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Molecular identification of keratinolytic actinomyces

NE Nagwa M. Abd El-Aziz
BK Bigad E. Khalil
HI Hayam Fouad Ibrahim
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DNA extraction: The extraction method was applied according to the manufacturer of (Applied Biotechnology Co., Egypt), the actinomyces isolate was identified based on partial sequencing of the 16S rRNA gene, using the universal primers as follow, forward primer 63f (5’- CAGGCCTAACACATGCAAGTC-3’) and reverse primer 1387R (5’- GGGCGGWGTGTACAAGGC-3’). PCR product was purified and sequenced as described previously [28]. PCR program was 95 °C for 3 min, 30 cycles of denaturation at 95 °C for 45 s, annealing at 56 °C for 45 s, and extension at 72 °C for 1 min /1kbp, final extension 72 °C for 5 min. PCR product was purified using MEGA Quick-Spin total fragment DNA purification kit as instructed by the manufacturer. Sequencing was performed by the Sanger method [29].The phylogenetic tree was constructed using MAFFT alignment [30]. https://mafft.cbrc.jp/alignment/server/phylogeny.html.

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