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Successful extraction and PCR amplification of Giardia DNA from formalin-fixed stool samples
- Lee, Mellesia F., Lindo, John F., Auer, Herbert, Walochnik, Julia
- Experimental parasitology 2019 v.198 pp. 26-30
- DNA, DNA fragmentation, Giardia, alcohols, crosslinking, deoxyribonucleases, enzyme activity, enzyme inhibition, feces, formalin, genes, genotyping, glutamate dehydrogenase, loci, pathogens, peptidase K, polymerase chain reaction, proteins, triose-phosphate isomerase
- 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.