中国媒介生物学及控制杂志 ›› 2012, Vol. 23 ›› Issue (6): 499-502.

• 论著 • 上一篇    下一篇

高分辨率熔解曲线分析检测家蝇cyp6d1基因突变

王英红, 孔庆鑫, 邱丽华, 韦凌娅, 沈林海, 倪晓平   

  1. 杭州市疾病预防控制中心消毒监测与媒介生物防治所, 浙江 杭州 310021
  • 收稿日期:2012-05-28 出版日期:2012-12-20 发布日期:2012-12-20
  • 通讯作者: 倪晓平,Email:nixp@sohu.com
  • 基金资助:
    浙江省卫生厅科研项目(2009A176)

Determination of cyp6d1 gene mutation in Musca domestica by high-resolution melting analysis

WANG Ying-hong, KONG Qing-xin, QIU Li-hua, WEI Ling-ya, SHEN Lin-hai, NI Xiao-ping   

  1. Hangzhou Center for Disease Control and Prevention, Hangzhou 310021, Zhejiang Province, China
  • Received:2012-05-28 Online:2012-12-20 Published:2012-12-20
  • Supported by:
    Supported by the Scientific Research Fund of Health Bureau in Zhejiang Province (No. 2009A176)

摘要: 目的 探讨家蝇溴氰菊酯抗性机制。方法 使用高分辨率熔解曲线(high-resolution melting, HRM)分析与半分析定量PCR相 结合的方法, 分析家蝇抗性基因cyp6d1突变和扩增情况。通过对扩增的PCR产物进行测序来验证HRM准确性。结果 与敏感品系相比, 抗 性品系cyp6d1基因的mRNA表达量显著增加(P=0.0002), 抗性品系cyp6d1基因的mRNA表达量是敏感品系的50.7倍;使用HRM对cyp6d1进 行突变分析, 将其分为3种基因型, 分别为A型(Tm=85.5℃)、B型(Tm=87.3℃)和C型(Tm=88.5℃);经测序, 该HRM分析结果 分别与3类基因序列型相对应。A型基因型为纯合的1087G、1101T和1155A(对应GenBank序列号U22367.1);C型基因型为纯合的1087A、 1101G和1155G;B型基因型为A型和C型的杂合型。生物信息学分析表明, 该突变为无意义突变。结论 杭州市家蝇抗性品系cyp6d1基因 mRNA表达量显著增加, 可能是家蝇抗性增加的原因之一。

关键词: 高分辨率熔解曲线, 家蝇, 抗性, 基因突变, cyp6d1

Abstract: Objective To investigate the mechanism of deltamethrin resistance in Musca domestica. Methods The mutation and amplification of cyp6d1, a deltamethrin resistance gene in M. domestica, were determined by high-resolution melting (HRM) analysis and semiquantitative PCR. The accuracy of HRM analysis was validated by sequencing of PCR products. Results The mRNA expression of cyp6d1 in deltamethrin-resistant strain was 50.7-fold that in deltamethrin-sensitive strain, showing significant difference between the two strains (P=0.0002). The PCR products were classified into three genotypes, type A (Tm=85.5℃), type B (Tm=87.3℃), and type C (Tm=88.5℃), according to the mutation analysis of cyp6d1 by HRM. The results from HRM corresponded to the sequences of the three enotypes. As for the genotypes, type A was homozygous 1087G, 1101T, and 1155A (GenBank No. U22367.1), type C was homozygous 1087A, 1101G, and 1155G, and type B was heterozygous for type A and type C. Bioinformatic analysis revealed that this mutation was non-sense mutation. Conclusion The mRNA expression of cyp6d1 is significantly increased in the deltamethrin-resistant strain of M. domestica in Hangzhou, China, which may be one of the mechanisms of deltamethrin resistance increase in M. domestica.

Key words: High-resolution melting, Musca domestica, Deltamethrin resistance, Gene mutation, cyp6d1

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