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Loop-mediated isothermal amplification (LAMP), a newly developed gene amplification method, combines rapidity, simplicity, and high specificity. Several tests have been developed based on this method, and simplicity is maintained throughout all steps, from extraction of nucleic acids to detection of amplification. In the LAMP reaction, samples are amplified at a fixed temperature through a repetition of two types of elongation reactions occurring at the loop regions: self-elongation of templates from the stem loop structure formed at the 3′-terminal and the binding and elongation of new primers to the loop region. The LAMP reaction has a wide range of possible applications, including point-of-care testing, genetic testing in resource-poor settings (such as in developing countries), and rapid testing of food products and environmental samples.

Background Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. Results Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in “single pot” reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. Conclusions LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.

The technique of loop-mediated isothermal amplification (LAMP) utilizes 4 (or 6) primers targeting 6 (or 8) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. In this study, a set of LAMP primers were designed targeting the prfA gene sequence of Listeria monocytogenes, and dimethyl sulfoxide (DMSO) as well as Touchdown LAMP were employed to increase the sensitivity and specificity of the LAMP reactions. The results indicate that the detection limit of this novel LAMP assay with the newly designed primers and additives was 10 fg per reaction, which is ten-fold more sensitive than a commercial Isothermal Amplification Kit and hundred-fold more sensitive than previously reported LAMP assays. This highly sensitive LAMP assay has been shown to detect 11 strains of Listeria monocytogenes, and does not detect other Listeria species (including Listeria innocua and Listeria invanovii), providing some advantages in specificity over commercial Isothermal Amplification Kits and previously reported LAMP assays by Tang, et al.

Fluorescence real-time LAMP assays were designed for the orf43 gene of CyHV-3 European genotype and the p4a gene of the CEV genogroup I. A third LAMP assay to detect the ef1a gene of the host common carp was designed as an internal control. The limit of detection was 102 and 103 viral copies under 25 min for CyHV-3 and CEV, respectively. The specificity of the CyHV-3 LAMP assay was 95.6% of 72 fish herpesviruses tested. Sixty-three non-lethal common carp mucus swabs were collected across 16 sites during disease investigations. DNA extractions were performed in under 10 min using the QuickExtract™ digestion buffer. The LAMP amplification of CyHV-3 DNA in mucus swabs from clinical cases was detected from 4 to 13 min in 13 sites, while a co-infection of CyHV-3 and CEV was confirmed by LAMP in a single site. The LAMP results agreed with the results of the reference laboratory. The common carp ef1a was amplified only in 61% of the mucus swabs collected, preventing its use as a robust internal control to distinguish false negatives from invalid tests. After further optimization, these tests could be implemented for border inspection posts surveillance and decentralizing testing during disease outbreaks.

The degree of detrimental effects inflicted on mankind by the COVID-19 pandemic increased the need to develop ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) POCT (point of care testing) to overcome the current and any future pandemics. Much effort in research and development is currently advancing the progress to overcome the diagnostic pressure built up by emerging new pathogens. LAMP (loop-mediated isothermal amplification) is a well-researched isothermal technique for specific nucleic acid amplification which can be combined with a highly sensitive immunochromatographic readout via lateral flow assays (LFA). Here we discuss LAMP-LFA robustness, sensitivity, and specificity for SARS-CoV-2 N-gene detection in cDNA and clinical swab-extracted RNA samples. The LFA readout is designed to produce highly specific results by incorporation of biotin and FITC labels to 11-dUTP and LF (loop forming forward) primer, respectively. The LAMP-LFA assay was established using cDNA for N-gene with an accuracy of 95.65%. To validate the study, 82 SARS-CoV-2-positive RNA samples were tested. Reverse transcriptase (RT)-LAMP-LFA was positive for the RNA samples with an accuracy of 81.66%; SARS-CoV-2 viral RNA was detected by RT-LAMP-LFA for as low as CT-33. Our method reduced the detection time to 15 min and indicates therefore that RT-LAMP in combination with LFA represents a promising nucleic acid biosensing POCT platform that combines with smartphone based semi-quantitative data analysis.

The recent outbreak of Zika virus (ZIKV) in the Americas and its devastating developmental and neurological manifestations has prompted the development of field-based diagnostics that are rapid, reliable, handheld, specific, sensitive, and inexpensive. The gold standard molecular method for lab-based diagnosis of ZIKV, from either patient samples or insect vectors, is reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The method, however, is costly and requires lab-based equipment and expertise, which severely limits its use as a point-of-care (POC) tool in resource-poor settings. Moreover, given the lack of antivirals or approved vaccines for ZIKV infection, a POC diagnostic test is urgently needed for the early detection of new outbreaks and to adequately manage patients. Loop-mediated isothermal amplification (LAMP) is a compelling alternative to RT-qPCR for ZIKV and other arboviruses. This low-cost molecular system can be freeze-dried for distribution and exhibits high specificity, sensitivity, and efficiency. A growing body of evidence suggests that LAMP assays can provide greater accessibility to much-needed diagnostics for ZIKV infections, especially in developing countries where the ZIKV is now endemic. This review summarizes the different LAMP methods that have been developed for the virus and summarizes their features, advantages, and limitations.

Airborne animal viral pathogens can rapidly spread and become a global threat, resulting in substantial socioeconomic and health consequences. To prevent and control potential epidemic outbreaks, accurate, fast, and affordable point-of-care (POC) tests are essential. As a proof-of-concept, we have developed a molecular system based on the loop-mediated isothermal amplification (LAMP) technique for avian metapneumovirus (aMPV) detection, an airborne communicable agent mainly infecting turkeys and chickens. For this purpose, a colorimetric system was obtained by coupling the LAMP technique with specific DNA-functionalized AuNPs (gold nanoparticles). The system was validated using 50 different samples (pharyngeal swabs and tracheal tissue) collected from aMPV-infected and non-infected chickens and turkeys. Viral detection can be achieved in about 60 min with the naked eye, with 100% specificity and 87.88% sensitivity for aMPV. In summary, this novel molecular detection system allows suitable virus testing in the field, with accuracy and limit of detection (LOD) values highly close to qRT-PCR-based diagnosis. Furthermore, this system can be easily scalable to a platform for the detection of other viruses, addressing the current gap in the availability of POC tests for viral detection in poultry farming. Key points •aMPV diagnosis using RT-LAMP is achieved with high sensitivity and specificity. •Fifty field samples have been visualized using DNA-nanoprobe validation. •The developed system is a reliable, fast, and cost-effective option for POCT. Graphical Abstract

Loop-mediated isothermal amplification (LAMP) is a widely used method for clinical diagnosis, customs quarantine, and disease prevention. However, the low catalytic activity of Bst DNA polymerase has made it challenging to develop rapid and reliable point-of-care testing. Herein, we developed a series of Bst DNA polymerase mutants with enhanced activity by predicting and analyzing the activity sites. Among these mutants, single mutants K431D and K431E showed a 1.93- and 2.03-fold increase in catalytic efficiency, respectively. We also created a chimeric protein by fusing the DNA-binding domain of DNA ligase from Pyrococcus abyssi (DBD), namely DBD-K431E, which enabled real-time LAMP at high temperatures up to 73 ℃ and remained active after heating at 70 ℃ for 8 h. The chimeric DBD-K431E remained active in the presence of 50 U/mL heparin, 10% ethanol, and up to 100 mM NaCl, and showed higher activity in 110 mM (NH4)2SO4, 110 mM KCl, and 12 mM MgSO4. Notably, it generated a fluorescence signal during the detection of Salmonella typhimurium at 2 × 102 ag/μL of genomic DNA and 1.24 CFU/mL of bacterial colony, outperforming the wild type and the commercial counterpart Bst 2.0. Our results suggest that the DBD-K431E variant could be a promising tool for general molecular biology research and clinical diagnostics.Key points• Residue K431 is probably a key site of Bst DNA polymerase activity• The chimeric DBD-K431E is more inhibitor tolerant and thermostable than Bst-LF• The DBD-K431E variant can detect Salmonella typhimurium at 102 ag/μL or 100 CFU/mL

ABSTRACT Sexually transmitted infections (STIs) remain a significant public health concern. Given the asymptomatic nature of many STIs, diagnostic testing is critical for determining the appropriate treatment, enabling effective tracing and reducing the risk of further transmission. Nucleic acid amplification tests (NAATs) are the most sensitive and the most widely used in well‐resourced settings. The majority of available NAATs are based on polymerase chain reaction (PCR), which requires highly trained personnel and costly equipment, making it impractical for resource‐limited settings. Loop‐mediated isothermal amplification (LAMP) has emerged as a simple, rapid, sensitive and low‐cost alternative for pathogen detection, particularly well‐suited for point‐of‐care tests (POCT). In this review, we evaluate LAMP assays reported in the literature for the detection of pathogens linked to the high incidence STIs prioritised by the World Health Organization (WHO) for POCT in 2023. These include Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, T. pallidum subspecies pallidum, as well as other common STIs such as herpes simplex virus, hepatitis B virus and human immunodeficiency virus (HIV). For each LAMP assay, we identified and summarised the key elements such as the type and number of tested clinical specimens, chosen target gene, detection system, reference test and clinical outcomes. We highlight the advantages and limitations of these assays and discuss the gaps that should be addressed to improve their applicability for POCT. LAMP assay for point‐of‐care tests.

Early and quick detection of pathogens are crucial for managing the spread of infections in the biomedical, biosafety, food, and agricultural fields. While molecular diagnostics can offer the specificity and reliability in acute infectious diseases, detection of pathogens is often slowed down by the current benchtop molecular diagnoses, which are time consuming, labor intensive, and lack the mobility for application at the point-of-need. In this work, we developed a complete on-farm use detection protocol for the plant-devastating anthracnose agent: Colletotrichum gloeosporioides. Our methods combined a simplified DNA extraction on paper that is compatible with loop-mediated isothermal amplification (LAMP), coupled with paper-based immunoassay lateral flow sensing. Our results offer simple, quick, easy, and a minimally instrumented toolkit for Colletotrichum gloeosporioides detection. This scalable and adaptable platform is a valuable alternative to traditional sensing systems towards on-the-go pathogen detection in food and agriculture, biomedical, and other fields.