Enhancing melting curve analysis for the discrimination of loop-mediated isothermal amplification products from four pathogenic molds: Use of inorganic pyrophosphatase and its effect in reducing the variance in melting temperature values
“…This indicated Bst 3.0 could be a useful model for studying non-specific amplification. We observed that early amplifying products corresponded to specific amplification events, and the later products corresponded to non-specific amplification, supporting our prediction that we could use T m as a proxy for sequence identity, as is common with PCR and has been used previously in LAMP (25)(26)(27)(28)(29).…”
Section: Bulk Lamp Studies Reveal Non-specific Products With High Melsupporting
Isothermal amplification assays, such as loopmediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.
“…This indicated Bst 3.0 could be a useful model for studying non-specific amplification. We observed that early amplifying products corresponded to specific amplification events, and the later products corresponded to non-specific amplification, supporting our prediction that we could use T m as a proxy for sequence identity, as is common with PCR and has been used previously in LAMP (25)(26)(27)(28)(29).…”
Section: Bulk Lamp Studies Reveal Non-specific Products With High Melsupporting
Isothermal amplification assays, such as loopmediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.
“…Nonspecific products as a result of undesired polymerase activity on oligonucleotide primers in PCR solution were easily differentiable by melt temperature from specific amplicons. This ability to distinguish products by melt peaks provides a method for screening false positives and potential for multiplexed assays that is unavailable with standard protocols for isothermal techniques like LAMP typically investigated for POC instrumentation 23 . Figure 5 Cartridge PCR and melt performance.…”
Gold standard quantitative nucleic acid tests for diagnosis of viral diseases are currently limited to implementation in laboratories outside of the clinic. An instrument for conducting nucleic acid testing at the point-of-care (POC) that is easily operable by the clinician would reduce the required number of visits to the clinic and improve patient retention for proper treatment. Here we present a droplet magnetofluidic (DM) platform, which leverages functionalized magnetic particles to miniaturize and automate laboratory assays for use in the clinic at the POC. Our novel thermoformed disposable cartridge coupled to a portable multiaxial magnetofluidic instrument enables real-time PCR assays for quantitative and sensitive detection of nucleic acids from crude biosamples. Instead of laborious benchtop sample purification techniques followed by elution and spiking into PCR buffer, the user simply injects the biosample of interest into a cartridge with magnetic particles and loads the cartridge into the instrument. We demonstrate the utility of our platform with hepatitis C virus (HCV) RNA viral load quantitation from blood serum in approximately 1 hour. Clinical serum samples (n = 18) were directly processed on cartridges with no false positives and a limit of detection of 45 IU per 10 µl sample injection.
“…Tone and co-workers investigated the influences of T m values of LAMP products and found that inorganic pyrophosphatase (PPase) reduced the T m value variances and allowed differentiation of pathogenic agents using an annealing curve analysis for the LAMP method [ 37 ]. However, annealing curve analysis is difficult to apply to the multiplex LAMP assay because products of different target genes were similar in structure and length, all of which were dumbbell-like structures.…”
Background: Acinetobacter baumannii is a health burden responsible for various nosocomial infections, and bacteremia in particular. The resistance of A. baumannii to most antibiotics including carbapenem has increased. OXA-23-producing A. baumannii is the chief source of nosocomial outbreaks with carbapenem-resistant A. baumannii. Successful antibiotic treatment relies on the accurate and rapid identification of infectious agents and drug resistance. Here, we describe a multiplex loop-mediated isothermal amplification (LAMP) assay for simultaneous and homogeneous identification for A. baumannii infection screening and drug-resistance gene detection. Methods: Four primer pairs were designed to amplify fragments of the recA gene of A. baumannii and the oxa-23 gene. The reaction with a 25 μl of final volume was performed at 63°C for 60 min. For comparative purposes, we used a traditional method of bacterial identification to evaluate assay efficacy. Results: The multiplex LAMP assay enables simultaneous and homogeneous detection of the recA gene of A. baumannii and the oxa-23 gene and requires less than 21 min with no pre-requisite for DNA purification prior to the amplification reaction. The detection is specific to A. baumannii, and the coincidence rate of the multiplex LAMP and the traditional method was 100%. Conclusions: Our data indicate that the multiplex LAMP assay is a rapid, sensitive, simultaneous and homogeneous method for screening of A. baumannii and its drug-resistance gene.
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