The effect of silica desiccation under different storage conditions on filter-immobilized environmental DNA

Objective Silica gel beads have promise as a non-toxic, cost-effective, portable method for storing environmental DNA (eDNA) immobilized on filter membranes. Consequently, many ecological surveys are turning to silica bead filter desiccation rather than ethanol preservation. However, no systematic evaluation of silica bead storage conditions or duration past 1 week has been published. The present study evaluates the quality of filter-immobilized eDNA desiccated with silica gel under different storage conditions for over a year using targeted quantitative real-time polymerase chain reaction (qPCR)-based assays. Results While the detection of relatively abundant eDNA target was stable over 15 months from either ethanol- or silica gel-preserved filters at − 20 and 4 °C, silica gel out-performed ethanol preservation at 23 °C by preventing a progressive decrease in eDNA sample quality. Silica gel filter desiccation preserved low abundance eDNA equally well up to 1 month regardless of storage temperature (18, 4, or − 20 °C). However only storage at − 20 °C prevented a noticeable decrease in detectability at 5 and 12 months. The results indicate that brief storage of eDNA filters with silica gel beads up to 1 month can be successfully accomplished at a range of temperatures. However, longer-term storage should be at − 20 °C to maximize sample integrity.

Alcohol keeps eDNA at the party longer

For a typical eDNA water study, water will be filtered on site, before prompt transfer to a laboratory for DNA extraction and required scientific analysis. In a setting where transport is quick and available, this is a straightforward process. However, many of our studies can occur in remote Australia where sample preservation presents many logistical challenges. Typically, we advise clients to store eDNA water filters after sampling below 4 °C to ensure minimal DNA degradation. For many clients however, field studies often occur in an isolated setting without adequate refrigeration facilities, and as such present challenges for this process. Rather than compromise on sample integrity, EnviroDNA conducted a pilot study into the use of alternate preservation methods on our most commonly used 0.22 mm Sterivex filters. With help from our friendly neighbourhood goldfish tank, our standard 4 °C protocol was compared to a variety of conditions including filled ethanol filters, flushed ethanol filters, lysis buffer and silica bead storage conditions at both 4 °C and room temperature. The study, conducted at various time points over 14-days, used qPCR to quantify the amount of DNA extracted from the filter. Our results revealed that storage within or using flushed ethanol, allowed the samples to be stored for longer time intervals at room temperature, with similar, or in some cases, improved DNA elutions. This protocol optimisation has allowed us to offer an alternate sample storage protocol for clients, expanding the availability and accessibility of eDNA biodiversity assessments around Australia.

A Sample-In-Answer-Out Microfluidic System for the Molecular Diagnostics of 24 HPV Genotypes Using Palm-Sized Cartridge

This paper proposes an automated microfluidic system for molecular diagnostics that integrates the functions of a traditional polymerase chain reaction (PCR) laboratory into a palm-sized microfluidic cartridge (CARD) made of polystyrene. The CARD integrates 4 independent microfluidic sample lanes, which can independently complete a sample test, and each sample lane integrates the 3 functional areas of the sample preparation area, PCR amplification area, and product analysis area. By using chemical cell lysis, magnetic silica bead-based DNA extraction, combined with multi-PCR-reverse dot hybridization with microarray, 24 HPV genotypes can be typing tested in CARD. With a custom-made automated CARD operating platform, the entire process can be automatically carried out, achieving sample-in-answer-out. The custom-made operation platform is developed based on a liquid handling station-type, which can automatically load off-chip reagents without placing reagents in CARD in advance. The platform can control six CARDs to work simultaneously, detect 24 samples at a time. The results show that the limit of detection of the microfluidic system is 200 copies/test, and the positive detection rate of clinical samples by this system is 100%, which is an effective method for detection of HPV.

Norovirus Extraction from Frozen Raspberries Using Magnetic Silica Beads

Human noroviruses (HuNoV) are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several HuNoV food-related outbreaks. However, the extraction of HuNoV RNA from frozen raspberries remains challenging. Recovery yields are low, and real-time quantitative reverse transcriptase PCR (RT-qPCR) inhibitors limit the sensitivity of the detection methodologies. A new approach using fine magnetic silica beads was developed for the extraction of HuNoV spiked on frozen raspberries. Relatively low recovery yields were observed with both the magnetic silica bead and the reference ISO 15216-1:2017 methods. High RT-qPCR inhibition was observed with the ISO 15216-1:2017 recommended amplification kit but could be reduced by using an alternative kit. Reducing RT-qPCR inhibition is important to limit the number of inconclusive HuNoV assays thus increasing the capacity to assess the HuNoV prevalence in frozen raspberries.

The Effect of Silica Desiccation Under Different Storage Conditions on Filter-Immobilized Environmental DNA

Objective Silica gel beads have promise as a non-toxic, cost-effective, portable method for storing environmental DNA (eDNA) immobilized on filter membranes. Consequently, many ecological surveys are turning to silica bead filter desiccation rather than ethanol preservation. However, no systematic evaluation of silica bead storage conditions or duration past one week has been published. The present study evaluates the quality of filter-immobilized eDNA desiccated with silica gel under different storage conditions for over a year using targeted quantitative real-time polymerase chain reaction (qPCR)-based assays. Results While the detection of relatively abundant eDNA target was stable over 15 months from either ethanol- or silica gel-preserved filters at -20 and 4oC, silica gel out-performed ethanol preservation at 23oC by preventing a progressive decrease in eDNA sample quality. Silica gel filter desiccation preserved low abundance eDNA equally well up to 1 month regardless of storage temperature (18, 4, or -20oC). However only storage at -20oC prevented a noticeable decrease in detectability at 5 and 12 months. The results indicate that brief storage of eDNA filters with silica gel beads up to one month can be successfully accomplished at a range of temperatures. However, longer-term storage should be at -20oC to maximize sample integrity.