Acta parasitologica · 2020
Evaluation of Different Oocyst DNA Extraction Methods for Cryptosporidium spp. Research in Environmental Samples.
СтатьяНазвание и аннотация: Перевод в очереди
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Moreira Neliane Cristina, Cabrine-Santos Marlene, de Oliveira-Silva Márcia Benedita
Аннотация
PURPOSE: The major problem of the PCR method for the search of protozoan cysts/oocysts in environmental samples is the presence of inhibitors. DNA extraction methods capable of removing inhibitory substances of environmental origin and recovering the DNA are decisive for the efficiency of PCR. This study aimed to compare the efficiency of different DNA extraction methods for the search by Cryptosporidium oocysts in water samples by molecular methods.
METHODS: DNA extraction from water samples was performed using four different methods. Two methods use a chaotropic buffer to extract DNA and promote the selective binding of DNA to a silica membrane (GuSCN-silica and GFX Kit). The other method is based on the lysis and digestion of the samples in buffer and proteinase K, adsorption of impurities by an "InhibitEX" insertion matrix and purification of the DNA by a silica column (QIAamp Kit). The fourth method uses ionic and non-ionic detergents and proteinase K, to solubilize and separate the DNA from proteins, and a paramagnetic resin for DNA purification in the presence of high concentrations of guanidine ions (MAGNEX DNA Kit). Nested-PCR was performed, and the Cryptosporidium SSU rDNA gene amplified.
RESULTS: The results demonstrated that MAGNEX and GFX commercial kits showed higher sensitivity, with detection of up to 100 oocysts/mL and 104 oocysts/mL respectively.
CONCLUSION: In conclusion, this study confirmed that for low-DNA environmental samples, extraction methods should include an efficient oocyst wall breaking step, and showed that the best Cryptosporidium DNA extraction methods are those that use paramagnetic resins.
METHODS: DNA extraction from water samples was performed using four different methods. Two methods use a chaotropic buffer to extract DNA and promote the selective binding of DNA to a silica membrane (GuSCN-silica and GFX Kit). The other method is based on the lysis and digestion of the samples in buffer and proteinase K, adsorption of impurities by an "InhibitEX" insertion matrix and purification of the DNA by a silica column (QIAamp Kit). The fourth method uses ionic and non-ionic detergents and proteinase K, to solubilize and separate the DNA from proteins, and a paramagnetic resin for DNA purification in the presence of high concentrations of guanidine ions (MAGNEX DNA Kit). Nested-PCR was performed, and the Cryptosporidium SSU rDNA gene amplified.
RESULTS: The results demonstrated that MAGNEX and GFX commercial kits showed higher sensitivity, with detection of up to 100 oocysts/mL and 104 oocysts/mL respectively.
CONCLUSION: In conclusion, this study confirmed that for low-DNA environmental samples, extraction methods should include an efficient oocyst wall breaking step, and showed that the best Cryptosporidium DNA extraction methods are those that use paramagnetic resins.