DNA Amplification Methods Research Articles

Evaluation of amplicons by AF4 as assistant for deep comprehension of loop-mediated isothermal amplification combined with lateral flow assay

Loop-mediated isothermal amplification (LAMP) is a rapid and efficient method for DNA amplification, producing concatemers of varying lengths (amplicons). This study explores the characterization of LAMP amplicons using asymmetric flow field-flow fractionation (AF4) and their realization in LAMP – lateral flow assay (LFA) for point-of-care diagnostics. We examined LAMP products from the invA gene of Salmonella enterica using two specific primer sets and three methods: fluorescent staining with SYBR Green, electrophoretic detection, and AF4. LAMP products labeled with biotin and fluorescein (introduced through primers, 12 combinations) were detected by LFA due to binding the labels. The efficiency of amplicon detection by test strips varied depending on the primer combination: the color intensities for the best and worst combinations differed by more than 7 times. AF4 enabled precise detection of components from primers to amplicons up to 500 bp and revealed differences influenced by labeling and primer combinations. An important advantage of AF4, which was demonstrated for the first time, was the characterization of native complexes of amplicons with receptors. The analysis of amplicons in complexes with streptavidin, anti-fluorescein antibodies, or both receptors together using AF4 revealed significant differences in the fractograms. The shape and maxima of the absorption peaks at 260 nm depended on retention time in the range from 20 to 35 min, indicating the formation of complexes of different compositions. AF4 and LFA showed that smaller amplicons (200-300 bp) primarily contribute to the binding of detected labels, while longer amplicons (1000-1500 bp) do not enhance the signal due to structural complexities. Our findings demonstrate the potential of AF4 as a tool for deep comprehension of LAMP processes and the composition of the formed amplicon-receptor complexes, offering valuable insights for enhancing LAMP-LFA systems. This work provides a foundation for the advanced application of AF4 for nucleic acid amplification technologies.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) method for Candida glabrata detection.

Loop-mediated isothermal amplification (LAMP) is a simple and rapid nucleic acid method for DNA amplification at a constant temperature. The "gold standard" culture method for yeast detection, has low sensitivity with severe consequences, increasing morbidity and mortality rates. Here, we report the development of a LAMP method for the specific detection of C. glabrata. The specific LAMP primers for C. glabrata detection were designed and evaluated. The LAMP assay accurately detected C. glabrata with no cross-reactivity with other Candida species. The developed molecular method would be a promising tool in the management of invasive candidiasis.

Multiplexed and Quantitative Imaging of Live‐Cell Membrane Proteins by a Precise and Controllable DNA‐Encoded Amplification Reaction

AbstractAmplifying DNA conjugated affinity ligands can improve the sensitivity and multiplicity of cell imaging and play a crucial role in comprehensively deciphering cellular heterogeneity and dynamic changes during development and disease. However, the development of one‐step, controllable, and quantitative DNA amplification methods for multiplexed imaging of live‐cell membrane proteins is challenging. Here, we introduce the template adhesion reaction (TAR) method for assembling amplifiable DNA sequences with different affinity ligands, such as aptamers or antibodies, for amplified and multiplexed imaging of live‐cell membrane proteins with high quantitative fidelity. The precisely controllable TAR enables proportional amplification of membrane protein targets with variable abundances by modulating the concentration ratios of hairpin templates and primers, thus allowing sensitive visualization of multiple membrane proteins with enhanced signal‐to‐noise ratios (SNRs) without disturbing their original ratios. Using TAR, we achieved signal‐enhanced imaging of six proteins on the same live‐cell within 1–2 h. TAR represents an innovative and programmable molecular toolkit for multiplexed profiling of membrane proteins in live‐cells.

Multiplexed and Quantitative Imaging of Live-Cell Membrane Proteins by a Precise and Controllable DNA-Encoded Amplification Reaction.

Amplifying DNA conjugated affinity ligands can improve the sensitivity and multiplicity of cell imaging and play a crucial role in comprehensively deciphering cellular heterogeneity and dynamic changes during development and disease. However, the development of one-step, controllable, and quantitative DNA amplification methods for multiplexed imaging of live-cell membrane proteins is challenging. Here, we introduce the template adhesion reaction (TAR) method for assembling amplifiable DNA sequences with different affinity ligands, such as aptamers or antibodies, for amplified and multiplexed imaging of live-cell membrane proteins with high quantitative fidelity. The precisely controllable TAR enables proportional amplification of membrane protein targets with variable abundances by modulating the concentration ratios of hairpin templates and primers, thus allowing sensitive visualization of multiple membrane proteins with enhanced signal-to-noise ratios (SNRs) without disturbing their original ratios. Using TAR, we achieved signal-enhanced imaging of six proteins on the same live-cell within 1-2 h. TAR represents an innovative and programmable molecular toolkit for multiplexed profiling of membrane proteins in live-cells.

Enhanced pyrophosphate detection: Utilizing oPD-derived carbon dots and Fe3+ interactions in a paper strip biosensor

The development of portable, cost-effective, and straightforward DNA biosensors holds immense importance in various fields, including healthcare, environmental monitoring, and food safety. This study contributes to the objective by introducing an innovative approach for synthesizing carbon dots (Cdots) with high quantum yield (QY) and remarkable selectivity for Fe3+ ions. Utilizing o-phenylenediamine as a precursor, the study achieved a straightforward and environmentally friendly synthesis method, enabling the efficient detachment of metal ions from the Cdot surface upon introducing pyrophosphate (PPi). The presence of surface hydroxyl and amino groups facilitated specific Fe3+ recognition. Employing D-optimal response surface methodology, the study optimized Cdot synthesis parameters, identifying temperature and heating time as critical factors influencing QY. Statistical analysis confirmed the model's reliability, predicting maximum QY of 48.8 % with minimal deviation from experimental results. Characterization studies revealed the amorphous nature of Cdots through HR-TEM, XRD, and FTIR analysis. Furthermore, the proposed LAMP/PPi biosensing technique demonstrated higher sensitivity, specificity, and repeatability, with negligible interference from common anions and efficacy across varying pH levels. The limit of detection (LOD) of 0.079 (±0.01) μM and the detection range of 0.1 μM–2 mM underscore the biosensor's practical utility. This study highlights a promising direction for developing paper-based LAMP/PPi biosensors with potential diagnostics and environmental monitoring applications. Significantly, the biosensing technique is applicable to any DNA amplification method generating pyrophosphate (PPi) as a by-product.

DNA-Templated Click Ligation Chain Reaction Catalyzed by Heterogeneous Cu2O for Enzyme-Free Amplification and Ultrasensitive Detection of Nucleic Acids.

Nucleic acids play a pivotal role in the diagnosis of diseases. However, rapid, cost-efficient, and ultrasensitive identification of nucleic acid targets still represents a significant challenge. Herein, we describe an enzyme-free DNA amplification method capable of achieving accurate and ultrasensitive nucleic acid detection via DNA-templated click ligation chain reaction (DT-CLCR) catalyzed by a heterogeneous nanocatalyst made of Cu2O (hnCu2O). This hnCu2O-DT-CLCR method is built on two cross-amplifying hnCu2O-catalyzed DNA-templated azide-alkyne cycloaddition-driven DNA ligation reactions that boast a fast reaction rate and a high DNA ligation yield in minutes, enabling rapid exponential amplification of specific DNA targets. This newly developed hnCu2O-DT-CLCR-enabled DNA amplification strategy is further integrated with two signal reporting mechanisms to achieve low-cost and easy-to-use biosensors: an electrochemical sensor through the conjugation of a methylene blue redox reporter to a DNA probe used in hnCu2O-DT-CLCR and a colorimetric sensor through the incorporation of the split-to-intact G-quadruplex DNAzyme encoded into hnCu2O-DT-CLCR. Both sensors are able to achieve specific detection of the intended DNA target with a limit of detection at aM ranges, even when challenged in complex biological matrices. The combined hnCu2O-DT-CLCR and sensing strategies offer attractive universal platforms for enzyme-free and yet efficient detection of specific nucleic acid targets.

Cost analyses for malaria molecular diagnosis for research planners in India and beyond

ABSTRACT Background Malaria elimination mandates early and accurate diagnosis of infection. Although malaria diagnosis is programmatically dependent on microscopy/RDTs, molecular diagnosis has much better diagnostic accuracy. Higher cost of molecular diagnoses is a recognized challenge for use at the point of care. Because funding is always a recognized constraint, we performed financial cost-analyses of available molecular platforms for better utilization of available budget. Methods Two strategies were applied to deduce the cost per sample. Strategy 1 included recurring components (RC) in minimum pack size, and biologist’s time whereas strategy 2 included only RC and non-recurring components and costs are calculated for sample sizes (1–1,000,000) to infer the sample size effect. Results Spin column-based manual DNA extraction (US$ 3.93 per sample) is the lowest-cost method, followed by magnetic bead-based automated, semi-automated, and PCI-based manual method. Further, DNA extraction cost per sample via spin column-based manual method and semi-automated method decreases with an increase in sample size up to 10,000. Real-time PCRs are ~ 2-fold more economical than conventional PCR, regardless of sample size. Conclusions This study is the first for malaria to estimate systematic molecular diagnosis financial costs. Kit-based and automated methods may replace conventional DNA extraction and amplification methods for a frugal high-throughput diagnosis.

Enhanced selective discrimination of point-mutated viral RNA through false amplification regulatory direct insertion in rolling circle amplification

Coronaviruses are single-stranded RNA viruses with high mutation rates. Although a diagnostic method for coronaviruses has been developed, variants appear rapidly. Low test accuracy owing to single-point mutations is one of the main factors in the failure to prevent the early spread of coronavirus infection. Although reverse transcription-quantitative polymerase chain reaction can detect coronavirus infection, it cannot exclude the possibility of false positives, and an additional multiplexing kit is needed to discriminate single nucleotide polymorphism (SNP) variants. Therefore, in this study, we introduced a new nucleic acid amplification method to determine whether an infected person has a SNP mutation using a lateral flow assay (LFA) as a point-of-care test. Unlike traditional DNA amplification methods, direct insertion into rolling circle amplification amplifies the target genes without false amplification. After SNP-selective nucleic acid amplification, nuclease enzymes are used to make double-stranded DNA fragments that the LFA can detect, where specific mismatched DNA is found and cleaved to show different signals when a SNP-type is present. Therefore, wild- and SNP-type variants can be selectively detected. In this study, the limit of detection was 400 aM for viral RNA, and we successfully identified a dominant SNP variant selectively. Clinical tests were also conducted.

On-Chip Magnetic Extraction of Circulating Cell-Free DNA from Biological Samples.

In recent years, the analysis of circulating cell-free DNA (cfDNA) containing tumor-derived DNA has emerged as a noninvasive means for cancer monitoring and personalized medicine. However, the isolation of cfDNA from peripheral blood has remained a challenge due to the low abundance and high fragmentation of these molecules. Here, we present a dynamic Magnetic ExTRactiOn (METRO) protocol using microfluidic fluidized bed technology to isolate circulating cfDNA from raw biological materials such as undiluted serum. This protocol maximizes the surface area for DNA binding within the chip in order to capture short DNA fragments. It uses only a few μL of sample and reagents. The protocol can be automated, and it is fully compatible with sensitive DNA amplification methods such as droplet-based digital PCR (ddPCR).

Evaluation of a Point-of-Need Molecular Diagnostic Tool Coupled with Rapid DNA Extraction Methods for Visceral Leishmaniasis.

A rapid, cost-effective, and simple nucleic acid isolation technique coupled with a point-of-need DNA amplification assay is a desirable goal for programmatic use. For diagnosis of Visceral Leishmaniasis (VL), Recombinase Polymerase Amplification (RPA) rapid tests for the detection of Leishmania DNA are versatile and have operational advantages over qPCR. To facilitate the delivery of the RPA test at point-of-need for VL diagnosis, we compared two rapid DNA extraction methods, SwiftDx (SX) and an in-house Boil and Spin (BS) method, coupled with RPA amplification, versus more widely used methods for DNA extraction and amplification, namely Qiagen (Q) kits and qPCR, respectively. A total of 50 confirmed VL patients and 50 controls, matched for age and gender, were recruited from Mymensingh, Bangladesh, a region highly endemic for VL. Blood samples were collected from each participant and DNA was extracted using Q, SX and BS methods. Following DNA extraction, qPCR and RPA assays were performed to detect L. donovani in downstream analysis. No significant differences in sensitivity of the RPA assay were observed between DNA extraction methods, 94.00% (95% CI: 83.45-98.75%), 90% (95% CI: 78.19-96.67%), and 88% (95% CI: 75.69-95.47%) when using Q, SX, and BS, respectively. Similarly, using qPCR, no significant differences in sensitivity were obtained when using Q or SX for DNA extraction, 94.00% (95% CI: 83.45-98.75%) and 92.00% (80.77-97.78%), respectively. It is encouraging that RPA and qPCR showed excellent agreement (k: 0.919-0.980) when different extraction methods were used and that the DNA impurities using BS had no inhibitory effect on the RPA assay. Furthermore, significantly higher DNA yields were obtained using SX and BS versus Q; however, a significantly higher parasite load was detected using qPCR when DNA was extracted using Q versus SX. Considering the cost, execution time, feasibility, and performance of RPA assay, rapid extraction methods such as the Boil and Spin technique appear to have the potential for implementation in resource-limited endemic settings. Further clinical research is warranted prior to broader application.

Identification of a novel family B DNA polymerase from Enterococcus phage IME199 and its overproduction in Escherichia coli BL21(DE3)

BackgroundIdentification and characterization of novel, faithful and processive DNA polymerases is a driving force in the development of DNA amplification methods. Purification of proteins from natural phages is often time-consuming, cumbersome and low yielding. Escherichia coli is a host bacterium widely used for the production of recombinant proteins, is the cell factory of choice for in vitro studies of phage protein function.ResultsWe expressed the gene encoding Enterococcus faecium phage IME199 DNA polymerase (IME199 DNAP) in Escherichia coli BL21(DE3), and characterized protein function. IME199 DNAP has 3′-5′ exonuclease activity, but does not have 5′-3′ exonuclease activity. In addition, IME199 DNAP has dNTP-dependent 5′-3′ polymerase activity and can amplify DNA at 15–35 °C and a pH range of 5.5–9.5. The amino acid residues Asp30, Glu32, Asp112 and Asp251 are the 3′-5′ exonuclease active sites of IME199 DNAP, while residues Asp596 and Tyr639 are essential for DNA synthesis by IME199 DNAP. More importantly, the IME199 DNAP has strand displacement and processive synthesis capabilities, and can perform rolling circle amplification and multiple displacement amplification with very low error rates (approximately 3.67 × 10–6).ConclusionsA novel family B DNA polymerase was successfully overproduced in Escherichia coli BL21(DE3). Based on the characterized properties, IME199 DNAP is expected to be developed as a high-fidelity polymerase for DNA amplification at room temperature.

C2CAplus: A One-Pot Isothermal Circle-to-Circle DNA Amplification System.

Rolling circle amplification (RCA) is a widely used DNA amplification method that uses circular template DNA as input and produces multimeric, linear single- or double-stranded DNA. Circle-to-circle amplification (C2CA) has further expanded this method by implementing product recircularization using restriction and ligation, leading to a higher amplification yield and enabling the generation of circular products. However, C2CA is a multistep, nonisothermal method, requiring multiple fluid manipulations and thereby compromises several advantages of RCA. Here, we improved C2CA to implement a one-pot, single step, isothermal reaction at temperatures ranging from 25 to 37 °C. Our C2CAplus method is simple, robust, and produces large quantities of product DNA that can be seen with the naked eye.

Smartphone-readable RPA-LFA for the high-sensitivity detection of Leishmania kDNA using nanophosphor reporters.

Early diagnosis of infectious diseases improves outcomes by enabling earlier delivery of effective treatment, and helps prevent further transmission by undiagnosed persons. We demonstrated a proof-of-concept assay combining isothermal amplification and lateral flow assay (LFA) for early diagnosis of cutaneous leishmaniasis, a vector-borne infectious disease that affects ca. 700,000 to 1.2 million people annually. Conventional molecular diagnostic techniques based on polymerase chain reaction (PCR) require complex apparatus for temperature cycling. Recombinase polymerase amplification (RPA) is an isothermal DNA amplification method that has shown promise for use in low-resource settings. Combined with lateral flow assay as the readout, RPA-LFA can be used as a point-of-care diagnostic tool with high sensitivity and specificity, but reagent costs can be problematic. In this work, we developed a highly-sensitive smartphone-based RPA-LFA for the detection of Leishmania panamensis DNA using blue-emitting [(Sr0.625Ba0.375)1.96Eu0.01Dy0.03]MgSi2O7 (SBMSO) persistent luminescent nanophosphors as LFA reporters. The greater detectability of nanophosphors allows the use of a reduced volume of RPA reagents, potentially reducing the cost of RPA-LFA. The limit of detection (LOD) of RPA with gold nanoparticle-based LFA readout is estimated at 1 parasite per reaction, but LOD can be 100-fold better, 0.01 parasites per reaction, for LFA based on SBMSO. This approach may be useful for sensitive and cost-effective point-of-care diagnosis and contribute to improved clinical and economic outcomes, especially in resource-limited settings.

P-724 The choice of genetic service provider can have significant implications for the outcome of IVF cycles using preimplantation genetic testing for aneuploidy (PGT-A)

Abstract Study question Does the choice of PGT-A provider have any impact on clinical results, such as rates of aneuploidy, pregnancy and miscarriage? Summary answer The laboratory providing PGT can have a significant impact on various important clinical outcomes. PGT-A providers are not equivalent and should be chosen with care. What is known already A growing number of IVF cycles include PGT-A to assist in the identification of euploid embryos for transfer to the uterus. All modern PGT laboratories utilise a method called next generation sequencing (NGS) to predict the copy number of each chromosome in embryo biopsy samples. Given this technical convergence, it is often supposed that genetic results will be similar regardless which PGT laboratory is used, and consequently a clinic choosing a genetics provider need only consider convenience, quality of support and price. Here we consider whether the choice of PGT-A provider might have more profound affects, potentially impacting clinical results. Study design, size, duration A large network of IVF clinics switched from PGT-A provider ‘A’ to provider ‘B’. The final 6 months of clinical data using provider A was compared to 6 months of data after the switch to B. There were no changes in any aspect of patient population, treatment protocols, or embryological practice occurred between the two time periods evaluated. Genetic results (aneuploidy/euploidy rates) and clinical outcomes (implantation, miscarriage, ongoing pregnancy/birth) were compared to identify any differences. Participants/materials, setting, methods Within the two time periods considered, 9,091 embryos underwent PGT-A with provider A and 10,281 using B. The average female age was 39.0 and 39.3, respectively. Embryos were biopsied at the blastocyst stage and the cells were shipped to the genetics laboratories. Provider A was in the same country as the IVF clinics, while provider B was located overseas. Both PGT-A laboratories used NGS, but employed different methods for DNA amplification, sequencing and data analysis. Main results and the role of chance A higher proportion of embryos were classified euploid by provider B in comparison to A (49.3% versus 39.8%; p < 0.0001). Differences in reported aneuploidy were particularly striking for younger patients (<38 years; 67.5% euploid according to B versus 52.5% reported by A; p < 0.0001). For patients over 37 years, the lower aneuploidy frequency reported by B resulted in fewer cycles with all embryos classified ‘abnormal’ and more cycles with a transfer (52.2% of 2115 cycles in this age group had a transfer, compared to 48.6% of 1915 cycles for A; p = 0.02). Having more embryos classified euploid also benefitted younger patients, providing opportunities to combine morphological selection with genetic evaluation. Improved selection may explain a higher ongoing pregnancy rate observed for patients <38 years (63.2% ongoing after the first embryo transfer for B versus 55.3% for A). Differing miscarriage rates were also observed (18.1% when using A versus 15.1% for B; p = 0.047). We speculate that higher rates of euploidy and pregnancy when using provider B might be a consequence of viable embryos being excluded due to incorrect classification as ‘abnormal’ by A. Additionally, failure to detect some aneuploidies, leading to transfer of abnormal embryos, might explain the higher miscarriage rate associated with A. Limitations, reasons for caution There were no detectable differences in the composition of patient populations receiving PGT-A from the two providers, and no apparent changes to protocols used during the two time periods assessed. Nevertheless, it must be acknowledged that the conclusions are based on a retrospective review of data, not a controlled study. Wider implications of the findings PGT-A and NGS are umbrella terms encompassing multiple methods with widely varying levels of validation and accuracy. Our results suggest that the choice of PGT-A provider has important implications for clinical results, affecting the number of transferrable embryos, pregnancy, and miscarriage rates. The observed differences likely reflect differing PGT-A accuracies. Trial registration number Not applicable

Deteksi Gen SLC30A8 pada Sampel Darah Penderita Diabetes Melitus Tipe 2 Dengan Metode Polymerase Chain Reaction

Background: The SLC30A8 gene is a zinc efflux transporter associated with insulin secretion expressed by the pancreas. The SLC30A8 gene causes abnormalities in insulin synthesis, maturation & secretion as well as abnormalities in the body's effectiveness in responding to glucose metabolism. Polymerase Chain Reaction (PCR) is a DNA amplification method that can be used to detect the presence of the SLC30A8 gene in type 2 diabetes mellitus patients. Methods: This study used a descriptive research method conducted at the Molecular Biology Laboratory of Sekolah Tinggi Ilmu Kesehatan Nasional. The sampling technique in this study used a total sampling technique on 25 type 2 diabetes mellitus patients who were members of the Prolanis Diabetes Mellitus Puskesmas Bulu Sukoharjo, Central Java. The detection of the SLC30A8 gene went through several stages, namely DNA isolation, qualitative testing of DNA isolates, quantitative testing of DNA isolates and PCR Results: The results showed the presence of the SLC30A8 gene in all blood samples from type 2 diabetes mellitus patients using the PCR method. The primer used with forward primer 5'-GGACGAAAGAGTTCCCATAGCG-3' and reverse primer 5'- ATAGCAGCATGTTTTGAAGGTGGC-3' attaches specifically to chromosome 8 with a product length of 429 bp. Conclusion: All blood samples of type 2 diabetes mellitus patients detected the SLC30A8 gene

Sensitive and selective DNA detecting electrochemical sensor via double cleaving CRISPR Cas12a and dual polymerization on hyperbranched rolling circle amplification

Electrochemical sensors are widely used for nucleic acid detection. However, they exhibit low sensitivity and specificity. To overcome these limitations, DNA amplification method is necessary. In this study, we introduced CRISPR (Clustered regularly interspaced short palindromic repeats) Cas12a-dependent hyperbranched rolling circle amplification (HRCA) into an electrochemical sensor platform. By resolving the existing false-positive issue of HRCA, CRISPR Cas12a determines the real positive amplification that able to enhance its sensitivity for extremely low concentrations of nucleic acids and specificity for single-point mutations. In detail, CRISPR Cas12a, which activates the nucleic acid amplification reaction, was used for both trans and cis cleavage for the first time. Finally, selectively amplified DNA was detected using a screen-printed electrode. Using the change in surface coverage by DNA, the electrochemical sensor detected a decrease in the redox signal. In summary, combining a novel DNA amplification method and electrochemical sensor platform, our proposed method compensates for the shortcomings of existing RCA and hyperbranched RCA, secures a high sensitivity of 10 aM, and overcomes false-positivity problems. Moreover, such creative applications of CRISPR Cas12a may lead to the expansion of its applications to other nucleic acid amplification methods.

Molecular Tools for Early Detection of Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Reports of the expansion of the Asia malaria vector Anopheles stephensi mosquito into new geographic areas are increasing, which poses a threat to the elimination of urban malaria. Efficient surveillance of this vector in affected areas and early detection in new geographic areas is key to containing and controlling this species. To overcome the practical difficulties associated with the morphological identification of immature stages and adults of An. stephensi mosquitoes, we developed a species-specific PCR and a real-time PCR targeting a unique segment of the second internal transcribed spacer lacking homology to any other organism. Both PCRs can be used to identify An. stephensi mosquitoes individually or in pooled samples of mixed species, including when present in extremely low proportions (1:500). This study also reports a method for selective amplification and sequencing of partial ribosomal DNA from An. stephensi mosquitoes for their confirmation in pooled samples of mixed species.

Mitochondrial DNA D-Loop Amplification and Sequencing for Species Differentiation in Milk.

Adulteration of dairy products, mainly through the substitution of high-quality milk for lower-quality milk, results in the production of low-value products, raising health, social, and economic concerns. As such, the development of methods to ensure dairy products' safety and quality is of great concern for governments and consumers. Although several methods have been developed for species differentiation in dairy products, their application and the establishment of reliable molecular markers for authentication purposes still need to be improved. In this chapter, we describe a low-cost, sensitive, fast, and reliable PCR-based method for mitochondrial D-loop DNA amplification for efficient detection of cattle milk in binary mixtures with sheep milk, thereby allowing the authentication of processed dairy products.

LT-RPA: An Isothermal DNA Amplification Approach for Improved Microsatellite Genotyping and Microsatellite Instability Detection.

Microsatellites are short tandem repeats of one to six nucleotides that are highly polymorphic and extensively used as genetic markers in numerous biomedical applications, including the detection of microsatellite instability (MSI) in cancer. The standard analytical method for microsatellite analysis relies on PCR amplification followed by capillary electrophoresis or, more recently, next-generation sequencing (NGS). However, their amplification during PCR generates undesirable frameshift products known as stutter peaks caused by polymerase slippage, complicating data analysis and interpretation, while very few alternative methods for microsatellite amplification have been developed to reduce the formation of these artifacts. In this context, the recently developed low-temperature recombinase polymerase amplification (LT-RPA) is an isothermal DNA amplification method at low temperature (32°C) that drastically reduces and sometimes completely abolishes the formation of stutter peaks. LT-RPA greatly simplifies the genotyping of microsatellites and improves the detection of MSI in cancer. In this chapter, we describe in detail all the experimental steps necessary for the development of LT-RPA simplex and multiplex assays for microsatellite genotyping and MSI detection, including the design, optimization, and validation of the assays combined with capillary electrophoresis or NGS.

Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting.

Loop-mediated isothermal amplification (LAMP) has proven to be easier to implement than PCR for point-of-care diagnostic tests. However, the underlying mechanism of LAMP is complicated and the kinetics of the major steps in LAMP have not been fully elucidated, which prevents rational improvements in assay development. Here we present our work to characterize the kinetics of the elementary steps in LAMP and show that: (i) strand invasion / initiation is the rate-limiting step in the LAMP reaction; (ii) the loop primer plays an important role in accelerating the rate of initiation and does not function solely during the exponential amplification phase and (iii) strand displacement synthesis by Bst-LF polymerase is relatively fast (125 nt/s) and processive on both linear and hairpin templates, although with some interruptions on high GC content templates. Building on these data, we were able to develop a kinetic model that relates the individual kinetic experiments to the bulk LAMP reaction. The assays developed here provide important insights into the mechanism of LAMP, and the overall model should be crucial in engineering more sensitive and faster LAMP reactions. The kinetic methods we employ should likely prove useful with other isothermal DNA amplification methods.