An Optimized Protocol for Myxosporidia (Cnidaria: Myxosporea) DNA Extraction for Molecular Studies
Published: 1 January 2020
Open Journal of Animal Sciences , Volume 10, pp 378-386; doi:10.4236/ojas.2020.103023
Abstract: 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 Labeo batesii 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.
Keywords: optimization / DNA extraction / parasites / Myxosporidia / Livak Protocol
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