基础医学与临床 ›› 2022, Vol. 42 ›› Issue (1): 139-144.

• 临床研究 • 上一篇    下一篇

CLIP-PCR直接检测血液中疟原虫雌性配子体特异转录产物s25 mRNA

杨珺源1,骈红茹2,杨明珠2,郑直2   

  1. 1. 中国医学科学院基础医学研究所 北京协和医学院基础学院
    2. 中国医学科学院基础医学研究所
  • 收稿日期:2021-10-08 修回日期:2021-11-22 出版日期:2022-01-05 发布日期:2022-01-05
  • 通讯作者: 杨珺源 E-mail:360598370@qq.com
  • 基金资助:
    国家科技重大专项项目;中国医学科学院医学与健康科技创新

Direct detection of Plasmodium female gametocyte-specific transcript s25 mRNA in blood by CLIP-PCR

  • Received:2021-10-08 Revised:2021-11-22 Online:2022-01-05 Published:2022-01-05

摘要: 目的 建立一种疟原虫雌性配子体的分子检测法。方法 根据恶性疟原虫和间日疟原虫雌性配子体特异性的s25 mRNA靶标分别设计序列特异性的捕获探针和连接探针。无需核酸提取,血液样本经过裂解液裂解后,释放出的目的RNA直接被捕获探针固定在96孔板上,洗去未结合探针,将同样结合在RNA靶标上的连接探针进行特异性连接,得到两端为特殊设计序列单链扩增模板。用通用引物进行实时荧光定量染料法扩增,或在尾部设计荧光探针序列,用通用引物和通用荧光探针进行实时荧光定量探针法扩增。评估方法的灵敏度、特异性和重复性,且与现有的RT-qPCR方法比较,并将其应用于临床样本的检测。结果 该方法具有较高的灵敏度和特异性,三者均可检测低至11拷贝的s25 mRNA靶标,且相比RT-qPCR该法操作更简便。该法可准确检测到疟疾患者血液中的雌性配子体。可在3h内完成对96个样本的检测。结论 开发了一种恶性疟原虫和间日疟原虫雌性配子体的检测技术,为疟疾传播控制、配子体大规模筛查提供了灵敏高效的方法。

关键词: 疟原虫, 配子体, 检测方法

Abstract: Objective To establish a molecular detection method for the Plasmodium female gametocyte. Methods Capture and ligation probes was specific designed based on Plasmodium falciparum and Plasmodium vivax femal gametocyte special RNA targets,which is s25 mRNA. After the whole blood is lysed, the RNA target is directly released and fixed on the 96-well plate with capture probes without extraction. Washing away the unbound probe, the ligation probes which also hybridized to the RNA target is specific ligated to form an single-strand amplification template with specific-designed sequence at both ends. The SYBR Green qPCR was carried out with universal primers and the TaqMan qPCR was carried out with universal primers and universal fluorescent probes. Evaluate the sensitivity, specificity and repeatability of this method and compared with the RT-qPCR methods. And it was also used to detect clinical samples. Results This method was successfully applied to detect s25 mRNA with high sensitivity and specificity. All three methods can detect as low as 11 copies of S25 mRNA targets, and this method is simpler than RT-qPCR. And it can accurately detect gametocytes in the blood of malaria patients. The detection of 96 samples can be completed within 3h. Conclusion Established a method for the femal gametocyte of Plasmodium falciparum and Plasmodium vivax detection which provides a sensitive and efficient method for large-scale disease screening of gametocytes and control of malaria transmission .

Key words: Plasmodium, gametocyte, detection method

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