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

文献类型： 外文期刊

作者： Guo, Xiao-Meng 1 ; Gao, Wen 2 ; Wang, Hai-Liang 2 ; Wongkhaluang, Prapatsorn 2 ; Taengchaiyaphum, Suparat 2 ; Xie, Guo-Si 2 ; Li, Chen 2 ; Zhao, Ruo-Heng 2 ; Sritunyalucksana, Kallaya 4 ; Huang, Jie 1 ;

作者机构： 1.Shanghai Ocean Univ, Coll Fisheries & Life Sci, Shanghai 201306, Peoples R China

2.Chinese Acad Fishery Sci, Yellow Sea Fisheries Res Inst, State Key Lab Mariculture Biobreeding & Sustainabl, Qingdao 266071, Shandong, Peoples R China

3.Laoshan Lab, Lab Marine Fisheries Sci & Food Prod Proc, Qingdao 266237, Shandong, Peoples R China

4.Natl Sci & Technol Dev Agcy NSTDA, Integrat Aquaculture Biotechnol Res Grp, Natl Ctr Genet Engn & Biotechnol BIOTEC, Aquat Anim Hlth Res Team AQHT, Bangkok 10400, Thailand

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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