Screening and Applications of qPCR Primers for apomucin Gene of Echinococcus multilocularis

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Screening and Applications of qPCR Primers for apomucin Gene of Echinococcus multilocularis

SU Meng1,2, GUO Xiao-la2, YANG Jing2, SHAO Zhong-wei2, DING Jun-tao3, XIANG Hai-tao1, LUO Xue-nong2,4, ZHENG Ya-dong2,4, SUN Xiao-lin1*   

  1. 1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;2 State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Lanzhou 730046, China;3 College of Life Science and Technology, Xinjiang University, Urumqi 830046, China;4 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China
  • Online:2016-10-30 Published:2016-11-09

Abstract: Objective To screen for the optimal qPCR primers for Echinococcus multilocularis apomucin gene(Em-apo) and analyze Em-apo expression. Methods Primers were designed based on 4 Em-apo sequences from GeneDB. Primer specificity and PCR efficiency were determined, based on which the optimal primer pairs were selected. Alterations of Em-apo expression in 1 000 E. multilocularis protoscoleces treated with albendazole(5 μg/ml) and insulin(100 ng/ml) were separately assessed using the selected primers. DMSO used in albendazole dilution and in PBS insulin dilution were used as the control. Results Specific primers for Em-apo-1, Em-apo-2/3, Em-apo-4 and actin were selected. qPCR melting curves revealed a single peak for each primer pair and an amplification efficiency from 95% to 101%. The qPCR showed increased expression of Em-apo-1(1.51±0.27), Em-apo-2/3 (1.39±0.30) and Em-apo-4(1.14±0.18) after albendazole treatment in comparison to the DMSO control(1.00)(P>0.05 among the three genes); and an unaltered Em-apo-1 expression, slightly decreased Em-apo-4 expression, and significantly decreased Em-apo-2/3 expression(0.73±0.09) after insulin treatment in comparison to the PBS control(P>0.05 among the three genes). Conclusion The selected specific primers for Em-apo genes can be used to analyze the gene expression by qPCR. Treatment with albendazole and insulin show certain effects on the expression of Em-apo genes in E. multilocularis protoscoleces.

Key words: Echinococcus multilocularis, apomucin gene, qPCR, Albendazole, Insulin