Detection Of Specific Sequences Research Articles

Engineering thermostable fluorescent DNA aptamer for the isothermal amplification of nucleic acids.

Isothermal amplification-based nucleic acid detection technologies have become rapid and efficient tools for molecular diagnostics. Sequence-specific monitoring methods are crucial for isothermal amplification, as they help identify the occurrence of extended primer dimers, which can lead to false positive results. Fluorescent aptamers are promising tools for real-time monitoring of isothermal amplification but are inherently limited by thermostability. Here, we report an engineered fluorescent DNA aptamer variant, named thermostable Lettuce (TS-Lettuce), with a 5°C higher melting temperature and 20 times greater fluorescence at 60°C, ideal for real-time monitoring of sequence-specific isothermal amplification. Using molecular dynamics simulations for structural analyses, we introduced mutations to wild-type Lettuce to redesign the non-core sequences of the aptamer structure for tightly stabilizing its folding, thereby enhancing thermostability. The TS-Lettuce offers greater versatility and ease of design for coupling with isothermal amplification for all-in-one nucleic acid detection. We demonstrated three applications of TS-Lettuce in isothermal amplification: fluorescent turn-off, fluorescent turn-on, and fluorescent aptamer switch, facilitating the sequence-specific detection of nucleic acids. In addition, the results generated by TS-Lettuce are visible to the naked eye, enhancing the utility of isothermal amplification reactions in resource-constrained areas. The thermostable fluorescent DNA aptamers can be further utilized in more isothermal amplification methods.

Simultaneous and Ultraspecific Optical Detection of Multiple miRNAs Using a Liquid Flow-Based Microfluidic Assay.

Recent studies have reported that the cause and progression of many diseases are closely related to complex and diverse gene regulation involving multiple microRNAs (miRNAs). However, most existing methods for miRNA detection typically deal with one sample at a time, which limits the achievement of high diagnostic accuracy for diseases associated with multiple gene dysregulations. Herein, we develop a liquid flow-based microfluidic optical assay for the simple and reliable detection of two different target miRNAs simultaneously at room temperature without any enzymatic reactions. This assay utilizes the catalytic hairpin assembly cycling reaction in a mixture containing four types of hairpin DNAs to amplify two different dimeric DNA probes, each of which specifically recognizes one of the two different target miRNAs. The resultant two dimeric DNA probes effectively hybridize with anchor DNA grafted into two outlet channels of a microfluidic device, thus enabling i-motif-driven compact DNA hydrogels to form in the channels under acidic conditions. With this setup, the presence of two target miRNAs can be confirmed by the naked-eye observation of red-colored gold nanoparticles encountering a flow blockage in the two outlet channels. Notably, the developed assay demonstrates sensitive and sequence-specific detection that can precisely distinguish a single base mismatch mutant miRNA within 1.5 h. Our assay thus has the potential to serve as a powerful sensing platform for the simple and simultaneous detection of multiple miRNAs in clinical diagnostics at room temperature without analytic equipment or enzymatic reactions.

Ion-Regulated Signal Amplification Optical Microfiber Interferometric DNA Sensor.

Genetic information sensors play a pivotal role in the biomedical field. The detection of deoxyribonucleic acid (DNA) is achieved experimentally using an optical microfiber interferometric sensor, which operates based on an ion-regulation sensitivity enhancement mechanism. The optical microfiber is fabricated by drawing optical fiber into a diameter of less than 10 μm via the melting and tapering technique. Leveraging the characteristics of monovalent cations can effectively promote the folding of G-rich single-stranded DNA (ssDNA) into stable G-quadruplex structures, enabling the detection of specific sequences of ssDNA at low concentrations. The results show an improvement of the linear detection range by 3 orders of magnitude, and with the introduction of the ion-regulation sensitivity enhancement mechanism, the limit of detection (LOD) value is 1.07 × 10-15 M. This optical microfiber interferometric sensing architecture is characterized by its simplicity and high sensitivity, positioning it as a formidable tool for diverse biosensing and analytical applications.

One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2.

Compared to quantitative real-time PCR (q-PCR), CRISPR-Cas-mediated technology is more suitable for point-of-care testing (POCT) and has potential for wider application in the future. Generally, the operational procedure of CRISPR-Cas-mediated diagnostic method consists of two independent steps, the reaction of signal amplification and the CRISPR-Cas-mediated signal detection. Complex multi-step procedures can easily lead to cross-contamination. To develop a convenient and rapid method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, we propose a MCTOP method (Multiple cross displacement amplification-CRISPR-Cas12b-based testing in one-pot), which targets the open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) gene of SARS-CoV-2. This method combines MCDA isothermal amplification and CRISPR-Cas-mediated sequence-specific detection into a one-pot reaction. The optimal reaction was achieved with isothermal amplification of 40 min and CRISPR-Cas-based detection of 15 min, both at 64°C. Then, the results can be visualized by the real-time fluorescence instrument and also lateral flow biosensor. The lowest detection limit of the proposed method is 10 copies of each of target sequences, and it has no cross-reactivity with non-SARS-CoV-2 templates. In a clinical test of 70 pharyngeal swab samples, MCTOP assay showed a specificity of 100% and sensitivities of 98 and 96% for the real-time fluorescence instrument and lateral flow biosensor, respectively. The MCTOP developed in this study is a rapid, convenient, highly sensitive, and specific method for SARS-CoV-2 nucleic acid detection. It can be used as an effective point-of-care testing (POCT) tool for clinical diagnosis and epidemiologic surveillance of SARS-CoV-2 infections, especially suitable for the basic, field and clinical laboratory.

Sensor Arrays for Electrochemical Detection of PCR-Amplified Genes Extracted from Cells Suspended in Environmental Waters.

Ecological surveys of living things based on DNAs from environmental samples are attractive. However, despite simplicity of water sampling from the target environment, it is still necessary to transport the samples to the laboratory for DNA analysis based on skillful next-generation sequencers. To perform DNA-oriented surveys based on a simple protocol without any special training, we demonstrated, in this study, the detection of genes from cell-containing environmental waters using gene sensor arrays that require no DNA labeling and no external indicators. Cell-suspended PBS or river water were used as models of environmental waters containing living things, and DNA samples were prepared by PCR amplification. Ferrocene-terminated probes were synthesized and immobilized on an electrode array to develop a sensor array. The sensor array showed a large response to a target DNA complementary to the probe and no response to a mismatched DNA, indicating sequence-specific detection. For DNA samples prepared from the cells in PBS, they showed good responses similar to those for the target DNA. They also significantly detected DNA samples from the cells in river water at a general environmental concentration (38 cells mL-1) with 28-fold larger responses than those for 0 cells mL-1.

Programmable DNA-based nanostructure sensors for mutation detection coupled with asymmetric amplification in precision genome profiling

We introduce a DNA-based nanostructure sensor for the rapid, precise identification of single-nucleotide polymorphisms (SNPs), a key factor in determining disease susceptibility and tailoring medical treatment. The proposed two-state sensor design facilitates multiplexed, sequence-specific detection of double-stranded genomic DNA (gDNA), overcoming traditional obstacles presented by base pairing interference. Utilizing detector strands with binding penalties, the sensor achieves high SNP specificity, discernible by distinct gel electrophoresis band positions. Coupled with asymmetric PCR (asy-PCR), our integrated system amplifies genetic profiling effectiveness, especially in clinical biopsies, enhancing gDNA detection sensitivity and specificity for concurrent multiple mutation identifications. This cost-effective method ($0.41 per test) streamlines mutation screening, enabling high-throughput analysis with limited equipment—requiring only a single staining agent and a unified electrophoresis apparatus. Validated clinically with 70 colorectal cancer samples, the system boasts a 98.57% sensitivity and 95.71% specificity for KRAS mutations, surpassing traditional PCR detection and uncovering low-frequency mutations untraceable by direct sequencing. Our findings suggest this asy-PCR enhanced DNA-based nanostructure sensor system has the potential to revolutionize genetic diagnostics, offering expansive applications in detecting viral variants and microbial strains, thus advancing personalized genetic testing.

Fluorescent sensing platform based on two-dimensional layered double hydroxides and exonuclease I for DNA and microRNA detection

Interfacial and interlayer interactions between layered double hydroxides (LDHs) and nucleic acids offer LDHs great potential to absorb and detect nucleic acids. Herein, we demonstrate a simple, rapid, and versatile fluorescent sensing platform based on two-dimensional LDHs and exonuclease I (Exo I) for the selective analysis of DNA and microRNA. ZnNiAl-LDHs were utilized as quenchers for FAM labelled on single-stranded DNA (ssDNA) probes via dynamic quenching with high fluorescence quenching ability. The quenching mechanism was explored by means of spectral analysis, characterization experiments, DNA adsorption and desorption studies, zeta potential analysis, and molecular dynamics simulations. The driving forces for the adsorption of LDHs toward fluorophore-labelled nucleic acids were confirmed to be electrostatic interaction, van der Waals force, and anion exchange for the first time. In order to distinguish the formed target/probe duplexes from FAM-ssDNA probes, Exo I was introduced to specifically hydrolyze FAM-ssDNA probes. The fabricated fluorescent sensing platform for nucleic acids detection shows a low detection limit of 0.08 nM. Finally, this sensor was effectively utilized for the detection of DNA and microRNA in actual biological and environmental samples, achieving recoveries ranging from 96.4 % to 105 %, indicating high accuracy. This work proposed a promising sensing platform for sequence-specific nucleic acids detection, and provides a new perspective on two-dimensional LDHs as structural supports and biomolecular reservoirs for biomedical applications.

Ligation-driven hyperbranched DNA assembly for label-free and sequence-specific measurement of RNA N6-methyladenosine in human breast tissues

N6-methyladenosine (m6A) is a critical post-transcriptional regulator, and it is linked to various biological functions (e.g., degradation, export, and translation) and multiple human diseases (e.g., cancers, immune disorders, and neurodegenerative diseases). The current methods for sequence-specific detection of m6A usually suffer from radioactive risk, large sample consumption, washing/separation steps, and false-positive signal. Herein, we develop a ligation-driven hyperbranched DNA assembly-based fluorescent biosensor for label-free and sequence-specific detection of m6A. In this assay, the unmethylated RNA can be identified and cleaved by m6A-sensitive endoribonuclease MazF. The retained intact m6A-RNA is complementary to the padlock probe to form a circular DNA, subsequently initiating ligation-driven hyperbranched rolling circle amplification (HRCA). The obtained HRCA products can be simply quantified with SYBR Gold as a fluorescent indicator. This method can be homogeneously carried out in an isothermal, label-free and antibody-free manner. The limit of detection can reach 0.079 pM. Moreover, this method can differentiate even 0.01 % m6A level in excess coexisting interferences, monitor cellular m6A RNA level with single-cell sensitivity, and differentiate m6A level in breast tumor tissues and normal adjacent tissues, with promising applications in m6A-releated epigenetic research and clinical diagnosis.

Multiplexing LAMP Assays: A Methodological Review and Diagnostic Application.

The loop-mediated isothermal amplification (LAMP) technique is a great alternative to PCR-based methods, as it is fast, easy to use and works with high sensitivity and specificity without the need for expensive instruments. However, one of the limitations of LAMP is difficulty in achieving the simultaneous detection of several targets in a single tube, as the methodologies that allow this rely on fluorogenic probes containing specific target sequences, complicating their adaptation and the optimization of assays. Here, we summarize different methods for the development of multiplex LAMP assays based on sequence-specific detection, illustrated with a schematic representation of the technique, and evaluate their practical application based on the real-time detection and quantification of results, the possibility to visualize the results at a glance, the prior stabilization of reaction components, promoting the point-of-care use, the maximum number of specific targets amplified, and the validation of the technique in clinical samples. The various LAMP multiplexing methodologies differ in their operating conditions and mechanism. Each methodology has its advantages and disadvantages, and the choice among them will depend on specific application interests.

Sulfur incorporation into nucleic acids accelerates enzymatic activity

DNAzymes play a crucial role in biosensors for signal detection, but specific structures are required for diagnostic applications, complicating their design. This study reports a novel approach to the development of DNAzymes by incorporating sulfur atoms into nucleic acids via phosphorothioate (PS) bonds. Unlike traditional DNAzymes that rely on specific structures such as G-quadruplexes, this approach leads to high enzymatic activity without structural constraints. Computational analysis reveals that the change in the electron density of the nucleobases due to PS modification enhances interactions within the DNAzyme-H2O2-hemin complex, accelerating the rate-determining step and improving enzymatic activity. Systematic guidelines for the development of non-sequence constrained DNAzymes are provided by investigating the effect of the number of PS modifications, the length of the DNA, and various nucleobase combinations. α-thio-dNTP, a monomer containing PS, exhibits no observable enzymatic activity, but enzymatic activity is recorded for single-stranded DNA (ssDNA) containing PS. However, when the ssDNA is transformed into double-stranded DNA (dsDNA), the bases that react with hemin are blocked by hydrogen bonding, reducing enzymatic activity. An enzymogenic signaling system that differentiates between ssDNA and dsDNA is subsequently developed for sequence-specific colorimetric detection, demonstrating significant promise for overcoming the limitations of conventional DNAzymes in molecular diagnosis.

Bioluminescent Intercalating Dyes for Ratiometric Nucleic Acid Detection.

Rapid and sensitive DNA detection methods that can be conducted at the point of need may aid in disease diagnosis and monitoring. However, translation of current assays has proven challenging, as they typically require specialized equipment or probe-specific modifications for every new target DNA. Here, we present Luminescent Multivalent Intercalating Dye (LUMID), off-the-shelf bioluminescent sensors consisting of intercalating dyes conjugated to a NanoLuc luciferase, which allow for nonspecific detection of double-stranded DNA through a blue-to-green color change. Through the incorporation of multiple, tandem-arranged dyes separated by positively charged linkers, DNA-binding affinities were improved by over 2 orders of magnitude, detecting nanomolar DNA concentrations with an 8-fold change in green/blue ratio. We show that LUMID is easily combined with loop-mediated isothermal amplification (LAMP), enabling sequence-specific detection of viral DNA with attomolar sensitivity and a smartphone-based readout. With LUMID, we have thus developed a tool for simple and sensitive DNA detection that is particularly attractive for point-of-need applications.

DNA-Aptamer-Based qPCR Using Light-Up Dyes for the Detection of Nucleic Acids.

Quantitative polymerase chain reaction (qPCR) is widely used in detection of nucleic acids, but existing methods either lack sequence-specific detection or are costly because they use chemically modified DNA probes. In this work, we apply a DNA aptamer and light-up dye-based chemistry for qPCR for nucleic acid quantification. In contrast to the conventional qPCR, in our method, we observe an exponential decrease in fluorescence upon DNA amplification. The qPCR method we developed produced consistent Ct vs log10 (DNA amount) standard curves, which have a linearfit with R2 value > 0.99. This qPCR technique was validated by quantifying gene targets from Streptococcus zooepidemicus (SzhasB) and Mycobacterium tuberculosis (MtrpoB). We show that our strategy is able to successfully detect DNA at as low as 800 copies/μL. To the best of our knowledge, this is the first study demonstrating the application of light-up dyes and DNA aptamers in qPCR.

Curcumin-capped gold nanorods as optical sensing platform for sequence specific detection of DNA based on their self-assembly

We are reporting curcumin-induced gold nanorods as an optical sensing platform for the detection of sequence-specific DNA target through their self-assembly. The combined effect of eco-friendly reducing agent (i.e., curcumin) and silver nitrate in a basic medium (i.e., pH 10) has been attributed for the formation of small gold nanorods (AuNRs) having approximate length and diameter i.e., 19.7 ± 0.8 nm and 6.0 ± 0.5 nm, respectively, and lower longitudinal surface plasmon resonance (SPR) enable to detect and analyse different biomarkers. Further, for evaluating cellular uptake of as-synthesized AuNRs, the cytotoxicity study has been carried out by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay on A549 cells and HEPG2 cell lines, respectively, and shown approximately similar cytotoxicity. Interestingly, as-synthesized optically and electronically active AuNRs based nanobiosensing platform enable to detect sequence-specific DNA targets with low level of detection limit i.e., LOD 8.6 ± 0.15 pM for complimentary target (CT) DNA with higher sensitivity and better selectivity. Finally, this study is suggesting a simplistic bio-mediated approach of tuning the shape and size of AuNRs for sensitive, selective and reliable nanobiosensing platform for sequence-specific DNA detection related to cancer cells.

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue DEAtC.

Sequence-specific fluorescent probes for RNA are widely used in microscopy applications such as fluorescence in situ hybridization and a growing number of newer approaches to live-cell RNA imaging. The sequence specificity of most of these approaches relies on differential hybridization of the probe to the correct target. Competing sequences with only one or two base mismatches are prone to causing off-target recognition. Here, we report the sequence-specific fluorescent detection of model RNA targets using a tricyclic cytidine analogue DEAtC that is included as a surrogate for natural cytidine in DNA probe strands and that reports directly on Watson-Crick base pairing. The DEAtC-containing DNA oligonucleotide probes exhibit an average 8-fold increase in fluorescence intensity when hybridized to matched RNA with DEAtC base paired with G and little fluorescence turn-on when DEAtC is base paired with A. Duplex structure determination by NMR, time-resolved fluorescence studies, and Stern-Volmer quenching experiments suggest that the combination of greater π stacking and narrower grooves in the A-form DNA-RNA heteroduplex provides additional shielding and favorable electronic interactions between bases, explaining why DEAtC's fluorescence turn-on response to RNA targets is typically 3-fold greater than for DNA targets.

Rational Design of Profile HMMs for Sensitive and Specific Sequence Detection with Case Studies Applied to Viruses, Bacteriophages, and Casposons.

Profile hidden Markov models (HMMs) are a powerful way of modeling biological sequence diversity and constitute a very sensitive approach to detecting divergent sequences. Here, we report the development of protocols for the rational design of profile HMMs. These methods were implemented on TABAJARA, a program that can be used to either detect all biological sequences of a group or discriminate specific groups of sequences. By calculating position-specific information scores along a multiple sequence alignment, TABAJARA automatically identifies the most informative sequence motifs and uses them to construct profile HMMs. As a proof-of-principle, we applied TABAJARA to generate profile HMMs for the detection and classification of two viral groups presenting different evolutionary rates: bacteriophages of the Microviridae family and viruses of the Flavivirus genus. We obtained conserved models for the generic detection of any Microviridae or Flavivirus sequence, and profile HMMs that can specifically discriminate Microviridae subfamilies or Flavivirus species. In another application, we constructed Cas1 endonuclease-derived profile HMMs that can discriminate CRISPRs and casposons, two evolutionarily related transposable elements. We believe that the protocols described here, and implemented on TABAJARA, constitute a generic toolbox for generating profile HMMs for the highly sensitive and specific detection of sequence classes.

Electroactive hydrolysis probe-based portable PCR platform for sequence-specific detection of nontyphoidal Salmonella drug resistance gene

Etaq-PCR, a portable real-time polymerase chain reaction platform, has been developed based on an electroactive hydrolysis probe design and immobilization-free electrochemical readout. The Etaq-PCR platform integrates cost effective commercial instruments and thermally stable cycling into a minimal device enabling real-time monitoring of PCR amplification. No complex electrode surface treatment or micro-fabrication is involved in the construction of the Etaq-PCR platform. Thus, one can easily construct a portable PCR platform via our design. Etaq-PCR achieved sufficient sensitivity, selectivity, and rapidness to meet the WHO ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid, and robust, equipment-free, and deliverable) for point-of-care diagnosis systems. It showed good tolerance to clinical sample matrix as it displayed 100% accuracy in a validation experiment testing a drug resistance gene of nontyphoidalSalmonella in 25 raw clinical isolated samples in comparison with the gold standard qPCR. All in all, Etaq-PCR is a portable, deployable, and cost-effective PCR platform suitable for clinical applications.

An amplification-free, 16S rRNA test for Neisseria gonorrhoeae in urine.

An amplification-free, nanopore-based nucleic acid detection platform has been demonstrated for rapid, 16S rRNA sequence-specific detection of Neisseria gonorrhoeae at 10-100 CFU mL-1 in human urine against background bacterial flora at 1000 CFU mL-1. Gonorrhea is a very common notifiable communicable disease, antibiotic resistant strains have emerged, and the rate of reported gonococcal infections continues to increase. Since rapid clinical identification of bacterial pathogens in clinical samples is needed to guide proper antibiotic treatment and to control disease spread, it is important to engineer rapid, sensitive, selective, and inexpensive point-of-care (POC) diagnostic devices for pathogens such as N. gonorrhoeae. Our detector technology is based on straightforward conductometric detection of sustained blockage of a glass nanopore. Charge neutral, complementary peptide nucleic acid probes are conjugated to polystyrene beads to capture N. gonorrhoeae 16S rRNA selectively. In the presence of an electric field applied externally through a glass nanopore, the PNA-microbead conjugates that acquire substantial negative charge upon target hybridization are driven to the smaller diameter nanopore. At least partial blockage of the nanopore results in a sustained drop in ionic current that can be measured easily with simple electronics. The ability to detect N. gonorrhoeae over the range of 10 to 100 CFU mL-1 spiked in human urine was demonstrated successfully with estimated sensitivity and specificity of ∼98% and ∼100%, respectively. No false positives were observed for the control group of representative background flora (E. coli, K. pneumoniae, and E. faecalis) at 1000 CFU mL-1. Also, N. gonorrhoeae at 50 CFU mL-1 was successfully detected against 1000 CFU mL-1 of background flora in urine. These results suggest that this amplification-free technology may serve as the basis for rapid, inexpensive, low-power detection of pathogens in clinical samples at the POC.

An Ultrasensitive PCR-Based CRISPR-Cas13a Method for the Detection of Helicobacter pylori.

The rapid and simple detection of Helicobacter pylori (H. pylori) is essential for its clinical eradication. Although various methods for detecting H. pylori have been well established, such as endoscopy in combination with histology or culture, rapid urease test (RUT) and molecular tests using clinical specimens, it is of great importance to develop an ultrasensitive and accurate nucleic acid detection platform and apply it to identify H. pylori. To meet these demands, a novel method based on PCR and CRISPR-Cas13a, called PCR-Cas13a, was developed and validated using the DNA of 84 clinical strains and 71 clinical specimens. PCR primers for the pre-amplification of conservative sequence and CRISPR RNA (crRNA) for the detection of specific sequence were designed according to the principle. The designed primers and crRNA were specific to H. pylori, and the assay showed a high degree of specificity compared with other common pathogens. Our detection system can screen H. pylori with a limit of 2.2 copies/μL within 30 mins after PCR amplification. Using a coincidence analysis with traditional methods, our method exhibited 100% accuracy for the detection of H. pylori. Furthermore, its diagnostic performance was compared, in parallel with a q-PCR. The PCR-Cas13a demonstrates 98% sensitivity and 100% specificity. Moreover, our approach had a lower limit of detection (LOD) than q-PCR. Herein, we present a diagnostic system for the highly sensitive screening of H. pylori and distinguish it from other pathogens. All the results demonstrated that this PCR-based CRISPR assay has wide application prospects for the detection of H. pylori and other slow-growth pathogens.

Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic

Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we repurpose the type III-A CRISPR complex from Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex generates two cyclic oligoadenylates (i.e., cA3 and cA4) that allosterically activate ancillary nucleases. We show that both Can1 and Can2 nucleases cleave single-stranded RNA, single-stranded DNA, and double-stranded DNA in the presence of cA4. We integrate the Can2 nuclease with type III-A RNA capture and concentration for direct detection of SARS-CoV-2 RNA in nasopharyngeal swabs with 15 fM sensitivity. Collectively, this work demonstrates how type-III CRISPR-based RNA capture and concentration simultaneously increases sensitivity, limits time to result, lowers cost of the assay, eliminates solvents used for RNA extraction, and reduces sample handling.

Efficient multiplexing and variant discrimination in reverse-transcription loop-mediated isothermal amplification with sequence-specific hybridization probes.

Loop-mediated isothermal amplification (LAMP) has proven a robust and reliable nucleic acid amplification method that is well suited for simplified and rapid molecular diagnostics. Various approaches have emerged for sequence-specific detection of LAMP products, but with limitations to their widespread utility or applicability for single-nucleotide polymorphism detection and multiplexing. Here we demonstrate the use of simple hybridization probes (as used for qPCR) that enable simple multiplexing and SARS-CoV-2 variant typing in reverse-transcription LAMP. This approach requires no modification to the LAMP primers and is amenable to the detection of single-nucleotide polymorphisms and small sequence changes, which is usually difficult in LAMP. By extending LAMP's ability to be utilized for multitarget and single-base change detection, we hope to increase its potential to enable more and better molecular diagnostic testing.