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_1_Akkermansia muciniphila as a Model Case for the Development of an Improved Quantitative RPA Microbiome Assay.PDF

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

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

HSV-2 PLNP LFA receiver operating characteristic (ROC) curves.

Each panel member was tested and analyzed in replicate (n = 2), by SeraCare, Inc, using HerpeSelect 2 ELISA from Focus Diagnostics, and the resulting absorbance mean was normalized to generate signal-to-cutoff ratios (reactivity ratios). Following SeraCare’s reported specifications, we considered panel members with average reactivity ratios < 1.0 and ≥1.0 as negative and positive, respectively, for human IgG antibodies specific to HSV-2. The panel was tested three times using the HSV-2 PLNP LFA (n = 63) with smartphone-based imaging and app readout. TLs above or below the range of quantitation of the iPhone app were given a value of 71 (the upper limit of quantitation) or 0, respectively. An HSV-2 PLNP LFA receiver operating curve (solid dark blue curve) was generated in MedCalc (version 18.2.1) using test line signal and positive cutoff values, to determine a test positive or negative. Each positive or negative test was then defined as true positive, true negative, false positive, or false negative when referenced to the gold standard method, HerpeSelect 2 ELISA. Based on the ROC analysis, a positive cutoff value of 8 for the TL of HSV-2 PLNP LFA produced the highest sensitivity (96.7%) while maintaining 100% specificity. The ROC curve and analysis gave a value of 0.997 for the area under the curve (AUC) with a 95% confidence interval of 0.937 and 1.00. The confidence region for the ROC curve is represented by the area between the light blue dotted lines.</p

Comparison of single measure intraclass correlations (ICC) in a two-way mixed-effects model for consistency of agreement using different combinations of imaging devices and image analysis tools (method A, B, C) for the same set of LFA strips.

For each method, the ICC is followed by the 95 percent confidence interval enclosed in brackets.</p

Representative images of the HSV PTH202 serum/plasma panel tested with the HSV-2 PLNP LFA.

The strips were imaged as a group on the FluorChem Platform (left side, panel members 1 to 21) or individually on the iPhone platform (right, panel member 2). The images capture a region that encompasses both the test line (TL) and control line (CL) on the test strip. Panel member 16 consistently showed a significantly weaker control line which was undetectable by the smartphone. For further investigation, Panel member 16 was re-tested at a higher dilution (20-fold instead of 3.5-fold), and the picture was added to the panel image (denoted by the asterisk).</p

Comparison of test metrics for HSV- 2 rapid tests based on the Seracare panel PTH202.

The results for each assay are based on the average of 2 or 3 replicates.</p

HSV-2 PLNP LFA schematic.

A sample is diluted and then mixed with PLNPs functionalized with goat anti-human IgGs to form human IgG-PLNP complexes that were dispensed onto the sample pad of the LFA strip. The anti-HSV human IgG PLNP complexes migrated up the membrane and were captured by recombinant HSV gG2 immobilized at the test line. The remaining uncaptured human IgG-PLNP complexes, whether specific to HSV2 gG2 or not, continued further up the strip until they were captured by goat anti-human IgGs immobilized at the control line.</p