Akkermansia muciniphila as a Model Case for the Development of an Improved Quantitative RPA Microbiome Assay

Abstract

Changes in the population levels of specific bacterial species within the gut microbiome have been linked to a variety of illnesses. Most assays that determine the relative abundance of specific taxa are based on amplification and sequencing of stable phylogenetic gene regions. Such lab-based analysis requires pre-analytical sample preservation and storage that have been shown to introduce biases in the characterization of microbial profiles. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification method that employs commercially available, easy-to-use freeze-dried enzyme pellets that can be used to analyze specimens rapidly in the field or clinic, using a portable fluorometer. Immediate analysis of diverse bacterial communities can lead to a more accurate quantification of relative bacterial abundance. In this study, we discovered that universal bacterial 16S ribosomal DNA primers give false-positive signals in RPA analysis because manufacturing host Escherichia coli DNA is present in the RPA reagents. The manufacturer of RPA reagents advises against developing an RPA assay that detects the presence of E. coli due to the presence of contaminating E. coli DNA in the reaction buffer (www.twistdx.co.uk/). We, therefore, explored four strategies to deplete or fragment extraneous DNA in RPA reagents while preserving enzyme activity: metal-chelate affinity chromatography, sonication, DNA cleavage using methylation-dependent restriction endonucleases, and DNA depletion using anti-DNA antibodies. Removing DNA with anti-DNA antibodies enabled the development of a quantitative RPA microbiome assay capable of determining the relative abundance of the physiologically-important bacterium Akkermansia muciniphila in human feces.

Microbial and host factors contribute to bloodstream infection in a pediatric acute lymphocytic leukemia mouse model

“…pH was measured using an Accumet AE150 instrument (Fisher Scientific, Pittsburgh, PA, USA). The presence of A. muciniphila was confirmed in fecal and intestinal samples using 5’-CAGCACGTGAAGGTGGGGAC-3’ forward, and 5’-CCTTGCGGTTGGCTTCAGAT-3’ reverse primers as described [ 22 ].…”

Screening for Selenomonas Noxia and Akkermansia Muciniphila from the Oral Cavity of Pediatric Patients

“…The primers synthesized from Fisher Scientific Company LLC were [6,20]: Forward primer-SN, 5'-TCT GGG CTA CAC ACG TAC TAC AAT G-3', 25 nt Reverse primer-SN, 5'-GCC TGC AAT CCG AAC TGA GA-3', 20 nt Forward primer-AM, 5'-CAG CAC GTG AAG GTG GGG-3', 18 nt Reverse primer-AM, 5'CCT TGG GGT TGG CTT CAG AT'-3', 2…”

Evaluation of a Metagenomic Next-Generation Sequencing Assay With a Novel Host Depletion Method for Pathogen Identification in Septic Patients

“…One widely recognised major hurdle in mNGS of blood samples is the overwhelming presence of human DNA, leading to vast wastage of sequencing real estate [11]. A few methods have been proposed for removing host DNA background, including differential lysis of human cells [12] and methylated human DNA removal [13]. Recently, host cell depletion device such as Devin fractionation filter has been made available for removing human nucleated cells.…”