Loop-mediated Isothermal Amplification Protocol Research Articles

End-point rapid detection of total and pathogenic Vibrio parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) in raw seafood using a colorimetric loop-mediated isothermal amplification-xylenol orange technique.

Vibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis in humans worldwide. To ensure seafood safety and to minimize the occurrence of seafood-borne diseases, early detection of total V. parahaemolyticus (pathogenic and non-pathogenic strains) and pathogenic V. parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) is required. This study further improved a loop-mediated isothermal amplification (LAMP) assay using xylenol orange (XO), a pH sensitive dye, to transform conventional LAMP into a one-step colorimetric assay giving visible results to the naked eye. LAMP-XO targeted rpoD for species specificity and tdh, trh1, and trh2 for pathogenic strains. Multiple hybrid inner primers (MHP) of LAMP primers for rpoD detection to complement the main primer set previously reported were designed by our group to maximize sensitivity and speed. Following the standard LAMP protocol, LAMP reaction temperature for rpoD, tdh, trh1, and trh2 detection was first determined using a turbidimeter. The acquired optimal temperature was subjected to optimize six parameters including dNTP mix, betaine, MgSO4, Bst 2.0 WarmStart DNA polymerase, reaction time and XO dye. The last parameter was done using a heat block. The color change of the LAMP-XO result from purple (negative) to yellow (positive) was monitored visually. The detection limits (DLs) of LAMP-XO using a 10-fold serial dilution of gDNA and spiked seafood samples were determined and compared with standard LAMP, PCR, and quantitative PCR (qPCR) assays. Subsequently, the LAMP-XO assay was validated with 102 raw seafood samples and the results were compared with PCR and qPCR assays. Under optimal conditions (65°C for 75 min), rpoD-LAMP-XO and tdh-LAMP-XO showed detection sensitivity at 102 copies of gDNA/reaction, or 10 folds greater than trh1-LAMP-XO and trh2-LAMP-XO. This level of sensitivity was similar to that of standard LAMP, comparable to that of the gold standard qPCR, and 10-100 times higher than that of PCR. In spiked samples, rpoD-LAMP-XO, tdh-LAMP-XO, and trh2-LAMP-XO could detect V. parahaemolyticus at 1 CFU/2.5 g spiked shrimp. Of 102 seafood samples, LAMP-XO was significantly more sensitive than PCR (P < 0.05) for tdh and trh2 detection and not significantly different from qPCR for all genes determined. The reliability of tdh-LAMP-XO and trh2-LAMP-XO to detect pathogenic V. parahaemolyticus was at 94.4% and 100%, respectively. To detect total and pathogenic V. parahaemolyticus, at least rpoD-LAMP-XO and trh2-LAMP-XO should be used, as both showed 100% sensitivity, specificity, and accuracy. With short turnaround time, ease, and reliability, LAMP-XO serves as a better alternative to PCR and qPCR for routine detection of V.parahaemolyticus in seafood. The concept of using a one-step LAMP-XO and MHP-LAMP to enhance efficiency of diagnostic performance of LAMP-based assays can be generally applied for detecting any gene of interest.

Detection of chronic toxoplasmosis in the brain of mice using loop-mediated isothermal amplification (LAMP) and conventional PCR

BackgroundToxoplasma gondii is a common protozoan parasite that infects approximately one-third of the world's population. It is a disease with multiple manifestations. In immunocompetent individuals, symptoms are mild and flu-like, whereas, in immunocompromised patients, it often results in severe morbidity and mortality. Thus, studies for developing a simple, rapid diagnostic tool for early detection of Toxoplasma are emerging. Molecular diagnosis is highly accurate and helpful in congenitally infected and immunocompromised patients. The loop-mediated isothermal amplification (LAMP) technique was invented to improve nucleic acid amplification efficacy in terms of sensitivity and specificity. Aim of the studyThe study aimed to validate a LAMP protocol for detecting Toxoplasma DNA in the brain homogenates from mice experimentally infected with Toxoplasma's ME-49 (cyst-forming type II) strain in comparison to PCR. MethodsIn this study, the target DNA fragment was the Toxoplasma 529-bp, repeated 200–300 copies/genome. The sensitivity of both LAMP and conventional PCR techniques was estimated in brain homogenates in experimental mice at eight weeks post-infection and compared to the histopathology data. ResultsThe LAMP reaction showed positive results in 18 of the 26 examined samples of brain homogenates. PCR showed the characteristic 529-bp band in 15 of the 26 examined samples. ConclusionThe LAMP showed a higher sensitivity over PCR in detecting Toxoplasma infection in brain homogenates of infected mice.

In-Field LAMP Detection of Flavescence Dorée Phytoplasma in Crude Extracts of the Scaphoideus titanus Vector

One of the most destructive diseases affecting grapevine in Europe is caused by Flavescence Dorée phytoplasma (FDp), which belongs to the 16Sr-V group and is a European Union quarantine pathogen. Although many molecular techniques such as loop-mediated isothermal amplification (LAMP) are widely used for the rapid detection of FDp in infected grapevine plants, there is no developed isothermal amplification assay for FDp detection in the insect vectors that are fundamental for the spread of the disease. For this reason, a simple in-field real-time LAMP protocol was optimized and developed for the specific detection of FDp in the insect vector Scaphoideus titanus. The LAMP assay was optimized to work with crude insect extracts obtained by manually shaking a single insect in a buffer for 5 min. Such a simple, sensitive, specific, economic, and user-friendly LAMP assay allowed the detection of FDp in S. titanus in less than half an hour, directly in the field. The developed insect tissue preparation procedure, combined with the LAMP protocol, promptly revealed the presence of FDp in infected S. titanus directly in the vineyards, allowing for monitoring of the spread of the pathogen in the field and to apply timely strategies required for the mandatory control of this pathogen.

DEVELOPMENT OF PARAMETER SETTING (LAMP) OF LOOP MEDIATED ISOTHERMAL AMPLIFICATION FOR ACCELERATED IDENTIFICATION OF BORDETELLA PETRII BACTERIA

The article is devoted to development of loop mediated isothermal amplification (LAMP) method for accelerated identification of little-studied bacteria Bordetella petrii, which are isolated from animals and humans. LAMP is based on identification of a conserved gene in a target group of microorganisms against which a series of four to six primers should be designed. The study protocol takes less than one hour and includes amplification of the target gene using Bst polymerase in isothermal conditions. Species-specific primers were designed to identify B. petrii bacteria, transcription regulator gene LysR was used in the studies. The specificity of the selected primers was validated by BLAST at National Center of Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov/). The researchers selected parameters for layout of the reaction, tested specificity of the developed protocol and appropriate amplification time, also they determined sensitivity of the developed LAMP protocol for identification of B. petrii bacteria. It was established that appropriate amplification result is recorded after 40-50 minutes; agarose gel electrophoresis indicates that sensitivity of the developed amplification protocol is 10 DNA copies. The developed method is specific, which is confirmed by a negative reaction on bacteria: Pseudomonas aeruginosa, Alcaligenes spp., Acinetobacter calcoaceticus, Aeromonas hydrophila, Citrobacter freundi, Escherichia coli, Klebsiella pneumoniae, Salmonella infantis, Yersinia enterocolitica, Staphylococcus aureus, Enterococcus faecalis, Bacillus sub.

Rapid detection of the E198A mutation of carbendazim-resistant isolates in Colletotrichum gloeosporioides by loop-mediated isothermal amplification

Apple bitter rot is a serious agricultural disease caused by Colletotrichum gloeosporioides. In recent years, carbendazim-resistant C. gloeosporioides strains bearing an E198A point mutation in the β-tubulin gene (GAG to GCG) have emerged, threatening global apple production. As such, rapidly detecting the presence of this E198A mutation in C. gloeosporioides isolates is essential in order to monitor the spread of this pathogen and to prevent outbreaks of disease. Herein, we developed a simple loop-mediated isothermal amplification (LAMP) approach to detecting the E198A mutation in C. gloeosporioides isolates from ‘Gala’ apple samples. This optimized LAMP protocol was sufficient to establish the E198A genotype of a given isolate following a 60 min incubation at 63 °C by using four specific primers. The results of this reaction could be interpreted visually based on a fluorescent yellow-green color change upon the addition of the SYBR Green I dye, and were additionally confirmed via gel electrophoresis. Importantly, this LAMP assay was capable of rapidly and reliably detecting apples that were infected with carbendazim-resistant isolates harboring this E198A mutation. In conclusion, this LAMP assay in this study can rapidly, specifically, and sensitively detect cases of apple bitter rot caused by C. gloeosporioides isolates harboring the E198A mutation.

Detection of Sri Lankan cassava mosaic virus by loop-mediated isothermal amplification using dried reagents

Recently, the widespread occurrence of Sri Lankan cassava mosaic virus (SLCMV), genus Begomovirus, family Geminiviridae, which causes a mosaic disease in cassava (Manihot esculenta Crantz) in South-East Asia have, become a serious economic issue. Since cassava is propagated through vegetative cuttings, a rapid virus diagnostic method is crucial for generating virus-free planting materials. In this study, a loop-mediated isothermal amplification (LAMP) assay using six primers was developed and validated for the rapid detection of SLCMV in cassava leaves. This SLCMV assay had a detection sensitivity that was up to 10,000 times higher than that of the conventional polymerase chain reaction assay and can detect the virus from symptomless stem cutting, which is a potential long-distance spreader of the virus. Furthermore, a practical LAMP protocol using stable dried reagents from a commercial kit was established so that the assay could be performed in the field by incubating the reactions in water at 60–65 °C instead of using a thermal cycler. The primer sequences and the LAMP protocol described here should be useful for the rapid and sensitive on-site detection of SLCMV.

Closed-tube field-deployable loop-mediated isothermal amplification (LAMP) assay based on spore wall protein (SWP) for the visual detection of Enterocytozoon hepatopenaei (EHP)

Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment.

Rapid Detection of Pityophthorus juglandis (Blackman) (Coleoptera, Curculionidae) with the Loop-Mediated Isothermal Amplification (LAMP) Method.

The walnut twig beetle Pityophthorus juglandis is a phloem-boring bark beetle responsible, in association with the ascomycete Geosmithia morbida, for the Thousand Cankers Disease (TCD) of walnut trees. The recent finding of TCD in Europe prompted the development of effective diagnostic protocols for the early detection of members of this insect/fungus complex. Here we report the development of a highly efficient, low-cost, and rapid method for detecting the beetle, or even just its biological traces, from environmental samples: the loop-mediated isothermal amplification (LAMP) assay. The method, designed on the 28S ribosomal RNA gene, showed high specificity and sensitivity, with no cross reactivity to other bark beetles and wood-boring insects. The test was successful even with very small amounts of the target insect’s nucleic acid, with limit values of 0.64 pg/µL and 3.2 pg/µL for WTB adults and frass, respectively. A comparison of the method (both in real time and visual) with conventional PCR did not display significant differences in terms of LoD. This LAMP protocol will enable quick, low-cost, and early detection of P. juglandis in areas with new infestations and for phytosanitary inspections at vulnerable sites (e.g., seaports, airports, loading stations, storage facilities, and wood processing companies).

Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass

The red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

In Vitro System for the Detection of Prostate Cancer Markers via Loop-Mediated Isothermal Amplification

Loop-mediated isothermal amplification (LAMP) of nucleic acids enables the detection of amplification products 10–20 min after the beginning of the reaction. An intraoperative method for the detection of metastases in lymph nodes based on LAMP, one-step nucleic acid amplification (OSNA), allows the rapid detection of the epithelial-specific marker gene KRT19 in lymph nodes. The standard protocol for OSNA was developed more than 10 years ago to detect breast cancer metastases in lymph nodes. Since then, a new version of the key enzyme involved in the reaction (Bst-polymerase) was obtained, but its use in OSNA has remained unexplored. Moreover, the time has come to apply OSNA to the detection of other cancer types, in particular, prostate cancer. The first step is to create an in vitro system that allows LAMP to be carried out on prostate-cancer cell lines. In this work, the LAMP protocol was developed for the new Bst 3.0 polymerase with optimized dNTP concentrations and without reverse transcriptase, and cell lines were selected (DU-145 for prostate cancer and Molt-4 for negative control). As a result, a new, in vitro system for the LAMP-based detection of prostate cancer with the marker gene KRT19 was developed.

Optimization of isothermal amplification method for Mycobacteriumtuberculosisdetection and visualization method for fieldwork

Background/aim Tuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure.Materials and methods Sputum samples were homogenized with 5% trypsin solution in CaCl2 to obtain DNA. DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB’s Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w/v agarose gel electrophoresis methods were used.ResultsOptimum amplification temperature for LAMP was found to be 71.4 °C. The detection limit of the method was 102 CFU/mL and sensitivity was determined 100% compared to five different Mycobacterium species. Conclusion The current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field.

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation.

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 °C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) Chapter 5.

LAMP and real-time PCR assays for rapid identification of Frankliniella intonsa

Frankliniella intonsa Trybom is a pest causing significant economic losses of crops cultivated in greenhouses and fields, especially losses of ornamental and horticultural plants. This species is responsible for causing direct damage to plants as well as for transmitting viruses, including the tomato spot wilt virus (TSWV), which is considered the most harmful. Because the proper identification of a pest species is crucial for plant protection, and morphological identification methods require good entomological expertise and are time consuming, molecular diagnostic techniques are a great alternative. Among the molecular diagnostic protocols available for F. intonsa detection, no protocols based on the loop-mediated isothermal amplification (LAMP) method or real-time PCR have been described thus far. Therefore, the aim of this study was to develop species-specific, fast and sensitive protocols based on these two approaches for the identification of F. intonsa. The real-time PCR and LAMP assays developed and described here gave positive results only for F. intonsa populations with a thrips DNA concentration as low as 10−3 ng/μl, proving that they are species specific and sensitive. No reaction products were observed for the negative controls. Moreover, the described LAMP protocol allows for the identification of F. intonsa without a DNA extraction step.

Loop mediated isothermal amplification (LAMP) assays as a rapid diagnostic for COVID-19

Recently, a novel coronavirus (SARS-CoV-2; coronavirus disease 2019, COVID-19) has emerged, rapidly spreading and severely straining the capacity of the global health community. Many nations are employing combinations of containment and mitigation strategies, where early diagnosis of COVID-19 is vital in controlling illness progression and limiting viral spread within the population. Thus, rapid and accurate methods of early detection are vital to contain COVID-19 and prevent further spread and predicted subsequent infectious waves of viral recurrence in future. Immediately after its initial characterization, Chinese and American Centers for Disease Control and Prevention (CDCs) rapidly employed molecular assays for detection of COVID-19, mostly employing real-time polymerase chain reaction (RT-PCR) methods. However, such methods require specific expensive items of equipment and highly trained analysts, requiring upwards of 4–8 h to process. These requirements coupled with associated financial pressures may prevent effective deployment of such diagnostic tests. Loop mediated isothermal amplification(LAMP) is method of nucleic acid amplification which exhibits increased sensitivity and specificity are significantly rapid, and do not require expensive reagents or instruments, which aids in cost reduction for coronavirus detection. Studies have shown the successful application of LAMP assays in various forms to detect coronavirus RNA in patient samples, demonstrating that 1–10 copies of viral RNA template per reaction are sufficient for successful detection, ~100-fold more sensitive than conventional RT-PCR methods. Importantly, studies have also now demonstrated the effectiveness of LAMP methodology in the detection of SARS-CoV-2 RNA at significantly low levels, particularly following numerous improvements to LAMP assay protocols. We hypothesise that recent advancements in enhanced LAMP protocols assay perhaps represent the best chance for a rapid and robust assay for field diagnosis of COVID-19, without the requirement of specialized equipment and highly trained professionals to interpret results. Herein, we present our arguments with a view to disseminate such findings, to assist the combat of this virus that is proving so devastating. We hope that this strategy could be applied rapidly, and confirmed for viability with clinical samples, before being rolled out for mass-diagnostic testing in these current times.

Loop-mediated isothermal amplification (LAMP) assays for the detection of abaca bunchy top virus and banana bunchy top virus in abaca

Abaca bunchy top virus (ABTV) and banana bunchy top virus (BBTV) are pathogens that infect abaca in the Philippines causing significant reduction in abaca fiber yield and quality. In this study, a sensitive loop-mediated isothermal amplification (LAMP) assay was developed for detecting ABTV and BBTV in abaca, Musa textilis Nee. Five primer sets were designed and tested to detect ABTV while three primer sets were designed and tested to detect BBTV. Out of which only one primer set for each virus was specific to their respective targets that is DNA-N for ABTV and DNA-S for BBTV for successful LAMP based detection applications. The concentrations of Bst DNA polymerase, dNTPs, incubation time and temperature were optimized for both viruses using either GelRed™ or SYBR™ Green I as detection dyes. The ABTV and BBTV LAMP assays were more sensitive than conventional PCR and were able to detect ABTV or BBTV in asymptomatic abaca samples. The developed LAMP protocols provide a simple, fast and accurate detection method that will be indispensable for the production of ABTV- and BBTV-free abaca tissue cultured plantlets. This is the first report of a LAMP-based assay for detection of ABTV and BBTV in abaca.

Система in vitro для детекции маркеров рака предстательной железы методом LAMP

Loop-mediated isothermal amplification (LAMP) of nucleic acids enables detection of amplification products in 10-20 min after the reaction begins. An intraoperative method for detection of metastases in lymph nodes is based on LAMP and is called one-step nucleic acid amplification (OSNA). It allows rapid detection of the epithelial-specific marker KRT19 in lymph nodes. The standard protocol for OSNA was developed more than 10 years ago. Since that, a new version of the key enzyme involved in the reaction (Bst-polymerase) was obtained, but its use in OSNA remains unexplored. Moreover, the time has come to apply OSNA to the detection of other cancer types, in particular prostate cancer. The first step is to create an in vitro system that allows LAMP to be carried out on prostate cancer cell lines. Here, LAMP protocol for the new Bst 3.0 polymerase with optimized dNTPs concentration and without reverse transcriptase was developed, and cell lines (DU-145 for prostate cancer and Molt-4 for negative control) were selected. In summary, a new in vitro system for LAMP-based detection of prostate cancer marker KRT19 was developed. LAMP, OSNA, prostate cancer This work was supported by a grant from the President of the Russian Federation for state support of young Russian scientists (MK-1906.2019.4).

A pilot evaluation of alternative procedures to simplify LAMP-based malaria diagnosis in field conditions

Highly-sensitive and field-friendly diagnostic tools are needed for accurate detection of low-density malaria infections. Although loop-mediated isothermal amplification (LAMP) fulfills these conditions, operational challenges are still encountered during pilot population screenings in remote settings when employing Loopamp™ MALARIA Pan/Pf detection kit (Eiken Chemical Co.). This study evaluates different procedures for the simplification of sample preparation and result reading steps of current LAMP protocols. The reference ‘Boil & Spin’ (B&S) pre-amplification procedure was compared to three alternative methods, along with a colorimetric staining protocol based on malachite green. Results suggested that the B&S supernatant transference step may be omitted without an impact on test performance, even when colorimetry was incorporated to facilitate results visualization. Procedures skipping centrifugation and/or heat-incubation were proved to be compatible with LAMP-based malaria DNA detection, but resulted in a low-to-moderate decrease in sensitivity and ambiguous result interpretation for the most straightforward protocol. Nevertheless, all simplified LAMP methods could still reach lower limits of detection than the currently used tools for malaria mass-screening (i.e. microscopy and rapid tests), indicating that these alternative strategies may deserve further consideration. This evaluation, therefore, demonstrates the feasibility of skipping some of the main procedural bottlenecks of LAMP-malaria protocols, a much-needed achievement to make point-of-care implementation of molecular diagnostics a reality.

Development and validation of a loop-mediated isothermal amplification assay-a rapid and sensitive detection tool for Mycobacterium avium subsp. paratuberculosis in small ruminants.

The aim of this study was to design an assay for the identification of Mycobacterium avium subsp. paratuberculosis (MAP) to be used in faeces and milk samples of small ruminants with a loop-mediated isothermal amplification (LAMP) system, as a time-saving and user-friendly method in contrast to real-time PCR. For the detection of MAP in milk and faeces of small ruminants, we developed a set of primers, specific for the target gene ISMap02. The analytical sensitivity of LAMP, when targeting ISMap02, showed a DNA detection limit of 10fgμl-1 . After performing spiking experiments with two MAP reference strains, DSM 44133 and ATCC 19698T , the limit of detection, using the LAMP protocol described herein were 3·8 MAP CFU per ml milk and 12·5 MAP CFU per gram faeces. All LAMP results during the establishment of the assay were compared to those of the real-time PCR results. An internal amplification control was incorporated into the assay to exclude false-negative results produced and had no significant negative impact on the analytical sensitivity. Validation of the assay was confirmed by testing field samples of faeces and revising the results with real-time PCR. Our study conducted the first MAP detection system with a LAMP targeting ISMap02. Due to the positive results we encourage the use of LAMP in combination with ISMap02, when detecting MAP in faeces samples, as an alternative to targeting other genes as f57 or IS900. Further research on MAP detection in different matrices like raw milk, tissue or sperm with this system is recommended. This study provides new achievements in MAP diagnostic. Especially small ruminants do not show signs of diarrhoea until the terminal stage of the illness. The greatest task in fighting MAP is to rule out animals, which shed MAP with faeces and milk before showing symptoms of Johne's disease. Worldwide there is a need to eradicate animals, which are low MAP shedders to stop the illness spreading in animal holdings. MAP detection with LAMP is time saving, easy to use, does not need expensive equipment, as, for example, PCR kits and can be used without access to laboratories. The target gene ISMap02 was shown to be a specific insertion element for MAP and is a reliable aim in future MAP detection studies.

Rapid Method for Detection of Staphylococcus aureus in Feces.

Staphylococcus aureus is an opportunistic human pathogen causing food-borne diseases. Therefore, the goal of the present work was to develop a robust protocol for identification of small amounts of S. aureus DNA in order to establish a rapid, sensitive, and simple method of detect whether this species is present in the stool of patients. A protocol was developed for extraction of DNA by Fe₃O₄ @SiO₂ microbeads and subsequent detection of S. aureus-specific DNA sequences by the loop-mediated isothermal amplification (LAMP). The LAMP technique was rapid and sensitive, which allowsed the detect for bacteria at concentrations as low as 10² cells/mL. This level of detection was superior to that seen for conventional PCR and real-time PCR. Moreover, the LAMP protocol did not require specialized expensive instrumentation. These features of the novel assay developed here indicate that it may serve as an effective tool for point-of-care diagnosis of acute S. aureus infections.

RpoB Targeted Loop-Mediated Isothermal Amplification (LAMP) Assay for Consensus Detection of Mycobacteria Associated With Pulmonary Infections.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid method which has been used to identify mycobacteria including Mycobacterium tuberculosis in clinical microbiology laboratory and point of care settings. Previously published LAMP protocols for detection of mycobacterial species used conventional specific primer and targeted the 16S rRNA, gyrB, and insertion sequence genes. We developed and evaluated a LAMP assay targeting a mycobacterial rpoB gene conserved sequence and incorporating degenerate primers. This assay allowed consensus detection of mycobacterial species from pure culture, clinical respiratory tract samples, and mycobacteria growth indicator tube (MGIT) liquid-based culture medium. A panel of twenty mycobacterial species were successfully detected at detection thresholds of 102 CFU/mL and 103 CFU/mL when respectively performed on pure culture suspension or sputum and MGIT broth. The inclusion of degenerate bases in LAMP primers increased the diversity of mycobacterial species identified by the assay without negatively affecting analytical sensitivity. LAMP-based consensus detection of multiple pathogens can be achieved with degenerate primers therefore allowing the design of rapid multi-disease screening assays. Despite high analytical sensitivity, species specificity and the advantageous operational characteristics of LAMP over PCR, challenges such as potential ambiguity in visual interpretation of results and occasional non-specific amplification precludes the implementation of novel LAMP assay in routine diagnostics both in centralized and point-of-care laboratory.