Ultrasensitive qPCR-Based Detection of Plasmodium falciparum in Pregnant Women Using Dried Blood or Whole Blood Pellet Samples Processed through Different DNA Extraction Methods

Highly sensitive molecular techniques for the detection of low-level Plasmodium falciparum parasitemia are highly useful for various clinical and epidemiological studies. However, differences in how blood samples are preserved, the quantity of blood stored, as well as genomic DNA extraction methods used may compromise the potential usefulness of these methodologies. This study compared diagnostic sensitivity based on microscopy and malaria rapid diagnostic tests (mRDTs), with quantitative polymerase chain reaction (qPCR) P. falciparum positivity of dried blood spots (DBS) or whole blood pellets (WBP) from pregnant women using different DNA extraction protocols (Chelex-saponin or a commercial kit). Samples from 129 pregnant women were analyzed, of which 13 were P. falciparum positive by mRDT and 5 by microscopy. By using extraction kit on WBP and on DBS, qPCR positivity was 27 (20.9%) and 16 (12.4%), respectively, whereas Chelex extraction on DBS only resulted in 4 (3.1%) P. falciparum positive samples. Thus, extraction using commercial kits greatly improve the likelihood of detecting P. falciparum infections.

Microvolume DNA extraction methods for microscale amplicon and metagenomic studies

Investigating the composition and metabolic capacity of aquatic microbial assemblages usually requires the filtration of multi-litre samples, which are up to 1 million-fold larger than the microenvironments within which microbes are predicted to be spatially organised. To determine if community profiles can be reliably generated from microlitre volumes, we sampled seawater at a coastal and an oceanic site, filtered and homogenised them, and extracted DNA from bulk samples (2 L) and microvolumes (100, 10 and 1 μL) using two new approaches. These microvolume DNA extraction methods involve either physical or chemical lysis (through pH/thermal shock and lytic enzymes/surfactants, respectively), directly followed by the capture of DNA on magnetic beads. Downstream analysis of extracted DNA using both amplicon sequencing and metagenomics, revealed strong correlation with standard large volume approaches, demonstrating the fidelity of taxonomic and functional profiles of microbial communities in as little as 1 μL of seawater. This volume is six orders of magnitude smaller than most standard operating procedures for marine metagenomics, which will allow precise sampling of the heterogenous landscape that microbes inhabit.

The update and optimization of an eDNA assay to detect the invasive rusty crayfish (Faxonius rusticus)

Environmental DNA (eDNA) is nuclear or mitochondrial DNA shed into the environment, and amplifying this DNA can serve as a reliable, noninvasive way to monitor aquatic systems for the presence of an invasive species. Assays based on the collection of eDNA are becoming increasingly popular, and, when optimized, can aid in effectively and efficiently tracking invasion fronts. We set out to update an eDNA assay to detect the invasive rusty crayfish, Faxonius rusticus. We tested for species specificity compared to other stream crayfish and field tested the assay at sites with known presence (N = 3) and absence (N = 4) in the Juniata River watershed in central Pennsylvania, USA. To maximize sensitivity, we field tested different storage buffers (Longmire’s buffer and ethanol), DNA extraction methods (Qiagen’s DNEasy and PowerWater kits), and quantitative polymerase chain reaction (qPCR) chemistries (TaqMan and SYBR green). Our assay confirmed the presence data and performed optimally when filter samples were stored in Longmire’s buffer, DNA was extracted with DNeasy Blood and Tissue Kit, and TaqMan qPCR chemistry was utilized. With proper sample processing, our assay allows for accurate, noninvasive detection of F. rusticus in streams.

A Simple, Cost-Effective, and Automation-Friendly Direct PCR Approach for Bacterial Community Analysis

Understanding bacterial interactions and assembly in complex microbial communities using 16S rRNA sequencing normally requires a large experimental load. However, the current DNA extraction methods, including cell disruption and genomic DNA purification, are normally biased, costly, time-consuming, labor-intensive, and not amenable to miniaturization by droplets or 1,536-well plates due to the significant DNA loss during the purification step for tiny-volume and low-cell-density samples.

Lake sediment DNA as a proxy in fish population studies: analytical challenges and opportunities

Sedimentary environmental DNA (sed-eDNA) coupled with metabarcoding is increasingly exploited for ecological studies, but application of the method to resolve fish dynamics in lakes still needs better validation. This study (1) evaluated the sed-eDNA yields from the commonly used DNeasy PowerSoil DNA Kit from mineral-rich and organic-rich sediments and (2) examined the viability of fish sed-eDNA recovery and detection in surface sediment samples from 13 Swedish mountain lakes, with organic contents of 18–52%, by using conventional PCR and droplet digital PCR. Based on concurrent fish-population surveys these lakes contain arctic char and brown trout. We show that, compared to other specifically designed lysis buffers, the DNeasy PowerSoil DNA Kit is less effective to recover DNA from organic-rich sediments and almost 50% of the extracted DNA was lost during purification steps. The amplification of fish sed-eDNA using conventional PCR with teleo primers failed to detect positive signals; whereas ddPCR assays enabled quantification of amplifiable DNA in all the extracts. However, further molecular cloning of the positive ddPCR droplets from one sediment sample revealed amplified sequences of unidentified origin that cannot be aligned well to fish. Thus the performance of the teleo primers for quantification of fish sed-eDNA detection requires further examination. For detection of fish sed-eDNA for ecological studies, we suggest that DNA extraction methods and primers should be carefully selected and the performance of ddPCR to detect DNA at low quantities needs to be further scrutinized to circumvent the pitfalls of false positives.

Evaluation of DNA extraction methods for quantifying Helicobacter pylori in water with PCR inhibitors

DNA extraction methods were evaluated to reduce PCR inhibitors and quantify Helicobacter pylori directly from water samples using real-time PCR. Three nucleic acid extraction methods were evaluated for different types of water samples. While the QIAamp DNA mini kit for tissue was suitable for DNA extraction from treated water, the QIAamp DNA stool mini kit was still efficient in analyzing samples from river water after heavy rain and with high concentration of PCR inhibitors. The FastDNA SPIN Kit for Soil could extract DNA effectively from microbes in river and stream waters without heavy rain. Immunomagnetic separation (IMS) was used prior to DNA extraction and was a useful tool for reducing PCR inhibitors in influent and stream samples. H. pylori in various waters could be quantified directly by real-time PCR while minimizing the effect of PCR inhibitors by an appropriate method through the evaluation of DNA extraction methods considering the characteristics of the matrix water. The findings of the present study suggest that the types or characteristics of water sample by source and precipitation are an important factor in detecting H. pylori and they can be applied when detecting and monitoring of other pathogens in water.