Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases · 2016
[Screening and Applications of qPCR Primers for apomucin Gene of Echinococcus multilocularis].
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Su Meng, Guo Xiao-la, Yang Jing, Shao Zhong-wei, Ding Jun-tao, Xiang Hai-tao, Luo Xue-nong, Zheng Ya-dong, Sun Xiao-lin
Аннотация
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.
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.