Development of a panel of recombinase polymerase amplification assays for detection of common bacterial urinary tract infection pathogens

Aims: To develop and evaluate the performance of a panel of isothermal real?time recombinase polymerase amplification (RPA) assays for detection of common bacterial urinary tract infection (UTI) pathogens. Methods and Results: The panel included RPAs for Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Enterococcus faecalis. All five RPAs required reaction times of under 12 min to reach their lower limit of detection of 100 genomes per reaction or less, and did not cross?react with high concentrations of nontarget bacterial genomic DNA. In a 50?sample retrospective clinical study, the five?RPA assay panel was found to have a specificity of 100% (95% CI, 78�0%) and a sensitivity of 89% (95% CI, 75�%) for UTI detection. Conclusions:The analytical and clinical validity of RPA for the rapid and sensitive detection of common UTI pathogens was established. Significance and Impact of the Study: Rapid identification of the causative pathogens of UTIs can be valuable in preventing serious complications by helping avoid the empirical treatment necessitated by traditional urine culture's 48�?h turnaround time. The routine and widespread use of RPA to supplement or replace culture?based methods could profoundly impact UTI management and the emergence of multidrug?resistant pathogens

Aptamer-Phage Reporters for Ultrasensitive Lateral Flow Assays

We introduce the modification of bacteriophage particles with aptamers for use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an in vivo biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin朾iotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from Staphylococcus aureus (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ?100 times lower than those of previously reported IgE assays

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

Rapid, Affordable, and Near-Patient Disease Detection

Point-of-care (POC) diagnostics allow decentralization of disease detection, with analyses performed near the patient, reducing the time-to-result to enable earlier detection and treatment, better monitoring and management of diseases. This work focuses on advancing POC testing by applying cutting-edge nanotechnology, portable devices, and rapid methods of detection to measure a wide range of protein- and nucleic acid-based biomarkers. Isothermal nucleic acid amplification methods facilitate rapid amplification, decreasing system complexity and cost compared to PCR. Among the common isothermal nucleic acid amplification techniques, Recombinase polymerase amplification (RPA) stands out as the least complex to design and optimize, and offers the fastest time to result. We developed and evaluated the performance of a panel of RPA assays for detection of common bacterial urinary tract infection (UTI) pathogens. We also developed a quantitative RPA microbiome assay capable of determining the relative abundance of the physiologically-important bacterium Akkermansia muciniphila in human feces by eliminating contaminating DNA from RPA reagents. Lateral Flow Assays (LFAs) are a standard format for POC tests, as they are user-friendly, rapid, and affordable. We applied a smartphone-based lateral-flow immunoassay (LFA) with persistent luminescent nanoparticles (nanophosphors) as reporters to the detection of genital herpes seroconversion and dengue infection. POC smartphone devices equipped with mobile healthcare applications allow the immediate upload of results to facilitate better tracking and monitoring and prevent the spread of outbreaks. However, keeping up with evolving smartphone platform designs and creating a test that performs similarly across multiple smartphone devices are significant challenges. Here, we describe the development of the first one-step lateral flow fluorescent immunoassay for the detection of Anti-Müllerian hormone (AMH), a marker for ovarian reserve, and demonstrate quantitative point-of-need AMH detection on the portable ESEQuant LR3 Fluorescent Reader. Streamlining diagnostic testing to a patient’s physician's office would reduce the inconvenience, turnaround time, and the cost of testing, allowing for more frequent monitoring.Biology and Biochemistry, Department o

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_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_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_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