中国寄生虫学与寄生虫病杂志

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境外输入性恶性疟原虫抗药性相关基因突变的检测

徐超,魏庆宽,李瑾,肖婷,尹昆,王用斌,孔祥礼,许艳,崔勇,孙慧,朱嵩,闫歌,黄炳成*   

  1. 山东省医学科学院,山东省寄生虫病防治研究所,山东省疟疾诊断参比实验室,济宁272033
  • 出版日期:2016-12-30 发布日期:2017-01-10
  • 基金资助:

    山东省自然科学基金(No. ZR2014YL036,No. 2012ZRC03040);山东省医药卫生科技发展计划项目(No. 2016WS0394);山东省医学科学院医药卫生科技创新工程;全球基金中国疟疾项目(No. 201201055);山东省医学科学院课题计划(No. 2014-59)

Detection of Genetic Mutations Associated with Drug Resistance in Imported Plasmodium falciparum

XU Chao, WEI Qing-kuan,LI Jin,XIAO Ting,YIN Kun,WANG Yong-bin,KONG Xiang-li,XU Yan, CUI Yong,SUN Hui,ZHU Song,YAN Ge,HUANG Bing-cheng*   

  1. Shandong Provincial Reference Laboratory for Malaria Diagnosis, Shandong Institute of Parasitic Diseases, Shandong Academy of Medical Sciences, Jining, 272033, China
  • Online:2016-12-30 Published:2017-01-10
  • Supported by:

    Supported by the Natural Science Fund of Shandong Province(No. ZR2014YL036, No. 2012ZRC03040), Projects of Medical and Health Technology Development Program in Shandong Province (No. 2016WS0394),The Innovation Project of Shandong Academy of Medical Sciences,China Malaria Project of the Global Fund to Fight AIDS, Tuberculosis, and Malaria(No. 201201055), and Project of Shandong Academy of Medical Sciences(No. 2014-59)

摘要:

目的 了解山东省输入性恶性疟原虫(Plasmodium falciparum)抗药性相关基因(PfcrtPfmdr1Pfdhfr和K13)的突变情况。 方法 采集山东省2014年自非洲劳务返乡的94例输入性恶性疟患者血样,提取血样中恶性疟原虫基因组DNA,分别根据恶性疟原虫PfcrtPfmdr1Pfdhfr和K13基因序列设计引物,进行巢式PCR扩增和测序,并对测序结果进行序列比对分析各基因的突变情况。 结果 94例输入性恶性疟患者自18个非洲国家劳务返乡。提取94份患者血样中的恶性疟原虫基因组DNA,除1份血样的DNA Pfcrt基因PCR扩增失败外,其余均扩增成功,并测序。结果显示,94份患者血样的恶性疟原虫Pfcrt基因K76T突变型、Pfmdr1基因N86Y突变型和Pfdhfr基因S108N突变型分别占36.6%(34/93)、21.3%(20/94)和98.9%(93/94),3种基因的突变型所占比例的差异有统计学意义(χ2=127.5,P<0.05)。未检测到K13基因C580Y的突变。6份患者血样(占6.5%)的恶性疟原虫同时携带K76T、N86Y和S108N突变,其中2例自利比里亚输入,4例分别自安哥拉、赤道几内亚、刚果和几内亚输入;28份血样(占30.1%)的恶性疟原虫同时携带K76T和S108N突变,14份血样(占15.1%)同时携带N86Y和S108N突变;44份血样(占47.3%)的恶性疟原虫仅携带S108N突变;1份血样的恶性疟原虫3个基因均未发生突变。 结论 2014年山东省输入性恶性疟原虫PfcrtPfmdr1Pfdhfr基因均有不同程度的突变,其中Pfdhfr基因的S108N突变型所占比例最高,未检测到K13基因的突变。

关键词: 恶性疟原虫, 输入性, 分子标记, 基因型, 突变

Abstract:

 Objective To investigate the mutation of genes associated with drug resistance(Pfcrt, Pfmdr1, Pfdhfr and K13) in imported Plasmodium falciparum in Shandong Province. Methods Blood was collected from 94 falciparum malaria cases who returned from Africa in 2014. Genomic DNA for P. falciparum was extracted from the blood samples and nested PCR was performed using primers specifically designed for Pfcrt, Pfmdr1, Pfdhfr and K13. The PCR products were sequenced. Gene mutations were analyzed by sequence alignment. Results The 94 imported cases were from 18 African countries. Nested PCR was successful on DNA from all the blood samples except for Pfcrt amplification in one sample. Sequence analysis revealed three types of mutations Pfcrt K76T (36.6%, 34/93), Pfmdr1 N86Y (21.3%, 20/94), and Pfdhfr S108N (98.9%, 93/94) (χ2=127.5, P<0.05). K13 C580Y mutation was not found. Co-occurrence of K76T, N86Y, and S108N was found in 6 blood samples (6.5%), which were imported from Liberia(2), Angola(1), Equatorial guinea(1), Congo(1), and Guinea(1). Co-occurrence of K76T and S108N mutations was found in 28 samples(30.1%), and that of N86Y and S108N in 14 samples (15.1%). Forty-four samples(47.3%) harbored S108N mutation only, and one sample was null for any of the mutations. Conclusion There are mutations in Pfcrt, Pfmdr1, and Pfdhfr in imported Plasmodium falciparum in Shandong Province. No mutation was found for the K13 gene.

Key words: Plasmodium falciparum, Imported malaria, Molecular marker, Genotype, Mutation