中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (44): 8245-8248.doi: 10.3969/j.issn.1673-8225.2010.44.019

• 移植与基因 transplantation and gene • 上一篇    下一篇

自行构建人类白细胞抗原DQA1位点基因分型寡核苷酸芯片性能评价：100例样本与PCR-SSP法的比较

王  彤1，王天骄2，彭  莉3，王  杰1   

  1. 1沈阳医学院基础医学部生物化学教研室，辽宁省沈阳市  110034； 2中国医科大学生物芯片中心，辽宁省沈阳市，110001；3辽宁省血液中心沈阳中心血站质量检验科，辽宁省沈阳市  110044
  • 出版日期:2010-10-29 发布日期:2010-10-29
  • 通讯作者: 王杰，博士，教授，副院长、硕士生导师。沈阳医学院基础医学部生物化学教研室，辽宁省沈阳市 110034 wangjie19932002@163.com
  • 作者简介:王彤★，女，1974年生，辽宁省沈阳市人，汉族，2004年中国医科大学毕业，硕士，讲师，主要从事生物芯片与生物信息学方面的研究。

Performance evaluation of human leucocyte antigen-DQA1 genotyping by fabricated low-density Oligonucleotide microarrays: Compared with PCR-SSP method in 100 samples

Wang Tong1, Wang Tian-jiao2, Peng Li3, Wang Jie1   

  1. 1 Department of Biochemistry and Molecular Biology, Shenyang Medical College, Shenyang  110034, Liaoning Province, China; 2 Biochip Center of China Medical University, Shenyang  110001, Liaoning Province, China; 3 Quanlity-Control Department of Shenyang Central Blood Station, Shenyang  110044, Liaoning Province, China
  • Online:2010-10-29 Published:2010-10-29
  • Contact: Wang Jie, Doctor, Professor, Master’s supervisor, Department of Biochemistry and Molecular Biology, Shenyang Medical College, Shenyang 110034, Liaoning Province, China wangjie19932002@163.com
  • About author:Wang Tong★, Master, Lecturer, Department of Biochemistry and Molecular Biology, Shenyang Medical College, Shenyang 110034, Liaoning Province, China

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摘要：

背景：人类白细胞抗原等位基因分型对器官移植和法医鉴定等有重要意义。目前的分型方法难以实现高通量和集成化，准确性和重复性也不理想。
目的：比较自行构建的寡核苷酸芯片与序列特异性引物-聚合酶链反应(polymerase chain reaction with sequence-specific primers，PCR-SSP)两种方法用于人类白细胞抗原DQA1基因分型的结果，以评价寡核苷酸芯片的性能。
方法：纳入2006-01/2009-03于中国医科大学附属第一医院血液科门诊就诊的患者100例。分别用等位基因特异的PCR-SSP和自行构建的寡核苷酸芯片对患者的人类白细胞抗原DQA1进行分型。寡核苷酸芯片分型采用荧光标记的组间特异性引物扩增基因组DNA，扩增后的产物与分型芯片的探针杂交，由杂交产生的荧光信号确定人类白细胞抗原DQA1位点基因型。比较两种方法分型所获得的结果，不吻合的样本经第三方测序验证。
结果与结论：100例临床样本经寡核苷酸芯片和PCR-SSP分型全部成功。分型结果的吻合率为94%。不吻合样本6例，其中4例PCR-SSP定型为杂合子，芯片分型全部为纯合子。经第三方验证，证实芯片的分型全部正确。另外2例不吻合的样本经测序，证实SSP分型错误1例，芯片分型错误1例。芯片的重复率为95%。说明自行构建的寡核苷酸芯片的特异性和灵敏度都能满足基因分型的需要，且稳定性较好。

关键词: 人类白细胞抗原DQA1, 寡核苷酸芯片, 序列特异性引物-聚合酶链反应, 基因分型, 细胞移植

Abstract:

BACKGROUND: The genotype of human leukocyte antigen (HLA) alleles has important effect on organ transplantation and medicolegal expertise. The presented genotyping can not reach high-flux or integration, without accuracy or repetitiveness.
OBJECTIVE: To evaluate the accuracy and reliability of self-fabricated oligoneucleotide array by comparing with polymerase chain reaction with sequence-specific primers (PCR-SSP) in identification of HLA-DQA1 alleles.
METHODS: A total of 100 clinical samples were enrolled in the study. HLA-DQA1 genotyping were performed by PCR-SSP and oligoneucleotide array, respectively. A pairs of group-specific primers were designed according to the sequence polymorphism of HLA-DQA1 exon two. The target DNA was asymmetrically amplified with the labeled sense primer. The labeled PCR products were hybridized with the specific allele typing probes immobilized on a glass support, and the signals were scanned by scanner and then analyzed. The discordant samples by arrays and PCR-SSP were verified by sequencing.
RESULTS AND CONCLUSION: All 100 samples have been genotyped by oligoneucleotide array and PCR-SSP successfully. The coincidence rate was 94%. Four homozygous samples typed by PCR-SSP were actually heterozygous by array. The other two unidentified samples were typed by sequencing. The results showed that a mistake for one sample was made by PCR-SSP or array. In reproducible tests, the signal reappear rate was 95%. That is, the self-fabricated oligoneucleotide array with ideal stability, specificity and sensitivity, which provide a suitable plateau for HLA-DQA1 genotyping domestically.

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