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

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

Development of Bioassays Based on Nucleic Acid Amplification and Next-Generation Sequencing

Adulteration and mis-labeling of honey to mask its true origin have become a global concern. Pollen microscopy, the current gold standard for identifying the geographical origins of honey, is very laborious and requires extensive training. In addition, filtered honey cannot be identified by pollen examination and can be spiked with pollen from a more favorable plant to disguise its origins. We targeted the nuclear ribosomal ITS2 region of plant DNA, which is known to support genus-level discrimination. We purified pollen-free DNA from honey, filtered or centrifuged to remove all pollens, using three different methods: (i) anti-dsDNA antibodies coupled to magnetic particles; (ii) Q Sepharose anion exchanger; and (iii) ceramic hydroxyapatite, Type I. The ITS2 region of the captured pollen-free DNA was PCR-amplified and subjected to next-generation sequencing (NGS). Q Sepharose showed the greatest capacity to capture trace pollen-free DNA and was applied to DNA isolation from two additional honey samples. Additionally, using pollen DNA barcoding and NGS, we have developed a method to authenticate Manuka honey, a high-value product native to New Zealand. We targeted the nuclear ribosomal ITS2 region of plant DNA of twenty-one different manuka samples. Using our in-house developed bioinformatics pipeline, we have successfully developed an NGS-based quantitative technique for measuring manuka DNA out of total plant DNA. Enrichment of trace pollen-free DNA from filtered honey samples opens a new approach to identify the true origins of filtered honey samples. The methods developed may be useful in other applications of trace DNA analysis. In another study, we developed an immuno-PCR-based diagnostic platform which couples detection antibodies to self-assembled, ultra-detectable DNA-avidin nanoparticles stabilized with poly(ethylene glycol) to link DNA amplification to target protein concentration. Electrostatic neutralization and steric cloaking of the PCR-amplifiable DNA labels by avidin and PEG coating reduces non-specific “stickiness” and enhances assay sensitivity. We further optimized the detectability of the nanoparticles by incorporating four repeats of a unique synthetic DNA PCR target into each nanoparticle. Using human chorionic gonadotropin hormone (hCG) as a model analyte, this platform was able to quantitate the target hCG protein in femtomolar concentrations using only standard laboratory equipment

Smartphone-Read Phage Lateral Flow Assay for Point-of-Care Detection of SARS-CoV-2 Infection

The COVID-19 pandemic has highlighted the urgent need for sensitive, affordable, and widely-accessible testing at the point-of-care. Here we report the development of a sensitive chemiluminescence-based smartphone-readable lateral flow assay for the detection of SARS-CoV-2 nucleoprotein using M13 phage conjugated with antibodies and HRP enzymes as LFA reporter particles. We screened 84 anti-nucleoprotein monoclonal antibody pairs in phage LFA and identified an antibody pair that gave an LoD of 25 pg/mL nucleoprotein in nasal swab extract using a FluorChem gel documentation system and 100 pg/mL when the test was imaged and analyzed by an in-house-developed smartphone reader. The smartphone-read LFA signals for positive clinical samples tested (N = 15, with known Ct) were statistically different (p < 0.001) from negative clinical samples tested (N = 11). The translation-ready phage LFA technology combined with smartphone chemiluminescence imaging can enable the timely development of ultrasensitive, affordable point-of-care testing platforms for SARS-CoV-2 and beyond

Image_2_Akkermansia muciniphila as a Model Case for the Development of an Improved Quantitative RPA Microbiome Assay.PDF

<p>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.</p

Image_5_Akkermansia muciniphila as a Model Case for the Development of an Improved Quantitative RPA Microbiome Assay.PDF

<p>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.</p

Image_3_Akkermansia muciniphila as a Model Case for the Development of an Improved Quantitative RPA Microbiome Assay.PDF

<p>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.</p

Image_4_Akkermansia muciniphila as a Model Case for the Development of an Improved Quantitative RPA Microbiome Assay.PDF

<p>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.</p