Loop-mediated isothermal amplification (LAMP) and reverse-transcription LAMP (RT-LAMP) (see Tomita, N.; Mori, Y.; Kanda, H.; Notomi, T. Loop-Mediated Isothermal Amplification (LAMP) of Gene Sequences and Simple Visual Detection of Products. Nat. Protoc. 2008, 3, 877-882 and Parida, M.; Sannarangaiah, S.; Dash, P. K.; Rao, P. V. L.; Morita, K. Loop Mediated Isothermal Amplification (LAMP): A New Generation of Innovative Gene Amplification Technique; Perspectives in Clinical Diagnosis of Infectious Diseases. Rev. Med. Virol. 2008, 18, 407-421) are powerful molecular techniques for rapid exponential amplification of nucleic acids under isothermal conditions without the need for expensive thermal cycling equipment and time-consuming protocols as required in PCR and RT-PCR assays (see Zhao, Y.; Chen, F.; Li, Q.; Wang, L.; Fan, C. Isothermal Amplification of Nucleic Acids. Chem. Rev. 2015, 115, 12491-12545).
Advantages such as simple reaction setup, short turnaround time, multiple available readout methods, high tolerance to common inhibitors, and possibility to bypass nucleic acid extraction make LAMP/RT-LAMP-based diagnostics especially attractive for inexpensive point-of-care (POC) testing and near-patient testing of emerging pathogens (see Wong, Y.-P.; Othman, S.; Lau, Y.-L.; Radu, S.; Chee, H.-Y. Loop-Mediated Isothermal Amplification (LAMP): A Versatile Technique for Detection of Micro-Organisms. J. Appl. Microbiol. 2018, 124, 626-643) and infectious diseases (see Mori, Y.; Notomi, T. Loop-Mediated Isothermal Amplification (LAMP): A Rapid, Accurate, and Cost-Effective Diagnostic Method for Infectious Diseases. J. Infect. Chemother. 2009, 15, 62-69).
However, the high sensitivity and specificity of LAMP/RT-LAMP reaction fundamentally depends on the meticulous design and optimization of a set of six primers which recognize eight distinct regions on the target sequence (see Moehling, T. J.; Choi, G.; Dugan, L. C.; Salit, M.; Meagher, R. J. LAMP Diagnostics at the Point-of-Care: Emerging Trends and Perspectives for the Developer Community. Expert Rev. Mol. Diagn. 2021, 21, 43-61). The necessity to completely redesign, screen, and optimize new sets of LAMP primers for each desired target makes it costly, time-consuming, and labor-intensive to develop high-performance LAMP/RT-LAMP assays in response to evolving pathogens and emerging infectious diseases.
Furthermore, to detect multiple targets, most state-of-the-art methods of multiplexed LAMP/RT-LAMP must rely on the inclusion of multiple orthogonal LAMP primer sets in the reaction mix (see Zhang, Y.; Tanner, N. A. Development of Multiplexed Reverse-Transcription Loop-Mediated Isothermal Amplification for Detection of SARS-CoV-2 and Influenza Viral RNA. Biotechniques 2021, 70, 167-174 and Jang, W. S.; Lim, D. H.; Yoon, J.; Kim, A.; Lim, M.; Nam, J.; Yanagihara, R.; Ryu, S.-W.; Jung, B. K.; Ryoo, N.-H.; et al. Development of a Multiplex Loop-Mediated Isothermal Amplification (LAMP) Assay for on-Site Diagnosis of SARS CoV-2. PLoS One 2021, 16, e0248042) as well as special readout mechanisms such as multiple fluorophore-quencher pairs (see Tanner, N. A.; Zhang, Y.; Evans, T. C. Simultaneous Multiple Target Detection in Real-Time Loop-Mediated Isothermal Amplification. Biotechniques 2012, 53, 81-89), microcapillaries (see Zhang, Y.; Zhang, L.; Sun, J.; Liu, Y.; Ma, X.; Cui, S.; Ma, L.; Xi, J. J.; Jiang, X. Point-of-Care Multiplexed Assays of Nucleic Acids Using Microcapillary-Based Loop-Mediated Isothermal Amplification. Anal. Chem. 2014, 86, 7057-7062 and Li, R.; Chen, J.; Zhang, X.; Cui, J.; Tao, S.; Yang, L. Mini-Disk Capillary Array Coupling with LAMP for Visual Detection of Multiple Nucleic Acids Using Genetically Modified Organism Analysis as an Example. J. Agric. Food Chem. 2020, 68, 899-906), lateral flow devices (see Zhu, X.; Wang, X.; Han, L.; Chen, T.; Wang, L.; Li, H.; Li, S.; He, L.; Fu, X.; Chen, S.; et al. Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Combined with Nanoparticle-Based Lateral Flow Biosensor for the Diagnosis of COVID-19. Biosens. Bioelectron. 2020, 166, 112437), or amplicon melting patterns (see Dong, J.; Xu, Q.; Li, C.; Zhang, C. Single-Color Multiplexing by the Integration of High-Resolution Melting Pattern Recognition with Loop-Mediated Isothermal Amplification. Chem. Commun. 2019, 55, 2457-2460) to interpret the reaction results. These implementations significantly increase the overall cost and complexity of the assays and may also lead to a higher risk of contamination (e.g., if the reaction vessel must be reopened post amplification for readout and analysis).
To simplify the implementation of multiplexed LAMP, a recent work proposed the idea of mediator-reporter set (see Becherer, L.; Bakheit, M.; Frischmann, S.; Stinco, S.; Borst, N.; Zengerle, R.; von Stetten, F. Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters. Anal. Chem. 2018, 90, 4741-4748), which enables the use of a single florescent hairpin reporter to visualize the amplification products generated from different LAMP reactions. While this prior design provides an efficient readout method for multiplexed LAMP, it still does not bypass the requirement to meticulously design, screen, and optimize a full set of LAMP primers for every single target involved in the assay.
Accordingly, there is an ongoing need for improved systems, methods, and associated workflows for LAMP/RT-LAMP, particularly for use in assays for responding to evolving pathogens and emerging infectious diseases.
The present disclosure is directed to universal LAMP/RT-LAMP assays for effective detection of distinct DNA/RNA targets. In conventional LAMP/RT-LAMP reactions, a set of six unique primers must be meticulously designed and optimized for each desired target sequence. Due to numerous design constraints on LAMP primers, the design and optimization of LAMP/RT-LAMP assays are usually difficult and require tedious empirical testing of multiple candidate primer sets for a desired target. As a result, the detection of additional targets requires the complete re-design, screening, and optimization of new sets of LAMP primers, which can be a very costly, time-consuming, and labor-intensive process.
The present disclosure integrates a novel transduction mechanism that functions upstream of a highly optimized LAMP reaction to effectively transduce the detection of any target DNA/RNA sequence into the rapid amplification of a universal LAMP template that is independent of the assay's target sequence. The transduction mechanism (
This LAMP reaction is highly optimized and does not need to be readjusted for detection of different targets. As a result, the present invention enables rapid development of universal one-pot molecular assays using just a single set of highly optimized LAMP primers to detect any desired set of distinct DNA or RNA sequences, with high specificity and sensitivity. The simplicity of the universal assay design facilitates fast deployment of inexpensive and accurate molecular diagnostics to enable widespread point-of-care/near-patient testing of current/emerging pathogens (e.g., viruses, bacteria, fungi), including infectious diseases of global public health importance such as Ebola, Zika, SARS-CoV-2, malaria, tuberculosis, dengue, yellow fever, etcetera. Other applications include analysis of environmental samples. For example, certain embodiments may be directed to environmental monitoring and assessment by analyzing water samples, soil samples, and/or other types of environmental samples.
Furthermore, the presently disclosed embodiments relate to designs and implementations of multiplexed universal LAMP/RT-LAMP assays with direct support for Boolean ‘Logic OR’ (
The novel transduction mechanism as described herein is broadly compatible with various isothermal amplification methods and readout techniques. In addition to molecular diagnostic applications, the proposed transduction mechanism can be broadly applied to the field of DNA and RNA computing to achieve simple universal transduction of input and output signals in complex molecular circuitries.
This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an indication of the scope of the claimed subject matter.
Various objects, features, characteristics, and advantages of the invention will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings and the appended claims, all of which form a part of this specification. In the Drawings, like reference numerals may be utilized to designate corresponding or similar parts in the various Figures, and the various elements depicted are not necessarily drawn to scale, wherein:
To substantially simplify the design and implementation of LAMP-based assays for effective detection of different targets, the present disclosure describes a simple scheme of universal LAMP/RT-LAMP, which leverages a novel transduction mechanism including three target-specific primers to rapidly initialize a highly optimized universal LAMP reaction upon the detection of any desired set of target DNA or RNA sequences. The coupling between the transduction reaction and the LAMP reaction is the target-triggered release of a universal ssDNA oligonucleotide that functions as one of the essential LAMP primers for a pre-designed, highly optimized LAMP reaction.
Distinct target nucleic acids can therefore be rapidly amplified with high specificity and sensitivity based on the design of only three primers as opposed to, in standard approaches, six specific LAMP primers for every target. In contrast to the state-of-the-art molecular assays that integrate multi-stage reactions with LAMP (see, e.g., Marciniak, J. Y.; Kummel, A. C.; Esener, S. C.; Heller, M. J.; Messmer, B. T. Coupled Rolling Circle Amplification Loop-Mediated Amplification for Rapid Detection of Short DNA Sequences. Biotechniques 2008, 45, 275-280), the presently described assays do not require additional enzymes or increases to the reaction time to achieve a high level of sensitivity and specificity on par with highly optimized LAMP/RT-LAMP assays.
The integration of the simple transduction mechanism with the universal LAMP reaction also facilitates direct implementations of Boolean logic operations during rapid detection of multiple nucleic acid targets. Various alternative design schemes of the ‘loaded primer’ are further described to enable flexible fine-tuning of the sensitivity, specificity, and multiplexity of the universal LAMP/RT-LAMP assays. Built-in support for Boolean logic computation also allows the use of a simple pH-based colorimetric readout (such as described in Tanner, N. A.; Zhang, Y.; Evans, T. C. Visual Detection of Isothermal Nucleic Acid Amplification Using PH-Sensitive Dyes. Biotechniques 2015, 58, 59-68, for example) or other relatively simple detection scheme for easy interpretation of the multiplexed test result by direct visual inspection.
A multiplexed assay with inherent support for Boolean logic computation offers numerous advantages for practical applications. For example, the multiplexed ‘Logic OR’ operation can be leveraged to improve the assay multiplexity (e.g., by simultaneously detecting multiple pathogens or multiple subtypes/mutants of a particular pathogen) or to enhance the assay sensitivity (e.g., by simultaneously detecting multiple genomic loci of a particular target pathogen). Similarly, the ‘Logic AND’ operation can be leveraged to improve the assay multiplexity (e.g., by detecting the co-presence of multiple pathogens or multiple subtypes/mutants of a particular pathogen) or to enhance the assay specificity (e.g., by detecting the co-presence of multiple genomic loci specific to a particular target pathogen).
The presently described assay enables universal rapid isothermal detection of nucleic acids (including but are not limited to DNA, ssDNA, dsDNA, RNA, mRNA, tRNA, microRNA, rRNA, siRNA, sgRNA, and nucleic acid analogues such as TNA, LNA, HNA, GNA) in either singleplex or multiplex assay format, offering higher sensitivity and specificity than PCR/RT-PCR while featuring simplified assay designs and lower development cost than conventional multiplex LAMP/RT-LAMP.
The universal assays as presently described are compatible with multiple readout methods, including but not limited to, colorimetric, fluorescence, chemiluminescence, electrochemical, turbidity, and the like. The presently described assays may also be employed in conjunction with readout mechanisms such as multiple fluorophore-quencher pairs, microcapillaries, lateral flow devices, or amplicon melting patterns.
In addition to applications in molecular diagnostics, the novel transduction mechanism proposed herein can be broadly applied to the field of DNA and RNA computing to easily enable universal transduction of input and output signals in complex molecular circuitries. For examples of such systems where the embodiments described herein may be utilized, see Song, X.; Reif, J. Nucleic Acid Databases and Molecular-Scale Computing. ACS Nano 2019, 13, 6256-6268 and Shah, S.; Wee, J.; Song, T.; Ceze, L.; Strauss, K.; Chen, Y.-J.; Reif, J. Using Strand Displacing Polymerase To Program Chemical Reaction Networks. J. Am. Chem. Soc. 2020, 142, jacs.0c02240.
It will be understood that the universal transduction mechanism proposed herein is also broadly compatible with various other isothermal amplification techniques in addition to LAMP, including but not limited to, strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), rolling cycle amplification (RCA), isothermal multiple displacement amplification (IMDA), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), nicking-enzyme amplification reaction (NEAR), single primer isothermal amplification (SPIA), and other variations of exponential amplification, linear amplification, and cascade amplification. See Zhao et al., Chem Rev. 2015 and Gill, P.; Ghaemi, A. Nucleic Acid Isothermal Amplification Technologies—A Review. Nucleosides, Nucleotides and Nucleic Acids 2008, 27, 224-243 for additional discussion of these techniques.
The illustrated transduction reaction includes these components: (1) a target strand that contains subsequences P2, P1, P3 (listed from 3′ to 5′ orientation) as three adjacent primer binding sites; (2) a loaded primer “A”, that includes a primer nucleic acid strand with subsequences Ū and
In particular, step (a) includes the hybridization reaction between the target strand and the loaded primer “A” via the primer binding site P1 on the target strand. Step (b) includes the polymerization reaction initialized from the 3′ end of
Scheme (b) illustrates the input and output of the transduction reaction based on a second design of the loaded primer A. In this scheme, the loaded primer A consists of a primer nucleic acid strand with multiple repeats of the subsequence Ū and a single subsequence
Scheme (c) illustrates the input and output of the transduction reaction based on a third design of the loaded primer A. In this scheme, the loaded primer A consists of a primer nucleic acid strand with a single subsequence
In
The present example utilizes the ssDNA ‘U’ released from the transduction reaction as the F3 primer in the universal LAMP reaction. However, depending on implementation preference, the ssDNA ‘U’ may be designed to serve as any one of the essential LAMP primers for optimal assay performance.
In
Step (a) illustrates the hybridization reaction between the universal template and the FIP primer via the primer binding site F2c on the universal template; step (b) illustrates the polymerization reaction initialized from the 3′ end of F2 of the nucleic acid complex produced from step (a); step (c) illustrates the hybridization reaction between the ssDNA ‘U’ released from the transduction reaction and the nucleic acid complex produced from step (b) via the primer binding site U on the universal template; step (d) illustrates the strand-displacing polymerization reaction initialized from the 3′ end of Ū of the nucleic acid complex produced from step (c), where the displaced ssDNA forms a nucleic acid complex with a loop structure at its 5′ end; step (e) illustrates the hybridizations of the BIP primer and the B3 primer with the nucleic acid complex produced from step (d) via the primer binding site B2c and B3c on the complex, respectively; step (f) illustrates the strand-displacing polymerization reaction initialized from the 3′ end of B2 of the nucleic acid complex produced from step (e) and the strand-displacing polymerization reaction initialized from the 3′ end of B3 of the nucleic acid complex produced from step (e), where the displaced ssDNA forms a nucleic acid complex with the loop structure at both ends; step (g) illustrates the hybridizations of the FIP primer, the BIP primer, the LoopF primer, and the LoopB primer with the nucleic acid complex produced from step (f) via the primer binding site F2c, B2c, FLP, and BLP on the complex, respectively; step (h) indicates the LAMP's self-primed auto-cycling amplification facilitated by the strand-displacing polymerization reactions initialized from the 3′ end of FIP, the 3′ end of LoopB, the 3′ end of BIP, and the 3′ end of LoopF of the nucleic acid complex produced from step (g), respectively.
In the “Transduction Reaction Components,” (1) illustrates the sequence composition of the first target strand, which contains subsequences P2, P1, P3 (listed from 3′ to 5′ orientation) as three adjacent primer binding sites, and (2) illustrates the three primers for detection of the first target strand, including: a loaded primer that includes a primer nucleic acid strand with subsequences Ū and
Also under “Transduction Reaction Components,” (3) illustrates the sequence composition of the second target strand, which contains subsequences Q2, Q1, Q3 (listed from 3′ to 5′ orientation) as three adjacent primer binding sites, and (4) illustrates the three primers for detection of the second target strand, including: a loaded primer that includes a primer nucleic acid strand with subsequences Ū and
The “Universal LAMP Reaction Components” illustrates components of the universal LAMP reaction for detection of two distinct targets based on ‘Logic OR’: (1) is the sequence composition of a pre-designed universal template for a highly optimized LAMP reaction, containing subsequences Ū, F2c, F1c, B1, B2, B3 (listed from 3′ to 5′ orientation) as six primer binding sites required for standard LAMP reactions; (2) is the pre-optimized set of LAMP primers for rapid amplification of the universal template from (1), including the F3 primer including a primer nucleic acid strand U (provided by the transduction reaction), the B3 primer including primer nucleic acid strand B3, the FIP primer including a primer nucleic acid strand with subsequences F1c and F2 (listed from 5′ to 3′ orientation), the BIP primer including a primer nucleic acid strand with subsequences B1c and B2 (listed from 5′ to 3′ orientation), the LoopF primer including a primer nucleic acid strand FLPc, and the LoopB primer including a primer nucleic acid strand BLPc; and (3) is a strand-displacing polymerase.
Standard, known reagents such as reaction buffer and nuclease-free water are not depicted but may be included. In this example, the ssDNA ‘U’ released from the transduction reaction functions as the F3 primer in the universal LAMP reaction. Depending on the implementation preference, the ssDNA ‘U’ may be designed to serve as any one of the essential LAMP primers for optimal assay performance.
Also under “Transduction Reaction Components,” (3) illustrates the sequence composition of the second target strand, which contains subsequences Q2, Q1, Q3 (listed from 3′ to 5′ orientation) as three adjacent primer binding sites, and (4) illustrates the three primers for detection of the second target strand, including: a loaded primer that includes a primer nucleic acid strand with subsequences
The “Universal LAMP Reaction Components” illustrates the components of the universal LAMP reaction for detection of two distinct targets based on ‘Logic AND’: (1) is the sequence composition of a pre-designed universal template for a highly optimized LAMP reaction. The template contains subsequences
Standard, known reagents such as reaction buffer and nuclease-free water are not depicted but may be included. In this example, the ssDNA ‘U1’ and the ssDNA ‘U2’ released from the transduction reaction function as the F3 primer and the B3 primer for the downstream universal LAMP reaction, respectively. Depending on the implementation preference, the ssDNA ‘U1’ and ‘U2’ may be designed to serve as any pair of the essential LAMP primers for optimal assay performance.
Step (a) illustrates the hybridization reaction between the universal template and the FIP primer via the primer binding site F2c on the universal template; step (b) illustrates the polymerization reaction initialized from the 3′ end of F2 of the nucleic acid complex produced from step (a); step (c) illustrates the hybridization reaction between the ssDNA ‘U1’ released from the transduction reaction and the nucleic acid complex produced from step (b) via the primer binding site
The ‘Logic OR’ embodiment shown in
For a ‘Logic AND’ operation, up to six primer subsets may be included, each directed to a different nucleic acid target and each configured to generate a different LAMP primer in the presence of its respective target nucleic acid. Up to six of such primer subsets may be included because each of the potentially generated LAMP primers can correspond to one of the six LAMP primers of the universal LAMP template. Some embodiments may instead use up to four of such primer subsets, excluding the LoopF and LoopB primers so that they can instead be added directly to ensure their function of ‘speeding up’ the LAMP reaction is fully utilized.
Some embodiments may be configured for a ‘Logic OR’ determination in combination with a ‘Logic AND’ determination. For example, an embodiment may include a set of AND relationships with one or more nested OR relationships. As an example, a primer subset (a1) may be configured to generate a LAMP primer (A) in the presence of its nucleic acid target, and optionally one or more different primer subsets (a2, a3, a4, etc.) each also configured to generate the LAMP primer (A) in the presence of its respective nucleic acid target. Any number of additional (a)-type primer subsets may be included, each being configured to generate the LAMP primer (A) in the presence of its respective nucleic acid target. The embodiment can also include a primer subset (b1) configured to generate a different LAMP primer (B) in the presence of its nucleic acid target, and optionally one or more different primer subsets (b2, b3, b4, etc.) each also configured to generate the LAMP primer (B) in the presence of its respective nucleic acid target. Any number of additional (b)-type primer subsets may also be included, each being configured to generate the LAMP primer (B) in the presence of its respective nucleic acid target.
Such an embodiment incorporates AND logic because the LAMP reaction will only proceed if both the LAMP primer (A) and LAMP primer (B) are generated. The embodiment also incorporates OR logic because the LAMP primer (A) will be generated if at least one of the nucleic acid targets associated with the (a)-type primer subsets are present, and the LAMP (B) primer will be generated if at least one of the nucleic acid targets associated with the (b)-type primer subsets are present.
Some embodiments may incorporate additional AND relationships. For example, one or more (c)-type primer subsets may be included, each configured to generate a LAMP primer (C) in the presence of its respective nucleic acid target. The LAMP reaction will only proceed if each of LAMP primers (A), (B), and (C) are generated. As described above, up to six (or in some embodiments up to four) LAMP primers are generated in this manner, and each LAMP primer may optionally include multiple primer subsets with an OR relationship.
While certain embodiments of the present disclosure have been described in detail, with reference to specific configurations, parameters, components, elements, etcetera, the descriptions are illustrative and are not to be construed as limiting the scope of the claimed invention.
Furthermore, it should be understood that for any given element of component of a described embodiment, any of the possible alternatives listed for that element or component may generally be used individually or in combination with one another, unless implicitly or explicitly stated otherwise.
In addition, unless otherwise indicated, numbers expressing quantities, constituents, distances, or other measurements used in the specification and claims are to be understood as optionally being modified by the term “about” or its synonyms. When the terms “about,” “approximately,” “substantially,” or the like are used in conjunction with a stated amount, value, or condition, it may be taken to mean an amount, value or condition that deviates by less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the stated amount, value, or condition. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Any headings and subheadings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims.
It will also be noted that, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” do not exclude plural referents unless the context clearly dictates otherwise. Thus, for example, an embodiment referencing a singular referent (e.g., “widget”) may also include two or more such referents.
It will also be appreciated that embodiments described herein may include properties, features (e.g., ingredients, components, members, elements, parts, and/or portions) described in other embodiments described herein. Accordingly, the various features of a given embodiment can be combined with and/or incorporated into other embodiments of the present disclosure. Thus, disclosure of certain features relative to a specific embodiment of the present disclosure should not be construed as limiting application or inclusion of said features to the specific embodiment. Rather, it will be appreciated that other embodiments can also include such features.
This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/191,590, filed on May 21, 2021, which is incorporated herein by this reference in its entirety.
Number | Date | Country | |
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63191590 | May 2021 | US |