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中国防痨杂志 ›› 2018, Vol. 40 ›› Issue (1): 63-67.doi: 10.3969/j.issn.1000-6621.2018.01.015

• 论著 • 上一篇    下一篇

荧光探针熔解曲线法检测结核患者N-乙酰基转移酶2基因型分布的应用评价

陈素婷,黄明翔,胡严杰,尚媛媛,梁倩,付育红,黄海荣()   

  1. 福建省福州肺科医院 福建医科大学临床教学医院检验科(黄明翔)
  • 收稿日期:2017-04-18 出版日期:2018-01-10 发布日期:2018-03-14
  • 通信作者: 陈素婷 E-mail:huanghairong@tb123.org
  • 基金资助:
    国家自然科学基金(31600107);首都卫生发展科研专项(2016-2-1041);北京市优秀人才项目(2015000021469G189)

The evaluation of the performance of fluorescence probe melting curve for NAT2 genotype detection in tuberculosis patients

Su-ting CHEN,Ming-xiang HUANG,Yan-jie HU,Yuan-yuan SHANG,Qian LIANG,Yu-hong FU,Hai-rong. HUANG()   

  1. *National Laboratory on Clinical Tuberculosis, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing 101149, China
  • Received:2017-04-18 Online:2018-01-10 Published:2018-03-14
  • Contact: Su-ting CHEN E-mail:huanghairong@tb123.org

摘要:

目的 评价荧光探针熔解曲线法检测结核病患者芳基胺N-乙酰基转移酶2(arylamine N-acetyltransferases 2,NAT2)基因多态性的性能。方法 选取于2015—2016年到福州肺科医院和北京胸科医院确诊的初治结核病患者作为研究对象,分别为371例和355例。收集研究对象外周血标本。应用荧光探针熔解曲线法[分析NAT2基因rs1801280(341T→G)、rs1799929(481C→T)、rs1799930(590G→A)和rs1799931(857G→A)等4个单核苷酸多态性(SNP)]和DNA测序分析法[分析NAT2基因的rs1801279 (191G→A)、rs1041983 (282C→T)、rs1801280(341T→C)、rs1799929 (481C→T)、rs1799930 (590G→A)、rs1208 (803A→G)和1799931 (857G→A)的7个SNP位点]对研究对象进行NAT2基因型分析,并根据NAT2基因型进行NAT2代谢类型的推断分析。结果 荧光探针熔解曲线法分析4个SNP位点与DNA测序分析法分析7个SNP位点推断NAT2代谢类型的结果完全一致。荧光探针熔解曲线法从DNA制备、PCR扩增和熔解曲线分析的整个检测过程需要2.5h。共检测到NAT2 *4/*4、NAT2 *5/*4、NAT2 *6/*4、NAT2 *7/*4、NAT2 *5/*11、NAT2 *6/*11、NAT2 *7/*11、NAT2 *5/*6、NAT2 *6/*7、NAT2 *5/*5、NAT2 *6/*6、NAT2 *7/*7、NAT2 *5/*7等13种NAT2基因型。福州肺科医院的结核病患者NAT2基因快乙酰化、中间乙酰化和慢乙酰化类型分别占37.20%(138/371)、42.32%(157/371)和20.49%(76/371);北京胸科医院的结核病患者中,快乙酰化、中间乙酰化和慢乙酰化类型分别占27.89%(99/355)、54.65%(194/355)、17.46%(62/355);两组人群的乙酰化类型分布差异有统计学意义(χ 2=11.37,P=0.003)。所有的快乙酰化代谢人群均携带NAT2 *4/*4基因型[100.00%(237/237)],而慢乙酰化代谢人群主要携带NAT2 *6和NAT2 *7的单倍型[90.22% (249/276)]。 结论 荧光探针熔解曲线法分析4个SNP位点检测NAT2基因型具有结果判读简便、准确、快速的优点,适用于中国地区人群的NAT2基因型的检测。

关键词: 结核, 核酸探针, 芳基胺N-乙酰转移酶, 基因型, 多态性, 单核苷酸

Abstract:

Objective To evaluate the performance of fluorescent probe melting curve method for the detection of the gene polymorphism of arylamine N-acetyltransferases 2 (NAT2) in tuberculosis (TB) patients in China.Methods From 2015 to 2016, the primary TB patients who visited Fuzhou Pulmonary Hospital of Fujian (n=371) or Beijing Chest Hospital (n=355) were selected. Peripheral blood samples of all patients were collected. NAT2 genotypes of all patients were analyzed by four-SNP genotype panels (rs1801280 (341T→G),rs1799929 (481C→T), rs1799930 (590G→A) and rs1799931 (857G→A)) using fluorescent probe melting curve method and seven-SNP genotype panels (rs1801279 (191G→A), rs1041983 (282C→T), rs1801280(341T→C), rs1799929 (481C→T), rs1799930 (590G→A), rs1208 (803A→G) and 1799931 (857G→A)) using DNA sequencing. The NAT2 acetylation phenotypes were deduced from the NAT2 genotypes.Results The NAT2 acetylation phenotype for each individual inferred from four-SNP genotype panels using fluorescent probes melting curve method is consistent with that from seven-SNP genotype panels using DNA sequencing. The whole procedure of fluorescent probes melting curve method including DNA preparation, PCR amplification and melting curve analysis only took 2.5 hours. A total of 13 genotypes of NAT2 gene were detected as follow: NAT2 *4/*4, NAT2 *5/*4, NAT2 *6/*4, NAT2 *7/*4, NAT2 *5/*11, NAT2 *6/*11, NAT2 *7/*11, NAT2 *5/*6, NAT2 *6/*7, NAT2 *5/*5, NAT2 *6/*6, NAT2 *7/*7 and NAT2 *5/*7. TB patients from Fuzhou Pulmonary Hospital of Fujian with NAT2 rapid, intermediate or slow acetylators accounted for 37.20% (138/371), 42.32% (157/371) and 20.49% (76/371), respectively, while patients with tuberculosis from Beijing Chest Hospital accounted for 27.89% (99/355), 54.65% (194/355) and 17.46% (62/355), respectively. The distribution of NAT2 acetylation phenotypes of the above two groups was significantly different (χ 2=11.37, P=0.003). All patients with NAT2 rapid acetylators carried NAT2 *4/NAT2 *4 genotype (100.00% (237/237)), and most of the patients with NAT2 slow acetylators carried NAT2 *6 and NAT2 *7 haplotype (90.22% (249/276)). Conclusion The four-SNP genotype panels using fluorescent probe melting curve method is applicable for NAT2 genotyping in TB patients in China, with the advantage of easy to interpret result, fast and accurate.

Key words: Tuberculosis, Nucleic acid probes, Arylamine N-acetyltransferase, Genotype, Polymorphism, Single nucleotide