Identification of <em>Clonorchis sinensis</em> Metacercariae Based on PCR Targeting Ribosomal DNA ITS Regions and <em>COX</em>1 Gene

›› 2014, Vol. 32 ›› Issue (3): 10-217-220.

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Identification of Clonorchis sinensis Metacercariae Based on PCR Targeting Ribosomal DNA ITS Regions and COX1 Gene

YANG Qing-li1，2，SHEN Ji-qing3，JIANG Zhi-hua2，YANG Yi-chao2，LI Hong-mei1，CHEN Ying-dan1，ZHOU Xiao-nong1*   

  1. 1 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention；Key Laboratory of Parasite and Vector Biology, MOH; WHO Collaborating Centre for Malaria, Schistosomiasis and Filariasis，Shanghai 200025，China；2 Center for Disease Control and Prevention of Guangxi Zhuang Autonomous Region，Nanning 530028，China；3 Department of Parasitology，Guangxi Medical University，Nanning 530021，China
  • Online:2014-06-30 Published:2014-09-17

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Abstract: Objective  To identify Clonorchis sinensis metacercariae using PCR targeting ribosomal DNA ITS region and COX1 gene.  Methods  Pseudorasbora parva were collected from Hengxian County of Guangxi at the end of May 2013. Single metacercaria of C. sinensis and other trematodes were separated from muscle tissue of P. parva by digestion method. Primers targeting ribosomal DNA ITS region and COX1 gene of C. sinensis were designed for PCR and the universal primers were used as control. The sensitivity and specificity of the PCR detection were analyzed.  Results  C. sinensis metacercariae at different stages were identified by PCR. DNA from single C. sinensis metacercaria was detected by PCR targeting ribosomal DNA ITS region and COX1 gene. The specific amplicans have sizes of 437/549, 156/249 and 195/166 bp, respectively. The ratio of the two positive numbers in PCR with universal primers and specific primers targeting C. sinensis ribosomal DNA ITS1 and ITS2 regions was 0.905 and 0.952, respectively. The target gene fragments were amplified by PCR using COX1 gene-specific primers. The PCR with specific primers did not show any non-specific amplification. However, the PCR with universal primers targeting ribosomal DNA ITS regions performed serious non-specific amplification.  Conclusion  C. sinensis metacercariae at different stages are identified by morphological observation and PCR method. Species-specific primers targeting ribosomal DNA ITS region show higher sensitivity and specificity than the universal primers. PCR targeting COX1 gene shows similar sensitivity and specificity to PCR with specific primers targeting ribosomal DNA ITS regions.

Key words: Clonorchis sinensis, Metacercaria, ITS region, COX1 gene, PCR