An Optimized Protocol for Myxosporidia (Cnidaria: Myxosporea) DNA Extraction for Molecular Studies  

作　　者：Guy Benoit Lekeufack-Folefack Bienvenu Feudjio-Dongmo Abraham Fomena Billy Tene-Fossog Murielle J. Wondji Guy Benoit Lekeufack-Folefack;Bienvenu Feudjio-Dongmo;Abraham Fomena;Billy Tene-Fossog;Murielle J. Wondji(Department of Animal Biology and Physiology, University of Yaoundé I, Yaoundé, Cameroon;Centre for Research in Infectious Diseases (CRID), Yaoundé, Cameroon;Department of Vector Biology, Liverpool School of Tropical Medicine, University of Liverpool, Liverpool, UK)

机构地区：[1]Department of Animal Biology and Physiology, University of Yaoundé I, Yaoundé, Cameroon [2]Centre for Research in Infectious Diseases (CRID), Yaoundé, Cameroon [3]Department of Vector Biology, Liverpool School of Tropical Medicine, University of Liverpool, Liverpool, UK

出　　处：《Open Journal of Animal Sciences》2020年第3期378-386,共9页动物科学期刊（英文）

摘　　要：Myxosporidia constitute a major group of fish parasites which have a significant negative impact on wild and cultured fish. The used of DNA in Myxosporidia studies has progressed rapidly over the last twenty years, especially in their identification and characterization as well as determination of species diversity and investigation of their evolutionary relationships. Extraction and isolation of pure and high quality DNA are essential for any molecular study, but constitute a challenge for many laboratories especially in low and middle income countries. Myxosporidia plasmodia filled with mature myxospores were isolated from different tissues of <span style="white-space:nowrap;"><i></span>Labeo batesii<span style="white-space:nowrap;"></i></span> Boulenger, 1911. DNA from myxosporidia myxospores were extracted using a Livak optimized DNAs extraction protocol. Four particular phases of the original protocol were optimized. Yield and absorbance ratios of extracted DNA were determined using spectrophotometer. DNA samples were used as template for the amplification of the 18S rDNA region and amplicons resolved on 1.5% agarose gel for determination of fragment sizes and purity evaluation. The concentration of extracted DNA from all Myxosporidia species ranged from 4.6 to 26 ng/μl with purity indices ranging from 1.88 to 2.12. We successfully amplified the 1050 bp DNA fragment as targeted. The intensity, thickness and clarity of the bands were evidences of non-degradation of DNA. The optimized Livak protocol is simple, low-cost and manageable. Regarding the quantity, purity and quality of extracted DNA, the optimized Livak protocol is highly recommended for Myxosporidia studies.Myxosporidia constitute a major group of fish parasites which have a significant negative impact on wild and cultured fish. The used of DNA in Myxosporidia studies has progressed rapidly over the last twenty years, especially in their identification and characterization as well as determination of species diversity and investigation of their evolutionary relationships. Extraction and isolation of pure and high quality DNA are essential for any molecular study, but constitute a challenge for many laboratories especially in low and middle income countries. Myxosporidia plasmodia filled with mature myxospores were isolated from different tissues of <span style="white-space:nowrap;"><i></span>Labeo batesii<span style="white-space:nowrap;"></i></span> Boulenger, 1911. DNA from myxosporidia myxospores were extracted using a Livak optimized DNAs extraction protocol. Four particular phases of the original protocol were optimized. Yield and absorbance ratios of extracted DNA were determined using spectrophotometer. DNA samples were used as template for the amplification of the 18S rDNA region and amplicons resolved on 1.5% agarose gel for determination of fragment sizes and purity evaluation. The concentration of extracted DNA from all Myxosporidia species ranged from 4.6 to 26 ng/μl with purity indices ranging from 1.88 to 2.12. We successfully amplified the 1050 bp DNA fragment as targeted. The intensity, thickness and clarity of the bands were evidences of non-degradation of DNA. The optimized Livak protocol is simple, low-cost and manageable. Regarding the quantity, purity and quality of extracted DNA, the optimized Livak protocol is highly recommended for Myxosporidia studies.

关 键 词：DNA Extraction Livak Protocol OPTIMIZATION Myxosporidia Parasites 

分 类 号：S93[农业科学—渔业资源]