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Licensed Unlicensed Requires Authentication Published by De Gruyter April 18, 2017

cDNA library construction of two human Demodexspecies

DongLing Niu, RuiLing Wang, YaE Zhao, Rui Yang, Li Hu, YuYang Lei and WeiChao Dan
From the journal Acta Parasitologica

Abstract

The research of Demodex, a type of pathogen causing various dermatoses in animals and human beings, is lacking at RNA level. This study aims at extracting RNA and constructing cDNA library for Demodex. First, P. cuniculiand D. farinaewere mixed to establish homogenization method for RNA extraction. Second, D. folliculorumand D. breviswere collected and preserved in Trizol, which were mixed with D. farinaerespectively to extract RNA. Finally, cDNA library was constructed and its quality was assessed. The results indicated that for D. folliculorum& D. farinae, the recombination rate of cDNA library was 90.67% and the library titer was 7.50 × 104 pfu/ml. 17 of the 59 positive clones were predicted to be of D. folliculorum; For D. brevis& D. farinae, the recombination rate was 90.96% and the library titer was 7.85 x104 pfu/ml. 40 of the 59 positive clones were predicted to be of D. brevis. Further detection by specific primers demonstrated that mtDNA cox1, cox3and ATP6 detected from cDNA libraries had 96.52%-99.73% identities with the corresponding sequences in GenBank. In conclusion, the cDNA libraries constructed for Demodexmixed with D. farinaewere successful and could satisfy the requirements for functional genes detection.


DongLing Niu and RuiLing Wang equally contributed to this paper and should be considered as first co-authors


Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant numbers 81271856 and 81471972).

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Received: 2016-11-16
Revised: 2016-12-20
Accepted: 2017-1-19
Published Online: 2017-4-18
Published in Print: 2017-6-1

© 2017 W. Stefański Institute of Parasitology, PAS