热带病与寄生虫学

热带病与寄生虫学 ›› 2021, Vol. 19 ›› Issue (4): 181-.

• 论著 •    下一篇

淡水鱼华支睾吸虫囊蚴 LAMP 检测方法的建立

朱海,汪奇志,孙成松,尹晓梅,汪峰峰,金郁,张世清   

  1. 安徽省血吸虫病防治研究所,安徽 合肥 230601
  • 出版日期:2021-08-20 发布日期:2021-08-26
  • 通信作者: 张世清,E-mail:zhangsq2820@ 163. com
  • 作者简介:朱海,男,硕士,检验师,研究方向:寄生虫病实验诊断。 E-mail:274853279@ qq. com
  • 基金资助:
    安徽省血吸虫病防治研究所青年科研基金项目(XF2018005)

Establishment of LAMP technique for detecting metacercariae in freshwater fish infected with Clonorchis sinensis

ZHU Hai,WANG Qi-zhi,SUN Cheng-song,YIN Xiao-mei,WANG Feng-feng,JIN Yu,ZHANG Shi-qing   

  1. Anhui Provincial Institute of Schistosomiasis Control,Hefei 230601,Anhui Province,China
  • Online:2021-08-20 Published:2021-08-26

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摘要： 目的 建立一种高效灵敏的环介导等温扩增(LAMP)方法检测淡水鱼中华支睾吸虫囊蚴。 方法 以华支睾吸虫 ITS2 基因序列为靶序列,合成 Cs-1、Cs-2、Cs-3 三组 LAMP 引物体系。 以华支睾吸虫囊蚴 DNA 样本为模板,日本血吸虫成虫、带绦虫成虫、蛔虫虫卵及其他吸虫囊蚴 DNA 样本为参考,比较三组引物体系的特异度;以不同浓度的华支睾吸虫成虫基因组 DNA 为模板,比较三组引物体系的灵敏度;用 LAMP 法及 PCR 法平行检测 16 份华支睾吸虫囊蚴样本和 23 份其他吸虫囊蚴样本,评价 LAMP 法的敏感性和特异性。 结果 三组引物体系的灵敏度及特异度以 Cs-3 为最优,以其建立的 LAMP 法反应体系,检测华支睾吸虫成虫 DNA 的最低检出浓度可达到 3. 33×10-4ng / μL,敏感性和特异性与 PCR 法一致,均为 100%。 结论 成功建立了一种可用于检测淡水鱼中华支睾吸虫囊蚴的 LAMP 方法,为淡水鱼寄生虫病监测工作提供了便捷高效的检测手段。

关键词: 环介导等温扩增技术, 华支睾吸虫, 囊蚴, 淡水鱼

Abstract: Objective To establish a loop-mediated isothermal amplification (LAMP) method for detecting the metacercariae in freshwater fish infected with Clonorchis sinensis. Methods ITS2 gene sequence of Clonorchis sinensis was used as the target sequence,and three sets of LAMP primers,including Cs-1,Cs-2 and Cs-3 were synthesized. The specificity of the three sets of primers was compared based on the samples of Clonorchis sinensis metacercaria as a template,and reference samples using DNA extracted from adult Schistosoma japonicum,adult tapeworms,roundworm eggs and metacercariae of other trematode. The sensitivity of the three sets of primers was compared in compliance with different concentration of genomic DNA from adult Clonorchis sinensis as a template. Finally,the sensitivity and specificity of LAMP were concurrently evaluated by comparing PCR findings in detecting the metacercariae in 16 samples of Clonorchis sinensis and 23 samples from other flukes. Results Of the three sets of primers,Cs-3 was the best in sensitivity and specificity. LAMP reaction system established on Cs-3 basis can reveal the DNA in adult Clonorchis sinensis by up to 3. 33×10-4 ng / μL at the minimal detection concentration,and the sensitivity and specificity were identical to PCR measurement,both of which were 100%. Conclusion A LAMP method for detecting metacercaria of Clonorchis sinensis in freshwater fish was successfully established,which can provide a quick and efficient detection approach to monitoring of parasitic diseases in freshwater fish.

Key words: LAMP, Clonorchis sinensis, Metacercaria, Freshwater fish

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