Reverse Transcription Loop-mediated Isothermal Amplification Research Articles

Application and Optimization of Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid SARS-CoV-2 Detection in Humans and Animals

Application and Optimization of Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid SARS-CoV-2 Detection in Humans and Animals

Development of colorimetric one-step reverse transcription loop-mediated isothermal amplification for rapid and visualized detection of tomato necrotic ringspot virus

Tomato necrotic ringspot virus (TNRV) stands out as one of the most destructive viruses affecting chili and tomato crops in Thailand. TNRV causes typical necrotic and chlorotic ringspot symptoms on both leaves and fruits. This study focused on the development of a colorimetric one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid and visualized virus detection in different host plants, including chili, tomato, and physalis, a weed found near tomato fields. The new set of LAMP primers was designed based on the sequence of the non-structural (NSs) protein and nucleocapsid (N) protein genes of TNRV. The assay was optimized to identify the optimum isothermal temperature and incubation period. Positive reactions were identified by a color change from pink to yellow, while negative reactions remained pink where the assay worked well at 65°C for 30 min. Specificity and sensitivity assays were conducted. The results revealed that the optimal incubation time for detecting LAMP products, along with colorimetric changes, was 30 min at 65°C. The specificity assay demonstrated where LAMP primers did not cross-react with other orthotospoviruses like capsicum chlorosis virus (CaCV), melon yellow spot virus (MYSV) and watermelon silver mottle virus (WSMoV). The sensitivity assay indicated that RT-LAMP detected TNRV from RNA diluted up to 10–7 (1.0 fg/μL). Evaluation of the colorimetric one-step RT-LAMP assay on naturally infected tomato, chili, and physalis samples confirmed its successful detection of TNRV.

Point-of-care test of blood Plasmodium RNA within a Pasteur pipette using a novel isothermal amplification without nucleic acid purification

BackgroundResource-limited regions face a greater burden of infectious diseases due to limited access to molecular tests, complicating timely diagnosis and management. Current molecular point-of-care tests (POCTs) either come with high costs or lack adequate sensitivity and specificity. To facilitate better prevention and control of infectious diseases in underserved areas, we seek to address the need for molecular POCTs that better align with the World Health Organization (WHO)’s ASSURED criteria—Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable to end users.MethodsA novel molecular POCT, Pasteur Pipette-assisted isothermal probe amplification (pp-IPA), was developed for malaria detection. Without any microfluidics, this method captures Plasmodium 18S rRNA in a modified Pasteur pipette using tailed genus-specific probes. After washing, the bound tailed probes are ligated to form a template for subsequent novel isothermal probe amplification using a pair of generic primers, bypassing nucleic acid extraction and reverse transcription. The method was assessed using cultured Plasmodium and compared with real-time quantitative reverse transcription PCR (RT-qPCR) or reverse transcription loop-mediated isothermal amplification (RT-LAMP) in clinical blood samples.ResultsThe entire assay is completed in 60–80 min with minimal hands-on time, using only a Pasteur pipette and a water bath. The pp-IPA’s analytical sensitivity is 1.28 × 10–4 parasites/μl, with 100% specificity against various blood-borne pathogens causing malaria-like symptoms. Additionally, pp-IPA needs only liquid-transfer skill for operation and the cost is around USD 0.25 per test, making it at least 300 times lower than mainstream POCT platforms.ConclusionsDesigned to improve the accessibility of molecular detection in resource-limited settings, pp-IPA’s simplicity, affordability, high sensitivity/specificity, and minimal equipment requirements make it a promising point-of-care pathogen identification tool in resource-constrained regions.Graphical

Towards the identification of body fluids using RT-LAMP isothermal amplification coupled with CRISPR-Cas12a

While often necessary in sexual assault cases, confirmatory identification of body fluids can be a lengthy and/or costly process. In particular, the detection of vaginal fluid and menstrual fluid in forensic casework is limited to endpoint reverse-transcription PCR to detect fluid-specific messenger RNA (mRNA) markers as there are no robust chemical or enzymatic techniques available for these fluids. Similarly, testing for rectal mucosa is not possible with standard methods, the presence of which would provide probative value in cases of alleged anal penetration, although mRNA-based markers have recently been described. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative technique that enables detection of mRNA at a single temperature (usually 60–65℃) for 10–30 minutes and has comparable sensitivity to PCR. We describe the coupling of RT-LAMP amplification (60℃ for 30 minutes) with CRISPR-mediated fluorescent detection of the body fluid specific mRNA markers MMP3 (menstrual fluid), CYP2B7P (vaginal material), TNP1 (spermatozoa), KLK2 (semen), and MUC12 (rectal mucosa). Following temperature optimization and final selection of RT-LAMP-CRISPR assays, their specificity across circulatory blood, buccal, menstrual fluid, vaginal material, semen, and rectal mucosa was assessed. Most assays were specific for their intended target body fluid, although MMP3 and CYP2B7P were detected in some rectal mucosa samples, the latter of which has been observed previously in the literature. A preliminary sensitivity assessment in target fluids was determined by a dilution series over six logs of RNA input. A range of assay approaches were investigated to develop a protocol suitable for use in a forensic screening laboratory. This included the determination of fluorescent assay results by eye, use of lyophilised reagents, and RT-LAMP and CRISPR reactions undertaken in one-tube in a lower resource setting.

CRISPR/Cas12 System-Based Assay for Rapid, Sensitive Detection of Rotavirus in Food Samples.

Foodborne viruses have become an important threat to food safety and human health. Among the foodborne viruses, group A rotavirus is the most important pathogen of diarrhea in autumn and winter. The field detection of rotavirus is crucial for the early control of infection and patient management. Quantitative real-time reverse transcription-polymerase chain reaction is the most widely used in virus detection. However, the technique relies on high-cost instruments and trained personnel, which limit its use in field detection. In this study, we developed accurate, realizable, and simple detection methods by combining optimized CRISPR (clustered regularly interspaced short palindromic repeats) Cas12 and reverse transcription loop-mediated isothermal amplification (RT-LAMP) (reverse transcription loop-mediated isothermal amplification) to reduce the requirements for temperature control and costly real-time fluorescence polymerase chain reaction instruments. We investigated two nucleic acid detection systems combining RT-LAMP with CRISPR Cas12a and RT-LAMP with CRISPR Cas12b and compared them with reverse transcription-quantitative polymerase chain reaction. The resulting detection system only needs a reaction temperature and in single tube to react for 60 min with the detection sensitivity of 38 copies/μL. Overall, this study developed an innovative method for the rapid detection of rotavirus in food samples, which will help to effectively identify food contaminated by pathogens and prevent human infections and economic losses caused by disease outbreaks.

Real-Time Visualization of HIV-1 RNA Detection Using Loop-Mediated Isothermal Amplification-Enabled Particle Diffusometry.

Isothermal nucleic acid amplification tests, NAATs, such as reverse transcription-loop-mediated isothermal amplification (RT-LAMP), offer promising capabilities to perform real-time semiquantitative detection of viral pathogens. These tests provide rapid results, utilize simple instrumentation for single-temperature reactions, support efficient user workflows, and are suitable for field use. Herein, we present a novel and robust method for real-time monitoring of HIV-1 RNA RT-LAMP utilizing a novel implementation of particle diffusometry (PD), a diffusivity quantification technique using fluorescent particles, to quantify viral concentration in nuclease-free water. We monitor changes in particle diffusion dynamics of 400 nm fluorescently labeled particles throughout the RT-LAMP of HIV-1 RNA in nuclease-free water, enabling measurement within 20 min and detection of concentrations as low as 25 virus particles per μL. Moreover, in a single-blind study, we demonstrate semiquantitative detection by accurately determining the initial concentration of an unknown HIV-1 RNA within a 10% absolute error margin. These results highlight the potential of real-time PD readout for quantifying HIV-1 RNA via RT-LAMP, offering promise for viral load monitoring of HIV and other chronic infections.

Comparing Typing Methods for Foot-and-Mouth Disease Virus Serotypes: Sandwich ELISA, Multiplex PCR, RT-LAMP and SYBR Green Real-time PCR

Background: Foot and Mouth Disease (FMD) poses a significant economic threat to the cattle population in Assam, with recurrent outbreaks occurring annually. Methods: This study aimed to compare different diagnostic techniques for the molecular detection and serotyping of Foot and Mouth Disease Virus (FMDV) outbreaks in the region. Epithelial tissue samples (n=29) were collected following standard procedures. SYBR Green real-time PCR targeting the 3D gene, sandwich enzyme-linked immunosorbent assay (S-ELISA), multiplex PCR (mPCR), and reverse transcription loop-mediated isothermal amplification (RT-LAMP) were utilized for detection and serotyping. Results: All 29 tissue samples tested positive for FMDV using SYBR Green real-time PCR, mPCR, and RT-LAMP, with 100% congruence in serotyping (20 serotype O, 9 serotype A). In contrast, S-ELISA detected only 86.21% of samples as positive (17 serotype O, 8 serotype A). Statistical analysis revealed significant differences between the sensitivity of S-ELISA and molecular techniques (p = 2.91×10-7).The findings underscore the superior sensitivity of SYBR Green real-time PCR, mPCR, and RT-LAMP, highlighting their potential for enhancing FMDV surveillance and control efforts in Assam.

Development and Validation of a Highly Sensitive RT-qLAMP Assay for Rapid Detection of SARS-CoV-2: Methodological Aspects.

Specific and reliable diagnostic methods are becoming increasingly essential to identify patients in light of the high transmission rate and the recent appearance of the new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For the specific detection of SARS-CoV-2, our quantitative reverse transcription loop-mediated isothermal amplification (RT-qLAMP) assay implementation demonstrates how flexible it can be with two readouts: visualized colorimetric and real-time fluorescence. Different factors were optimized to improve the reaction conditions, including temperature (60°C), assay runtime (60min), primers, MgSO4 (6mM), dNTPs (1mM), LAMP Buffer (1.2mM Tris-HCl), KCl (50mM), pH (8), and phenol red (10mM) concentrations. Regarding analytical sensitivity, the colorimetric RT-LAMP method detected samples with Ct values up to 29, while the RT-qLAMP assay identified up to Ct = 31. RT-qLAMP was evaluated on 40 clinical samples (25 positives and 15 negatives) for viral RNA detection. All negative samples were found negative through fluorescent reading in RT-qLAMP and quantitative reverse transcription PCR (RT-qPCR) assays. Twenty-three clinically positive samples demonstrated a positive RT-qLAMP reaction (up to Ct ≤ 31) with 92% clinical sensitivity, 100% clinical specificity, 100% positive predictive value (PPV), 88.24% negative predictive values (NPV), and 95% accuracy.

Optimization of Reverse Transcription Loop-Mediated Isothermal Amplification for In Situ Detection of SARS-CoV-2 in a Micro-Air-Filtration Device Format.

The Coronavirus disease 2019 (COVID-19) pandemic has supercharged innovation in the field of molecular diagnostics and led to the exploration of systems that permit the autonomous identification of airborne infectious agents. Airborne virus detection is an emerging approach for determining exposure risk, although current methods limit intervention timeliness. Here, we explore reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for one-pot detection of Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) (SCV2) run on membrane filters suitable for micro-air-filtration of airborne viruses. We use a design of experiments statistical framework to establish the optimal additive composition for running RT-LAMP on membrane filters. Using SCV2 liquid spike-in experiments and fluorescence detection, we show that single-pot RT-LAMP on glass fiber filters reliably detected 0.10 50% tissue culture infectious dose (TCID50) SCV2 per reaction (3600 E-gene copies) and is an order of magnitude more sensitive than conventional RT-LAMP.

A colorimetric reverse-transcription loop-mediated isothermal amplification method targeting the L452R mutation to detect the Delta variant of SARS-CoV-2

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered global difficulties for both individuals and economies, with new variants continuing to emerge. The Delta variant of SARS-CoV-2 remains most prevalent worldwide, and it affects the efficacy of coronavirus disease 2019 (COVID-19) vaccination. Expedited testing to detect the Delta variant of SARS-CoV-2 and monitor viral transmission is necessary. This study aimed to develop and evaluate a colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technique targeting the L452R mutation in the S gene for the specific detection of the Delta variant. In the test, positivity was indicated as a color change from purple to yellow. The assay’s 95% limit of detection was 57 copies per reaction for the L452R (U1355G)-specific standard plasmid. Using 126 clinical samples, our assay displayed 100% specificity, 97.06% sensitivity, and 98.41% accuracy in identifying the Delta variant of SARS-CoV-2 compared to real-time RT-PCR. To our knowledge, this is the first colorimetric RT-LAMP assay that can differentiate the Delta variant from its generic SARS-CoV-2, enabling it as an approach for studying COVID-19 demography and facilitating proper effective control measure establishment to fight against the reemerging variants of SARS-CoV-2 in the future.

Development of a visual detection method of porcine deltacoronavirus using loop-mediated isothermal amplification.

The emergence of porcine deltacoronavirus (PDCoV) presents a significant threat to both human and animal health due to its ability to cause highly contagious enteric diseases. This underscores the crucial need for timely and accurate diagnosis to facilitate effective epidemiological investigation and clinical management. This research aimed to establish a visual detection method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for PDCoV testing. In this study, six pairs of primers were designed according to the conserved sequences of PDCoV ORF1a/b genes. The primer sets and parameters that affect LAMP reaction were optimized. The visual RT-LAMP method was developed by incorporating methyl red into the optimized reaction system, it exclusively detected PDCoV without cross-reactivity with other viruses and the detection limits for PDCoV could reach 10 copies/μL. In comparison with RT-PCR for testing 132 clinical samples, the relative specificity and sensitivity of the visual RT-LAMP were found to be 99.2 and 100%, respectively, with a concordance rate of 99.2% and a kappa value of 0.959, indicating that the visual RT-LAMP is a reliable method for the application of PDCoV detection in clinical samples.

An automated syringe-based PoC RT-LAMP LFB platform for infectious disease detection from saliva

Decentralized Point-of-Care (PoC) diagnostics hold momentous potential for rapid and accessible viral infection disease detection. Presented is a unique design application of an easy-to-use (plug-and-play) platform for viral detection. The platform leverages a simplified multiplex Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Lateral Flow Biosensor (LFB) assay with a lyophilized master mix, eliminating the need for RNA isolation or special reporting equipment. A user-friendly Saliva Measuring Tube (SMT) ensures accurate saliva volume self-collection, and a Syringe-based PoC (SPoC) platform automates sample treatment, reagent mixing, and temperature control using readily available components and consumables. The platform’s performance was evaluated by multiplexed detection of the SARS-CoV-2 N2 target gene and human ACTB gene from saliva samples. The SPoC platform achieved a detection limit of spiked 500 copies/mL for SARS-CoV-2 and consistent internal control readout. The presented PoC system offers a promising initial step for further development toward a decentralized solution for viral infection testing.

Establishment of a Serology- and Molecular-Combined Detection System for Youcai Mosaic Virus and Its Application in Various Host Plants

The youcai mosaic virus (YoMV) can infect a diverse array of crop species, such as Raphanus sativus, Brassica napus, Solanum nigrum, and Rehmannia glutinosa, causing substantial economic damage. This study aimed to develop a rapid, sensitive, and economical diagnostic method for YoMV. We successfully expressed and purified the recombinant His-CPYoMV-YZ protein in E. coli BL21, which was used to immunize New Zealand White rabbits, generating high-titer polyclonal antibodies (PAb-CPYoMV-YZ). Additionally, a serological-based reverse transcription loop-mediated isothermal amplification (S-RT-LAMP) assay was refined, combining serological and molecular detection techniques to enhance practicality. Utilizing PAb-CPYoMV-YZ, we developed four techniques for detecting YoMV: Western blot, dot immunoblotting assay, enzyme-linked immunosorbent assay (ELISA), and S-RT-LAMP. YoMV isolates from various regions and hosts were analyzed. The results indicated that PAb-CPYoMV-YZ was highly effective in detecting YoMV across a range of hosts and isolates from diverse regions. This study fills an important gap in the serological detection of YoMV and provides a practical tool for on-site diagnosis and control of YoMV infection.

Comparison of PCR, Nested PCR, and RT-LAMP for Rapid Detection of Feline Calicivirus Infection in Clinical Samples.

Feline calicivirus (FCV) is a highly contagious virus that causes upper respiratory tract disease, commonly known as cat flu. It is widely distributed worldwide and poses a major threat to feline health. Therefore, it is essential to find an efficient and rapid method for detecting FCV. In this study, the colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay, using neutral red as an indicator, was developed and validated to target the ORF2 gene of FCV for the first time. Additionally, the study compared the diagnostic abilities of polymerase chain reaction (PCR), nested PCR, and RT-LAMP assays for detecting FCV in clinical samples. The optimized RT-LAMP amplification was carried out at 56.3 °C. The technique visually detected FCV within 70 min, with a limit of detection of 14.3 × 101 copies/µL, and showed no cross-reactivity with other feline pathogens. Out of 54 oropharyngeal swab samples, 17 tested positive for FCV using both nested PCR and RT-LAMP, while only one tested positive using conventional PCR. The positivity rate was higher with nested PCR and RT-LAMP (31.48%) compared to conventional PCR (1.85%). Consequently, these results demonstrated the effectiveness of the colorimetric RT-LAMP assay developed in this study as an alternative for diagnosing FCV in cats.

Point-of-Care Multiplex Detection of Respiratory Viruses.

The COVID-19 pandemic, in addition to the co-occurrence of influenza virus and respiratory syncytial virus (RSV), has emphasized the requirement for efficient and reliable multiplex diagnostic methods for respiratory infections. While existing multiplex detection techniques are based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) and extraction and purification kits, the need for complex instrumentation and elevated cost limit their scalability and availability. In this study, we have developed a point-of-care (POC) device based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that can simultaneously detect four respiratory viruses (SARS-CoV-2, Influenza A, Influenza B, and RSV) and perform two controls in less than 30 min, while avoiding the use of the RNA extraction kit. The system includes a disposable microfluidic cartridge with mechanical components that automate sample processing, with a low-cost and portable optical reader and a smartphone app to record and analyze fluorescent images. The application as a real point-of-care platform was validated using swabs spiked with virus particles in nasal fluid. Our portable diagnostic system accurately detects viral RNA specific to respiratory pathogens, enabling deconvolution of coinfection information. The detection limits for each virus were determined using virus particles spiked in chemical lysis buffer. Our POC device has the potential to be adapted for the detection of new pathogens and a wide range of viruses by modifying the primer sequences. This work highlights an alternative approach for multiple respiratory virus diagnostics that is well-suited for healthcare systems in resource-limited settings or at home.

Rapid, Simultaneous Detection of Various Biological Toxin Genes Using Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification(RT-LAMP)

Rapid, early, accurate detection and identification of the various pathogenic agents associated with the development of biological weapons is critical in preventing loss of life and limiting the impact of these organisms when used against civilian or military targets. The aim of this study was to produce a system for the simple, rapid, accurate and simultaneous detection and identification of Ricin, Botulinum toxin B and Staphylococcal enterotoxin B as a proof of principle for developing field appropriate reverse transcription loop-mediated isothermal amplification systems for the accurate identification of potential biological threats. These systems were designed to facilitate the identification of potential threats even in remote or resource-limited locations.

Aptamer-associated colorimetric reverse transcription loop-mediated isothermal amplification assay for detection of dengue virus.

Dengue is a neglected tropical disease of significant epidemiological importance in tropical and subtropical countries. Current diagnostics for this infection present challenges, such as cross-reactivity in serological tests. Finding ways to enhance the diagnosis of this disease is crucial, given the absence of specific treatments. An accurate, simple, and effective diagnosis contributes to the improved management of infected individuals. In this context, our work combines molecular biology techniques, such as isothermal loop amplification, with aptamers to detect the dengue virus in biological samples. Our method produces colorimetric results based on a color change, with outcomes available in less than 2 hours. Moreover, it requires simpler equipment compared to molecular PCR tests.

Enhanced Point-of-Care SARS-CoV-2 Detection: Integrating RT-LAMP with Microscanning.

The COVID-19 pandemic has highlighted the urgent need for rapid and accurate diagnostic methods for various infectious diseases, including SARS-CoV-2. Traditional RT-PCR methods, while highly sensitive and specific, require complex equipment and skilled personnel. In response, we developed an integrated RT-LAMP-MS assay, which combines rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) with microscanning (MS) technology for detecting SARS-CoV-2. The assay uses magnesium pyrophosphate formed during LAMP amplification as a visual marker, allowing direct observation via microscopy without the need for additional chemical indicators or probes. For the SARS-CoV-2/IC RT-LAMP-MS assay, the sample-LAMP reagent mixture was added to a microchip with SARS-CoV-2 primers and internal controls, then incubated at 62 °C for 30 min in a heat block, followed by amplification analysis using a microscanner. In clinical tests, the RT-LAMP-MS assay showed 99% sensitivity and 100% specificity, which is identical to the RT-LAMP results and comparable to the commercial AllplexTM SARS-CoV-2 assay results. Additionally, the limit of detection (LOD) was determined to be 10-1 PFU mL-1 (dynamic range: 103~10-1 PFU mL-1). The assay delivers results in 30 min, uses low-cost equipment, and demonstrates 100% reproducibility in repeated tests, making it suitable for point-of-care use in resource-limited settings.

Reverse transcriptase loop-mediated isothermal amplification assisted with localized surface plasmon resonance of gold nanorods for viability assessment of Lactobacillus acidophilus in yogurt

In light of the growing production and consumption of functional foods, it is crucial to develop new methods to assess the authenticity of these products and prevent fraud and mislabeling. In this research, by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and the localized surface plasmon resonance (LSPR) effect of gold nanorods (GNRs), the viability of the probiotic bacteria has been evaluated. The RT-LAMP-GNR method effectively amplifies and identifies bacterial 16S-rRNA within 60 min. The efficiency of the RT-LAMP method was assessed by its implementation using RNA isolated from Lactobacillus acidophilus strain (L. acidophilus) and commercial probiotic yogurts. The method's limit of detection (LoD) was 104 bacterial cells per ml, and its sensitivity was 10−5 ng/ml RNA.

Double domain fusion improves the reverse transcriptase activity and inhibitor tolerance of Bst DNA polymerase

To enhance the DNA/RNA amplification efficiency and inhibitor tolerance of Bst DNA polymerase, four chimeric Bst DNA polymerase by fusing with a DNA-binding protein Sto7d and/or a highly hydrophobic protein Hp47 to Bst DNA polymerase large fragment. One of chimeric protein HpStBL exhibited highest inhibitor tolerance, which retained high active under 0.1 U/μL sodium heparin, 0.8 ng/μL humic acid, 2.5× SYBR Green I, 8 % (v/v) whole blood, 20 % (v/v) tissue, and 2.5 % (v/v) stool. Meanwhile, HpStBL showed highest sensitivity (93.75 %) to crude whole blood infected with the African swine fever virus. Moreover, HpStBL showed excellent reverse transcriptase activity in reverse transcription loop-mediated isothermal amplification, which could successfully detect 0.5 pg/μL severe acute respiratory syndrome coronavirus 2 RNA in the presence of 1 % (v/v) stools. The fusion of two domains with different functions to Bst DNA polymerase would be an effective strategy to improve Bst DNA polymerase performance in direct loop-mediated isothermal amplification and reverse transcription loop-mediated isothermal amplification detection, and HpStBL would be a promising DNA polymerase for direct African swine fever virus/severe acute respiratory syndrome coronavirus 2 detection due to simultaneously increased inhibitor tolerance and reverse transcriptase activity.