Abstract:
:Extracting genomic DNA of pathogenic agents from formalin-fixed specimens is inherently difficult. Storage of samples in formalin results in nucleic acid cross-linking and DNA fragmentation. In this study, DNA was extracted from 45 Giardia-positive stool samples stored in formalin and subjected to PCR amplification targeting the triose phosphate isomerase (tpi), beta gardin (bg) and glutamate dehydrogenase (gdh) genes. Samples were rehydrated by using a descending alcohol series before DNA extraction using a commercial kit. This was followed by EDTA-mediated inhibition of DNase activity and prolonged treatment with proteinase K to digest contaminating proteins. DNA was amplified at rates of 64.4% (29/45) at the tpi, 40% (18/45) at the bg and 20% (9/45) at the gdh loci as seen on nested PCR. DNA quality was subsequently tested in a genotyping experiment which produced high-quality sequences at the tpi (41.2%; 12/29) bg (50%; 9/18), and gdh (22.2%; 2/9) loci and enabled differentiation of Giardia strains at the subtype level. The modified extraction protocol was effective at removing inhibitors and reversing cross-linking of DNA. However, PCR amplification was limited to short fragments of DNA which resulted in highest success rate on amplification of the shortest (334 bp) gene fragment tested.
journal_name
Exp Parasitoljournal_title
Experimental parasitologyauthors
Lee MF,Lindo JF,Auer H,Walochnik Jdoi
10.1016/j.exppara.2019.01.010subject
Has Abstractpub_date
2019-03-01 00:00:00pages
26-30eissn
0014-4894issn
1090-2449pii
S0014-4894(18)30198-Xjournal_volume
198pub_type
杂志文章abstract::In order to examine the role of IFN-gamma, TNFRp55 and iNOS in inflammatory reaction during toxoplasmosis, IFN-gamma(-/-), TNFRp55(-/-) and iNOS(-/-) mice were experimentally infected with Toxoplasma gondii ME-49 strain. The organs of the mice were evaluated for histology and immunohistochemistry in detection of tissu...
journal_title:Experimental parasitology
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journal_title:Experimental parasitology
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pub_type: 杂志文章
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journal_title:Experimental parasitology
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