Lack of specificity associated with using molecular beacons in loop mediated amplification assays.

BACKGROUND: Loop mediated isothermal amplification of nucleic acid templates is a rapid, sensitive and specific method suitable for molecular diagnostics. However the complexity of primer design and the number of primers involved can lead to false positives from non-specific primer interactions. Standard methods of LAMP detection utilise the increasing concentrations of DNA or inorganic pyrophosphate and therefore lack specificity for identifying the desired LAMP amplification. Molecular beacons used in PCR reactions are target specific and may enhance specificity with LAMP. RESULTS: We present a potential molecular beacon approach to LAMP detection targeting the single stranded region between loops, and test this for LAMP molecular beacons targeting the 35S promoter and NOS terminator sequences commonly used in GM crops. From these studies we show that molecular beacons used in LAMP, despite providing a change in fluorescent intensity with amplification, appear not to anneal to specific target sequences and therefore target specificity is not a benefit of this method. However, molecular beacons demonstrate a change in fluorescence which is indicative of LAMP amplification products. We identify the LAMP loop structure as likely to be responsible for this change in signal. CONCLUSIONS: Molecular beacons can be used to detect LAMP amplification but do not provide sequence specificity. The method can be used to determine effectively LAMP amplification from other primer-driven events, but does not discriminate between different LAMP amplicons. It is therefore unsuitable for multiplex LAMP reactions due to non-specific detection of LAMP amplification.

Optimisation of choline testing using Florence Iodine reagent, including comparative sensitivity and specificity with PSA and AP tests.

The detection of semen in forensic science is essential in cases of sexual assault but can be problematic in the absence of spermatozoa. Choline is known to occur in high concentrations in seminal fluid and the Florence Iodine test for its detection has been used in forensic science for many years, however very little is documented regarding its sensitivity and specificity in forensic casework. This paper describes the optimisation of the choline Florence Iodine test (FI) and investigates the sensitivity and specificity of the test against different body fluids, food and drink substances, cleaning products and laboratory chemicals. Comparative testing against Acid Phosphatase (AP) and Prostate Specific Antigen (PSA Seratec®) tests is described and shows that the FI test has greater specificity than the PSA test which cross reacts with a number of body fluids.

Molecular Beacons - Loop-Mediated Amplification (MB-LAMP).

High specificity has been demonstrated in polymerase chain reaction (PCR) with the use of molecular beacons (MBs) to detect amplified sequences containing mutations or single-nucleotide polymorphisms (SNPs). MBs have been adapted for use with the isothermal nucleic acid amplification technology loop-mediated amplification (LAMP) by targeting single-stranded loop sequences under optimized conditions to enable applications such as plant genotyping. LAMP has several benefits over PCR, such as rapid amplification, single-temperature reaction conditions enabling low-cost equipment, and robustness to certain PCR inhibitors. However, and despite the increased number of primers required, the specificity of LAMP is limited, and false positive results can be problematic. In this chapter, design considerations for molecular beacons in LAMP assays are described, as well as a method for MB-LAMP amplification and detection, with an example of gene sequences in genetically modified (GM) maize samples.

VarLOCK: sequencing-independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance.

The COVID-19 pandemic demonstrated the need for rapid molecular diagnostics. Vaccination programs can provide protection and facilitate the opening of society, but newly emergent and existing viral variants capable of evading the immune system endanger their efficacy. Effective surveillance for Variants of Concern (VOC) is therefore important. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. Here we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to provide rapid and sensitive discrimination of SARS-CoV-2 VOCs that can be used at point of care, implemented in the pipelines of small or large testing facilities, and even determine the proportion of VOCs in pooled population-level wastewater samples. This technology complements sequencing efforts to allow facile and rapid identification of individuals infected with VOCs to help break infection chains. We show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants and the rapid adaptability of the technique for new and emerging VOCs.

GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use.

BACKGROUND: There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device. RESULTS: Loop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. CONCLUSIONS: LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure quantification at low target concentrations.

Optimized Loop-Mediated Isothermal Amplification (LAMP) Allows Single Copy Detection Using Bioluminescent Assay in Real Time (BART).

The bioluminescent assay in real time (BART) is a molecular diagnostic tool for the detection of nucleic acid amplification by recording light output. The key component for BART is a thermostable luciferase derived from the firefly Photinus pyralis. Coupling BART detection with the isothermal amplification method loop-mediated isothermal amplification (LAMP) enables rapid molecular diagnostic results using simple equipment. LAMP-BART provides quantitative results from a closed tube and is appropriate to microliter standard tests and nanoliter microfluidic assays. In this chapter, we introduce a protocol to amplify and detect genetic markers using LAMP with BART. Furthermore, we provide advice to optimize LAMP assays for high sensitivity and specificity and to eliminate the incidence of "false positive" results which can occur from the components of the assay. The optimization of genetically modified (GM) maize by targeting the nopaline synthase terminator (NOSt) and 35S promoter (35Sp) sequences is described.

Full Dynamic Range Quantification using Loop-mediated Amplification (LAMP) by Combining Analysis of Amplification Timing and Variance between Replicates at Low Copy Number.

Quantification of nucleic acid targets at low copy number is problematic with the limit of detection at 95 percent confidence predicted to be 3 molecules or higher for quantitative PCR. Conversely the accuracy of digital PCR is diminished at higher concentrations of template approaching 100 percent positive partitions, with the Poisson distribution showing that an average of only 3 molecules per partition represents an amplification frequency of greater than 95 percent. Therefore a full range of template concentrations cannot be quantified accurately with these methods alone without dilution. Here we report the development of quantification metrics for use with loop-mediated amplification (LAMP) as a bridge between concentrated and dilute template concentrations. The basis for this is that real-time monitoring of LAMP reactions either by bioluminescent reporting (BART) or by fluorescent dye binding shows increasing variation in timings between replicates at low copy number due to the LAMP amplification mechanism. This effect increases with decreasing copy number, closely associated with the amplification frequency. The use of an artificial template showed that the increasing variation is not linked to the use of displacement primers during the initiation of amplification and is therefore a fundamental feature of the LAMP initiation event. Quantification between 1 and 10 copies of a template was successfully achieved with a number of methods with a low number of replicates with the strongest correlation to timing variance. These ultra-quantification methods for LAMP amplification either singularly or in combination have potential in a full dynamic range quantification strategy based on LAMP, in a closed tube, undiluted sample molecular diagnostic.

Bioluminescent detection of isothermal DNA amplification in microfluidic generated droplets and artificial cells.

Microfluidic droplet generation affords precise, low volume, high throughput opportunities for molecular diagnostics. Isothermal DNA amplification with bioluminescent detection is a fast, low-cost, highly specific molecular diagnostic technique that is triggerable by temperature. Combining loop-mediated isothermal nucleic acid amplification (LAMP) and bioluminescent assay in real time (BART), with droplet microfluidics, should enable high-throughput, low copy, sequence-specific DNA detection by simple light emission. Stable, uniform LAMP-BART droplets are generated with low cost equipment. The composition and scale of these droplets are controllable and the bioluminescent output during DNA amplification can be imaged and quantified. Furthermore these droplets are readily incorporated into encapsulated droplet interface bilayers (eDIBs), or artificial cells, and the bioluminescence tracked in real time for accurate quantification off chip. Microfluidic LAMP-BART droplets with high stability and uniformity of scale coupled with high throughput and low cost generation are suited to digital DNA quantification at low template concentrations and volumes, where multiple measurement partitions are required. The triggerable reaction in the core of eDIBs can be used to study the interrelationship of the droplets with the environment and also used for more complex chemical processing via a self-contained network of droplets, paving the way for smart soft-matter diagnostics.

Reduced False Positives and Improved Reporting of Loop-Mediated Isothermal Amplification using Quenched Fluorescent Primers.

Loop-mediated isothermal amplification (LAMP) is increasingly used in molecular diagnostics as an alternative to PCR based methods. There are numerous reported techniques to detect the LAMP amplification including turbidity, bioluminescence and intercalating fluorescent dyes. In this report we show that quenched fluorescent labels on various LAMP primers can be used to quantify and detect target DNA molecules down to single copy numbers. By selecting different fluorophores, this method can be simply multiplexed. Moreover this highly specific LAMP detection technique can reduce the incidence of false positives originating from mispriming events. Attribution of these events to particular primers will help inform and improve LAMP primer design.

Optimised LAMP allows single copy detection of 35Sp and NOSt in transgenic maize using Bioluminescent Assay in Real Time (BART).

Loop-mediated amplification (LAMP) has been widely used to amplify and hence detect nucleic acid target sequences from various pathogens, viruses and genetic modifications. Two distinct types of primer are required for LAMP; hairpin-forming LAMP and displacement. High specificity arises from this use of multiple primers, but without optimal conditions for LAMP, sensitivity can be poor. We confirm here the importance of LAMP primer design, concentrations and ratios for efficient LAMP amplification. We further show that displacement primers are non-essential to the LAMP reaction at certain concentrations providing accelerating loop primers are present. We investigate various methods to quantify DNA extracts from GM maize certified reference materials to calculate the target copy numbers of template presented to the LAMP reaction, and show that LAMP can amplify transgenic promoter/terminator sequences in DNA extracted from various maize GM events using primers designed to target the 35S promoter (35Sp) or NOS terminator (NOSt) sequences, detection with both bioluminescence in real-time (BART) and fluorescent methods. With prior denaturation and HPLC grade LAMP primers single copy detection was achieved, showing that optimised LAMP conditions can be combined with BART for single copy targets, with simple and cost efficient light detection electronics over fluorescent alternatives.