General Steps During Isolation of DNA and RNA

Gautam, Akash

doi:10.1007/978-3-030-94230-4_2

Cited by 1 publication

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“…It is 40 nm thick and contains an inner filamentous and an outer glycoprotein layer [ 31 ]. When PK was used separately or in combination, the amount of extracted DNA increased, especially for E. coli and C. parvum , as the PK eliminates interfering proteins in the solution and protects free DNA by inactivating DNases [ 32 ]. Moreover, the protease degrades the filamentous layer of the oocyst wall resulting in an increase in extraction efficiency for C. parvum [ 31 ].…”

Section: Discussionmentioning

confidence: 99%

Rapid Reverse Purification DNA Extraction Approaches to Identify Microbial Pathogens in Wastewater

et al. 2023

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Wastewater monitoring became a promising solution in the early detection of outbreaks. Despite the achievements in the identification of pathogens in wastewater using real-time PCR, there is still a lack of reliable rapid nucleic acid extraction protocols. Therefore, in this study, samples were subjected to alkali, proteinase K and/or bead-beating followed by reverse purification magnetic beads-based separation. Wastewater samples spiked with S. aureus, E. coli and C. parvum were used as examples for Gram-positive and -negative bacteria and protozoa, respectively. All results were compared with a spin column technology as a reference method. Proteinase K with bead beating (vortexing with 0.1 mm glass beads for three minutes) was particularly successful for bacterial DNA extraction (three- to five-fold increase). The most useful extraction protocol for protozoa was pre-treatment with proteinase K (eight-fold increase). The selected methods were sensitive as far as detecting one bacterial cell per reaction for S. aureus, ten bacterial cells for E. coli and two oocysts for C. parvum. The extraction reagents are cold chain independent and no centrifuge or other large laboratory equipment is required to perform DNA extraction. A controlled validation trial is needed to test the effectiveness at field levels.

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Section: Discussionmentioning

confidence: 99%