Chitinase and proteinase K treatments enhance the DNA yield of microsporidium Ecytonucleospora hepatopenaei spores

文献类型： 外文期刊

第一作者： Guo, Xiao-Meng

作者： Guo, Xiao-Meng;Huang, Jie;Guo, Xiao-Meng;Gao, Wen;Wang, Hai-Liang;Wongkhaluang, Prapatsorn;Taengchaiyaphum, Suparat;Xie, Guo-Si;Li, Chen;Zhao, Ruo-Heng;Huang, Jie;Guo, Xiao-Meng;Gao, Wen;Wang, Hai-Liang;Wongkhaluang, Prapatsorn;Taengchaiyaphum, Suparat;Xie, Guo-Si;Li, Chen;Zhao, Ruo-Heng;Huang, Jie;Sritunyalucksana, Kallaya

作者机构：

关键词： Microsporidium; Spore; DNA extraction; Chitinase

期刊名称：JOURNAL OF INVERTEBRATE PATHOLOGY （ 影响因子：2.4； 五年影响因子：3.0 ）

ISSN： 0022-2011

年卷期： 2024 年 207 卷

页码：

收录情况： SCI

摘要： Microsporidium Ecytonucleospora hepatopenaei (EHP) spores were purified from the hepatopancreas of Penaeus vannamei infected with EHP by percoll density gradient centrifugation and differential centrifugation. The EHP spores contain a thick chitin wall and might not rupture using the routine DNA extraction protocol. In this study, three enzymes were used, including chitinase, proteinase K, and DNase I. Chitinase or proteinase K digestions caused weakened fluorescence of chitin showing by a blurred edge of EHP spores stained with calcofluor white under a fluorescence microscope. Different combinations of these enzymes followed by DNA extraction with phenol-chloroform from EHP spores showed significant increases in the copy number of the EHP SSU gene per spore. The combination of the chitinase and proteinase K treatments resulted 4.46 f 1.07 copies/spore detected, which is 31.6 f 20.7 folds of no treatment groups, accounting to (55.7 f 13.4)% of the total copies of the gene in the spore. The additional treatment with chitinase to the conventional extraction protocol with a proteinase K digestion step for feces and hepatopancreas samples of P. vannamei resulted in a significant difference in EHP copies in the DNA of (83.8 f 64.1)% and (55.3 f 88.0)% increases. The study proved that chitinase and proteinase K treatment enhance the DNA extraction from microsporidian spores resulting in high yield.

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