Establishment and application of a multiplex recombinase-aided isothermal amplification technique for identifying Echinococcus granulosus and Echinococcus multilocularis

doi:10.12140/j.issn.1000-7423.2020.03.09

Abstract

Abstract:

Objective To establish a multiplex recombinase-aided isothermal amplification (mRAA) method for rapid detection of Echinococcus granulosus G1 (EgG1) and E. multilocularis (Em) DNA. Methods The mRAA rapid detection method for Echinococcus was established by designing primers based on the mitochondrial genes of EgG1 and Em as target sequences. The sensitivity of the mRAA method was assessed by amplifications of EgG1 and Em genomic DNA at concentrations of 10.00, 5.00, 1.00, 0.50, 0.10, 0.05, and 0.01 ng/μl and of pMD19-T (Simple) recombiant plasmids at 10⁵, 10⁴, 10³, 10² and 10 copies/μl. The specificity of the mRAA method was evaluated by using genomic DNA of Taenia saginata, T. asiatica, T. multiceps, Dipylidium caninum, Toxocara canis, Trichuris trichiura Linnaeus, Giardia lamblia, Fasciola hepatica, Paragonimus westermani, Fasciola gigantica and Clonorchis sinensis as templates for amplification. To verify the reliability and feasibility, optimized mRAA method was used to examine three fecal samples of dogs with simulated infection of mixed EgG1 and Em. 19 liver tissue samples of infected animals(10 with EgG1 infection and 9 with Em infection) collected from field, 7 fecal samples of infected dogs (5 EgG1 infected and 2 Em infected) collected from field. Results The established mRAA method could specifically amplify the EgG1 and Em mitochondrial gene fragments with the sequence lengths of 250 and 500 bp, respectively. The detactable limit of EgG1 and Em genomic DNA by mRAA was 2 pg/μl, and that of the EgG1 and Em recombinant plasmids was 200 copies/μl. The mRAA method showed negative results of amplification on genomic DNA of T. saginata, T. asiatica, T. multiceps, D. caninum, T. canis, T. trichiura Linnaeus, G. lamblia, F. hepatica, P. westermani, F. gigantica and C. sinensis. It was demonstrated that the mRAA method successfully detected EgG1 and Em DNA from the fecal samples of dogs with simulated infection of mixed EgG1 and Em, the liver tissue of infected field animals, and fecal samples of infected field dogs. Conclusion The mRAA method displays evident sensitivity, specificity and reliability, and could be used for rapid detection of EgG1 and Em DNA.

Key words: Echinococcus granulosus G1, Echinococcus multilocularis, Recombinase-aided isothermal amplification technique, Multiplex amplification

CLC Number:

R383.33

ZHOU Hong-rang, MAO Guang-yao, WANG Xiao-ling, CHEN Mu-xin, YU Qing, WANG Ying, Ai Lin, XIAO Ning. Establishment and application of a multiplex recombinase-aided isothermal amplification technique for identifying Echinococcus granulosus and Echinococcus multilocularis[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(3): 310-316.

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