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

Abstract

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.

Rapid Identification of Bacterial Composition in Wastewater by Combining Reverse Purification Nucleic Acid Extraction and Nanopore Sequencing

“…Results of real-time PCR showed that bead beating was crucial to break the thick, homogeneous cell wall of the Gram-positive bacterium S. aureus. , In the case of E. coli, the thin cell wall could be damaged by all procedures, and no significant variation in extraction efficacy was detected .…”

“…For the assessment of an appropriate extraction method, different buffer and pretreatment options were tested in both real-time PCR and nanopore sequencing. 21 An overview of all of the evaluated extraction procedures can be found in Table 1. For extraction (I), the protocol of the SwiftXDNA kit (Xpedite Diagnostics GmbH, Hallbergmoos, Germany) was applied.…”

“…For the assessment of an appropriate extraction method, different buffer and pretreatment options were tested in both real-time PCR and nanopore sequencing . An overview of all of the evaluated extraction procedures can be found in Table .…”

“…The bottleneck of sequencing is the correct preparation of the sample as the results can vary widely depending on the used nucleic acid extraction method. − A recent report showed that the detection of bacterial DNA in wastewater can be influenced by the extraction procedure used . The degraded or decreased DNA amount can influence the nanopore sequencing outcomes .…”

“…The evaluated extraction method is based on the principle of reverse purification, which has proven to be a valuable method at the PON due to few operating steps and little equipment required . Upon cell lysis, impurities and cell debris are bound to magnetic beads and are then attracted to a magnet.…”