Loop-mediated Isothermal DNA Amplification Research Articles

Use of the 23S rRNA gene as a target template in the universal loop-mediated isothermal amplification (LAMP) of genomic DNA from phytoplasmas.

Plant-pathogenic bacteria cause numerous diseases in host plants and can result in serious damage. Timely and accurate diagnostic techniques are, therefore, crucial. While advances in molecular techniques have led to diagnostic systems able to distinguish known plant pathogens at the species or strain level, systems covering larger categories are mostly lacking. In this study, a specific and universal LAMP-based diagnostic system was developed for phytoplasmas, a large group of insect-borne plant-pathogenic bacteria that cause significant agricultural losses worldwide. Targeting the 23S rRNA gene of phytoplasma, the newly designed primer set CaPU23S-4 detected 31 'Candidatus Phytoplasma' tested within 30 min. This primer set also showed high specificity, without false-positive results for other bacteria (including close relatives of phytoplasmas) or healthy plants. The detection sensitivity was ~10,000 times higher than that of PCR methods for phytoplasma detection. A simple, rapid method of DNA extraction, by boiling phytoplasma-infected tissues, was developed as well. When used together with the universal LAMP assay, it enabled the prompt and accurate detection of phytoplasmas from plants and insects. The results demonstrate the potential of the 23S rRNA gene as a versatile target for the LAMP-based universal detection of bacteria at the genus level and provide a novel avenue for exploring this gene as molecular marker for phytoplasma presence detection.IMPORTANCEPhytoplasmas are associated with economically important diseases in crops worldwide, including lethal yellowing of coconut palm, "flavescence dorée" and "bois noir" of grapevine, X-disease in stone fruits, and white leaf and grassy shoot in sugarcane. Numerous LAMP-based diagnostic assays, mostly targeting the 16S rRNA gene, have been reported for phytoplasmas. However, these assays can only detect a limited number of 'Candidatus Phytoplasma' species, whereas the genus includes at least 50 of these species. In this study, a universal, specific, and rapid diagnostic system was developed that can detect all provisionally classified phytoplasmas within 1 h by combining the LAMP technique targeting the 23S rRNA gene with a simple method for DNA extraction. This diagnostic system will facilitate the on-site detection of phytoplasmas and may aid in the discovery of new phytoplasma-associated diseases and putative insect vectors, irrespective of the availability of infrastructure and experimental resources.

Optimization of the molecular detection assay of Salmonella (MDS®) for drag swab samples of aviaries.

Detection of Salmonella sp. is important for the broiler chicken production chain because it is one microorganisms involved in food-borne diseases. Thus, this study performed the optimization of a technique of Loop-mediated isothermal DNA amplification (LAMP) through the 3MTM Molecular Detection Assay 2: Salmonella (MDS®), in accordance with Ordinance number 126 of the Ministry of Agriculture, for the detection of Salmonella sp. in drag swab. The methodology followed ISO 16140-2: 2016, with the analysis naturally contaminated drag swab samples collected from broiler aviaries and artificially contaminated with salmonella ATCCs. Of the 300 samples processed in protocol A (pre-enrichment tetrathionate broth (TT)), 45 were positive for Salmonella sp., 242 negative, one false-positive, and 12 false-negative, while of the 300 samples analyzed in protocol B (pre-enrichment brain-heart infusion broth (BHI)), 40 were positive, 256 negative, one false-positive, and three false-negative. The result for protocol A was a sensitivity of 79%, specificity of 99.6%, Positive Predictive Value (PPV) of 98%, and Negative Predictive Value (NPV) of 95%; and for protocol B, 93% sensitivity, 99.6% specificity, 98% PPV, and 99% NPV. Both protocols were associated with the reference method (p>0.05), concluding that the MDS® can be used for the qualitative detection of Salmonella sp.

Loop-Mediated Isothermal Amplification of DNA (LAMP) as an Alternative Method for Determining Bacteria in Wound Infections.

The increasing number of patients with chronic wounds requires the development of quick and accurate diagnostics methods. One of the key and challenging aspects of treating ulcers is to control wound infection. Early detection of infection is essential for the application of suitable treatment methods, such as systemic antibiotics or other antimicrobial agents. Clinically, the most frequently used method for detecting microorganisms in wounds is through a swab and culture on appropriate media. This test has major limitations, such as the long bacterial growth time and the selectivity of bacterial growth. This article presents an overview of molecular methods for detecting bacteria in wounds, including real-time polymerase chain reaction (rtPCR), quantitative polymerase chain reaction (qPCR), genotyping, next-generation sequencing (NGS), and loop-mediated isothermal amplification (LAMP). We focus on the LAMP method, which has not yet been widely used to detect bacteria in wounds, but it is an interesting alternative to conventional detection methods. LAMP does not require additional complicated equipment and provides the fastest detection time for microorganisms (approx. 30 min reaction). It also allows the use of many pairs of primers in one reaction and determination of up to 15 organisms in one sample. Isothermal amplification of DNA is currently the easiest and most economical method for microbial detection in wound infection. Direct visualization of the reaction with dyes, along with omitting DNA isolation, has increased the potential use of this method.

1329. Pediatric Group A Streptococcus (GAS) Pharyngitis Incidence has Decreased During the COVID-19 Pandemic

Abstract Background The COVID-19 pandemic has affected respiratory disease epidemiology. Measures to reduce transmission of SARS-CoV-2 (e.g., masking, social distancing, school closures) have reduced the incidence of common infectious diseases. At least one early report described changes in rates of pediatric GAS pharyngitis. We sought to determine the difference in GAS pharyngitis rates before and after the onset of the pandemic at our children’s hospital. Methods Clinical pharyngeal specimens tested for GAS by rapid antigen tests (RADTs) or LAMP methodology (loop-mediated isothermal amplification of GAS DNA) from January 2019 to March 2022 at our children’s hospital were analyzed. Clinician’s evaluating patients for GAS pharyngitis perform a RADT, when positive the patient is treated for GAS pharyngitis. When the RADT is negative, a LAMP test is performed. Patients were considered test-positive if either test was positive regardless of clinical symptoms. Annual positivity rates were compared using statistical process control (SPC) charts with both 2019 and 2020 as baseline years (i.e., 2019 vs 2020-3/2022; 2020 vs 2021-3/2022). The change in GAS incidence rate (positive patients/1000 tested) between years was assessed by chi-square tests. Results 9,896 patients had throat swabs tested for GAS from January 2019 through March 2022. Using SPC with 2019 as a baseline, there was a 15.1% decrease in mean GAS positivity through 3/2022. With 2020 as baseline, there was a 13.0% decrease in mean GAS positivity through 3/2022. The incidence of positive GAS tests decreased significantly: 2019 vs. 2020-3/2022 (x2 = 84.97, p ≤0.00001) and 2020 vs. 2021-3/2022 (x2 = 154.01, p ≤0.00001). Figure 1. Conclusion Using throat swab results, the incidence of pediatric GAS pharyngitis at our hospital has decreased significantly during the COVID-19 pandemic. Implications: Pandemic-related risk mitigation measures, such as masking, social distancing, and school closures, were associated with significantly decreased incidence of GAS pharyngitis. It is likely that GAS colonization and invasive infections were also affected. Disclosures All Authors: No reported disclosures.

Improvements to the Rapid Detection of the Marine Pathogenic Bacterium, Vibrio harveyi, Using Loop-Mediated Isothermal Amplification (LAMP) in Combination with SYBR Green.

The common methods that are presently used to identify Vibrio harveyi include microscopic examination and biochemical, immunological and PCR-based assays. These methods require technical expertise, and can be time-consuming. A rapid method is required for the high-throughput screening of large number of samples. As such, we have developed a rapid, simple yet sensitive and specific detection method based on the use of the loop-mediated isothermal amplification (LAMP) of DNA. A set of six primers, i.e., two outer, two inner and two loop primers, was designed based on the in silico analysis of a large pool of 39 strains of the toxR gene sequence of V. harveyi. The addition of the loop primers decreased the reaction time of the LAMP by more than half. Furthermore, with the application of SYBR Green, the result can be obtained as quickly as in 10 to 15 min without the need of gel electrophoresis. The specificity of the method primers was then determined by performing LAMP with Vibrio and non-Vibrio samples. LAMP has a greater sensitivity than PCR reaction. The sensitivity of PCR was at 0.6 pg concentration of V. harveyi recombinant plasmid DNA standard, while LAMP was able to detect lower amounts even at 0.6 fg. The development of the LAMP assay will provide a valuable tool for the high-throughput rapid detection of V. harveyi contamination both in laboratories and in the field.

Electrochemical DNA biosensor coupled to LAMP reaction for early diagnostics of cervical precancerous lesions

Electrochemical (EC) biosensors are potentially useful tools for detection of DNA and RNA cancer biomarkers. Gold screen-printed electrodes (AuSPEs) are especially popular due to their easy manufacturing and surface modifications, making them useful candidates in biosensor fabrication. On the other hand, biosensors based on AuSPEs are still not often applied to clinical samples from cancer patients or compared with routine laboratory methods. Therefore, we tried to address these challenges by developing AuSPE-based biosensor for detection of DNA from two highly oncogenic types of human papillomavirus (HPV), HPV16 and HPV18, that cause cervical cancer in women. We coupled loop-mediated isothermal amplification (LAMP) of HPV DNA from fifteen clinical samples, with the capture of amplified products at the surface of the AuSPE and with final EC detection in a multielectrode format. We show excellent selectivity of designed primers and probes by comparing HPV-positive and HPV-negative cancer cell lines, and most importantly, very good sensitivity and specificity of the proposed biosensor for detection of clinical samples when compared to the PCR used as a gold standard.

Paper functionalization for detection of Plasmodium falciparum DNA using square waves voltammetry

Malaria elimination is a major goal to be reached in the next decade. Significant progress were made in the past, and the prevalence decreased in many areas while the positive trend stalled in the last years. The exact number of asymptomatic carriers of Plasmodium parasites is unknown since this population is not detected by conventional diagnosis methods and participate in the maintenance of transmission. Molecular methods to detect low parasitemia are not available at point-of-care in low-income countries of malaria endemic areas. Adaptation of molecular methods such as loop-mediated isothermal amplification of DNA may provide effective tools but it required simplification of DNA detection. Square waves voltammetry, easily imbedded in small device such as cell phone, was largely described for DNA detection but support for reaction was an issue to address. Here we used an efficient functionalization method of paper-based material to facilitate the interactions between isothermal amplification product and methylene blue for easy-to-use DNA detection. The proof-of-concept of qualitative detection of very low parasitemia from malaria infected patients using newly chemically treated paper for square waves voltammetry was obtained with a sensitivity and specificity of 100% and a limit-of-detection of 0.1 parasite. μL−1 corresponding to a parasitemia of 0.000002%.

Real-time fluorescence and colorimetric identification of bulbus fritillariae using recombinase assisted loop-mediated isothermal DNA amplification (RALA).

Bulbus Fritillariae (BF) is a kind of herbal medicine widely used in many countries including China, Japan, Korea, and so on. Among the known BF medicinal materials, Bulbus Fritillariae cirrhosae (BF cirrhosae) was reported to have the best curative effect. Due to the limited resources of BF cirrhosae, a lot of adulterants have emerged in the market, impairing the market order, resource development, and above all, clinical efficacy. Here, a novel nucleic acid amplification technique, Recombinase Assisted Loop-mediated isothermal DNA Amplification (RALA), was used to establish a real-time fluorescence isothermal molecular authentication method for five commonly used BF drugs. Moreover, this RALA-based assay can also be developed as a colorimetric detection method for on-site detection. Both real-time fluorescence and visual methods could detect as low as 0.1% genuine targets in the mixed samples. In summary, we report an isothermal detection system for five kinds of BF circulating in the market, providing a new choice for the molecular identification of BF drugs and showing promise in the laboratory testing as well as field identification of other herbal medicines.

A Loop-Mediated Isothermal DNA Amplification (LAMP) Assay for Detection of the Clubroot Pathogen Plasmodiophora brassicae.

Clubroot caused by Plasmodiophora brassicae is a serious threat to cruciferous crops around the world. The resting spores of P. brassicae are a primary source of infection and can survive in soil for many years. Detection of resting spores in soil is essential for forecasting clubroot prevalence. Detection of P. brassicae has been relying on plant bioassays or PCR-based methods. The loop-mediated isothermal DNA amplification (LAMP) is a promising approach for microorganism detection with the advantage of high sensitivity, accuracy, and convenience in viewing. In this study, we developed a LAMP assay for detection of P. brassicae in soil, roots, and seeds. This method can detect P. brassicae at a minimal amount of 1 fg of plasmid DNA or 10 resting spores in the soil. Compared with conventional PCR, the LAMP was more sensitive in detection of P. brassicae at the lower levels in soil samples. In conclusion, we elaborated a sensitive, accurate, and easy-to-use LAMP assay to detect P. brassicae, which will facilitate sustainable clubroot management and planning.

Digital Microfluidics-Powered Real-Time Monitoring of Isothermal DNA Amplification of Cancer Biomarker.

We introduce a digital microfluidics (DMF) platform specifically designed to perform a loop-mediated isothermal amplification (LAMP) of DNA and applied it to a real-time amplification to monitor a cancer biomarker, c-Myc (associated to 40% of all human tumors), using fluorescence microscopy. We demonstrate the full manipulation of the sample and reagents on the DMF platform, resulting in the successful amplification of 90 pg of the target DNA (0.5 ng/µL) in less than one hour. Furthermore, we test the efficiency of an innovative mixing strategy in DMF by employing two mixing methodologies onto the DMF droplets—low frequency AC (alternating current) actuation as well as back-and-forth droplet motion—which allows for improved fluorescence readouts. Fluorophore bleaching effects are minimized through on-chip sample partitioning by DMF processes and sequential droplet irradiation. Finally, LAMP reactions require only 2 µL volume droplets, which represents a 10-fold volume reduction in comparison to benchtop LAMP.

Rapid Molecular Diagnostics in the Field and Laboratory to Detect Plant Pathogen DNA in Potential Insect Vectors.

A variety of sensitive and specific molecular diagnostic assays has been described for detecting nucleic acids in biological samples that may harbor pathogens of interest. These methods include very rapid, isothermal nucleic acid amplification methods that can be deployed outside of the laboratory environment, such as loop-mediated isothermal DNA amplification (LAMP) and recombinase-polymerase amplification (RPA). However, all molecular diagnostic assays must be preceded by nucleic acid extraction from the biological samples of interest, which provides suitable template molecules for the assays. To exploit the features of the amplification assays and be utilized outside of the lab, these methods must be rapid and avoid the need for typical laboratory chemicals and equipment. We describe a protocol for the extraction of DNA from field-collected insects that can be implemented at the point of collection and used to detect the presence of DNA sequences from potential plant pathogens that may be vectored by the insects. This protocol provides template DNA that is suitable for PCR, LAMP, and RPA. The FTA PlantSaver card-based DNA extraction product was also confirmed to amplify the mitochondrial cytochrome oxidase 1 (CO1) universal barcode that could later be sequenced to identify any insect. Lastly, we provide an example using field-collected insects, Neokolla (Graphocephala) heiroglyphica, and demonstrate the detection of the plant pathogen Xylella fastidiosa in carrier insects using PCR, RPA, and LAMP.

Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?

In 1998, when the PCR technique was already popular, a Japanese company called Eiken Chemical Co., Ltd. designed a method known as the loop-mediated isothermal amplification of DNA (LAMP). The method can produce up to 109 copies of the amplified DNA within less than an hour. It is also highly specific due to the use of two to three pairs of primers (internal, external, and loop), which recognise up to eight specific locations on the DNA or RNA targets. Furthermore, the Bst DNA polymerase most used in LAMP shows a high strand displacement activity, which eliminates the DNA denaturation stage. One of the most significant advantages of LAMP is that it can be conducted at a stable temperature, for instance, in a dry block heater or an incubator. The products of LAMP can be detected much faster than in standard techniques, sometimes only requiring analysis with the naked eye. The following overview highlights the usefulness of LAMP and its effectiveness in various fields; it also considers the superiority of LAMP over PCR and presents RT-LAMP as a rapid diagnostic tool for SARS-CoV-2.

Thermal analysis of loop mediated isothermal DNA amplification (LAMP) based point-of-care diagnostic device

Loop-Mediated Isothermal Amplification reaction or LAMP has been identified as a feasible DNA amplification process. Paper-based LAMP diagnostic devices offer several advantages for point-of-care (POC) applications. LAMP occurs at a constant temperature of higher limits, which remains a major challenge on-site. This calls for an efficient thermal design which is beneficial in maintaining a constant high temperature at POC locations. The present study reports thermal design and energy consumption analysis of a paper-based LAMP POC device for the first time using CFD analysis. The POC design consists of a rectangular porous membrane embedded on a rectangular solid material, heated from the side walls. Isothermal and discrete heating with different horizontal and vertical clearance configurations are used as a design parameter. CFD analysis of different thermal configurations is carried out in COMSOL Multiphysics platform to solve the energy and convection–diffusion-reaction equation in porous media. The thermal analysis demonstrated that two discrete heating configurations with high horizontal clearance are effective in reducing energy consumption. It was inferred that two portable AA batteries are sufficient for the POC device operation. Overall, the current work successfully demonstrates the energy optimization and speed of operation of the POC medical device by detailed thermal analysis.

Selection of a suitable paper membrane for Loop Mediated Isothermal DNA amplification reaction (LAMP) in a point-of-care diagnostic kit – Experimental and CFD analysis

Paper-based point-of-care (POC) devices have attracted tremendous attention because of low-cost, simple fabrication, and portability. LAMP-based POC platform comprises a paper substrate on which the DNA amplification reaction occurs. The quality of the paper substrate plays a critical role in performing an efficient LAMP reaction and for effective visualization of the amplification result. The major objective of the current work is to replace the frequently utilized nitrocellulose (NC) membrane by a cost-effective and high quality paper membrane viz. Whatman filter paper (WF) for rapid detection of E. coli via LAMP reaction. Isolated DNA is added on the paper membranes, which undergo LAMP reaction. The effect of initial DNA concentration and time on LAMP amplification reaction rate is analyzed on paper membranes via image analysis for the first time. Overall study inferred that WF is suitable for effective naked-eye detection and greater extent of LAMP reaction (generation of 19 968 DNA copies).

Evaluation of highly sensitive diagnostic tools for the detection of P. falciparum in pregnant women attending antenatal care visits in Colombia

BackgroundIn low transmission settings early diagnosis is the main strategy to reduce adverse outcomes of malaria in pregnancy; however, microscopy and rapid diagnostic tests (RDTs) are inadequate for detecting low-density infections. We studied the performance of the highly sensitive-RDT (hsRDT) and the loop mediated isothermal DNA amplification (LAMP) for the detection of P. falciparum in pregnant women.MethodsA cross-sectional study was conducted in two malaria-endemic municipalities in Colombia. We screened pregnant women in the context of an antenatal care program in health facilities and evaluated five tests (microscopy, conventional RDT, hsRDT, LAMP and nested polymerase chain reaction-PCR) for the detection of P. falciparum in peripheral blood, using a quantitative reverse transcription PCR (qRT-PCR) as the reference standard. Diagnostic performance of hsRDT and LAMP were compared with routine testing.ResultsThe prevalence of P. falciparum was 4.5% by qRT-PCR, half of those infections were subpatent. The sensitivity of the hsRDT (64.1%) was slightly better compared to microscopy and cRDT (59 and 53.8% respectively). LAMP had the highest sensitivity (89.7%) for detecting P. falciparum and the ability to detect very low-density infections (minimum parasite density detected 0.08 p/μL).ConclusionsThere is an underestimation of Plasmodium spp. infections by tests routinely used in pregnant women attending antenatal care visits. LAMP methodology can be successfully implemented at local hospitals in malaria-endemic areas. The relevance of detecting and treating this sub-patent P. falciparum infections in pregnant women should be evaluated.Trial registrationClinicalTrials.gov, Identifier: NCT03172221, Date of registration: May 29, 2017.

A Dual-Sensing Thermo-Chemical ISFET Array for DNA-Based Diagnostics.

This paper presents a 32×32 ISFET array with in-pixel dual-sensing and programmability targeted for on-chip DNA amplification detection. The pixel architecture provides thermal and chemical sensing by encoding temperature and ion activity in a single output PWM, modulating its frequency and its duty cycle respectively. Each pixel is composed of an ISFET-based differential linear OTA and a 2-stage sawtooth oscillator. The operating point and characteristic response of the pixel can be programmed, enabling trapped charge compensation and enhancing the versatility and adaptability of the architecture. Fabricated in 0.18 μm standard CMOS process, the system demonstrates a quadratic thermal response and a highly linear pH sensitivity, with a trapped charge compensation scheme able to calibrate 99.5% of the pixels in the target range, achieving a homogeneous response across the array. Furthermore, the sensing scheme is robust against process variations and can operate under various supply conditions. Finally, the architecture suitability for on-chip DNA amplification detection is proven by performing Loop-mediated Isothermal Amplification (LAMP) of phage lambda DNA, obtaining a time-to-positive of 7.71 minutes with results comparable to commercial qPCR instruments. This architecture represents the first in-pixel dual thermo-chemical sensing in ISFET arrays for Lab-on-a-Chip diagnostics.

An LED-Driven AuNPs-PDMS Microfluidic Chip and Integrated Device for the Detection of Digital Loop-Mediated Isothermal DNA Amplification.

The sensitive quantification of low-abundance nucleic acids holds importance for a range of clinical applications and biological studies. In this study, we describe a facile microfluidic chip for absolute DNA quantifications based on the digital loop-mediated isothermal amplification (digital LAMP) method. This microfluidic chip integrates a cross-flow channel for droplet generation with a micro-cavity for droplet tiling. DNA templates in the LAMP reagent were divided into ~20,000 water-in-oil droplets at the cross-flow channel. The droplets were then tiled in the micro-cavity for isothermal amplification and fluorescent detection. Different from the existing polydimethylsiloxane (PDMS) microfluidic chips, this study incorporates gold nanoparticles (AuNPs) into PDMS substrate through silica coating and dodecanol modification. The digital LAMP chip prepared by AuNPs-PDMS combines the benefits of the microstructure manufacturing performance of PDMS with the light-to-heat conversion advantages of AuNPs. Upon illumination with a near infrared (NIR) LED, the droplets were stably and efficiently heated by the AuNPs in PDMS. We further introduce an integrated device with a NIR heating unit and a fluorescent detection unit. The system could detect HBV (hepatitis B virus)-DNA at a concentration of 1 × 101 to 1 × 104 copies/μL. The LED-driven digital LAMP chip and the integrated device; therefore, demonstrate high accuracy and excellent performance for the absolute quantification of low-abundance nucleic acids, showing the advantages of integration, miniaturization, cost, and power consumption.

Recombinase assisted loop-mediated isothermal DNA amplification.

Polymerase chain reaction (PCR) and isothermal amplification methods such as LAMP and RPA are widely used for genetic detection. However, there are some shortcomings of these methods such as dependence on thermocycler instruments for PCR, complexity of primer design, the possibility for nonspecific amplification in LAMP and complexity of components in RPA. We develop a novel isothermal DNA detection system named Recombinase Assisted Loop-mediated Amplification (RALA). Recombinase from Thermus thermophilus (TthRecA) was used to open target double-stranded DNA to initiate loop-mediated amplification under isothermal conditions, which simplified the primer design and circumvented pre-denaturation. A FRET sensor named ProofMan and a proofreading enzyme Pfu were introduced to produce fluorescence signals by cleaving the sensor from the 3' end. Consequently, sequence-specific detection based on the RALA system was achieved, and even a single nucleotide polymorphism (SNP) could be identified. By introducing additional loop primers, the fast RALA version can amplify 102 DNA targets in 30 minutes. In addition to high sensitivity and specificity, the flexibility of choosing different reporting sensors makes this method versatile in either quantitative or qualitative DNA detection.

Rapid diagnostics of genital herpes by loop-mediated isothermal amplification method with fluorescent detection

Introduction. Due to the high clinical significance of herpesvirus diseases, the searching of fast and effective methods for their diagnosis remains relevant.The aim of the study was to evaluate the diagnostic efficiency of the loop-mediated isothermal amplification of DNA with real-time fluorescent detection (RT-LAMP) with SYTO-82 dye on a model of herpes simplex virus (HSV) infection.Materials and methods. A total of 44 urogenital swabs containing type 1 and type 2 HSV DNA and 43 swabs without HSV DNA, including 33 samples containing the DNA of cytomegalovirus, Epstein-Barr virus and herpesvirus type 6, were studied. For RT-LAMP, Bst 2.0 WarmStart DNA polymerase, SYTO-82 dye, LAMP primers were used.Results. The high efficiency of HSV DNA detection in the RT-LAMP reaction with SYTO-82 dye was shown. RT-LAMP in optimal conditions allowed to reduce reaction time for 2-3 times compared to real-time PCR (to 35 minutes). Analytical sensitivity of HSV type 1 and 2 detection in RT-LAMP was 103 copies of DNA/ml. The diagnostic sensitivity and specificity of the RT-LAMP diagnosis of HSV infection were 96% and 100%, respectively.Discussion. RT-LAMP method has a high sensitivity and specificity comparable to RTPCR, while the risk of false positive results obtaining is minimal.Conclusion. Thus, the reaction of RT-LAMP with SYTO-82 dye allows quickly, with high sensitivity and specificity to detect HSV DNA in clinical material and can be considered as a promising point-of-care testing method.

A Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of Treponema pallidum subsp. pertenue

Abstract Objectives The eradication of yaws, a childhood disease caused by Treponema pallidum subsp. pertenue, is constrained by the lack of rapid, accurate diagnosis. We sought to develop a molecular point-of-care test for the diagnosis of yaws. Methods A loop-mediated isothermal amplification (LAMP) assay with primers targeting the conserved gene, tp0967, with visual detection by lateral flow test strip was developed. The assay was optimized by varying the concentrations of reagents as well as the temperature and time of the reaction. The limit of detection and selectivity of the assay were evaluated. Subsequently, 63 clinical samples from yaws-infected lesions were used to determine the sensitivity of the assay from both unextracted and DNA extracted swab samples as compared to the current molecular testing protocol (CDC PCR assay) as the gold standard. A further five clinical samples from lesions containing PCR-confirmed syphilis pathogen (Treponema pallidum subsp. pallidum) were tested to ensure specificity of the assay for yaws alone. Results The developed LAMP assay was found to be optimal when run at 65oC for 30 minutes. The limit of detection was 2.7*104 copies DNA per mL. Out of the 63 yaws samples tested, the CDC assay, extracted DNA LAMP assay, and unextracted DNA using the LAMP assay resulted in 12, 14, and 8 positive results, respectively. None of the syphilis samples tested positive in any of the assays. Conclusion We show the development of a fast and sensitive LAMP assay for Treponema pallidum subsp. pertenue detected by lateral flow test strip. Using extracted DNA, the assay sensitivity is on par with gold standard detection. Further, the assay can be adapted to minimal sample processing required for in-field detection without DNA extraction.