶兴林侯栋朱惠霞张东琴选取温敏花椰菜不育系与杂交组合的高可育和高不育单株构建基因池利用对随机引物对其进行标记同时采用正交设计对其反应体系及扩增条件进行优化试验结果表明在反应体系中含引物