Xiao-Meng Guo , Wen Gao , Hai-Liang Wang , Prapatsorn Wongkhaluang , Suparat Taengchaiyaphum , Guo-Si Xie , Chen Li , Ruo-Heng Zhao , Kallaya Sritunyalucksana , Jie Huang
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引用次数: 0
Abstract
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 ± 1.07 copies/spore detected, which is 31.6 ± 20.7 folds of no treatment groups, accounting to (55.7 ± 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 ± 64.1)% and (55.3 ± 88.0)% increases. The study proved that chitinase and proteinase K treatment enhance the DNA extraction from microsporidian spores resulting in high yield.
期刊介绍:
The Journal of Invertebrate Pathology presents original research articles and notes on the induction and pathogenesis of diseases of invertebrates, including the suppression of diseases in beneficial species, and the use of diseases in controlling undesirable species. In addition, the journal publishes the results of physiological, morphological, genetic, immunological and ecological studies as related to the etiologic agents of diseases of invertebrates.
The Journal of Invertebrate Pathology is the adopted journal of the Society for Invertebrate Pathology, and is available to SIP members at a special reduced price.