Loop-mediated Isothermal Amplification Kit Research Articles

Study on producing a LAMP kit for diagnosis of small liver fluke Clonorchis sinensisinfection in humans

Background: Clonorchis sinensis is one of the three species of small liver flukes that cause infections in humans. C. sinensis is found mainly in several Asian countries and Northern, Vietnam. The Loop-mediated isothermal amplification (LAMP) with a sensitivity and specificity comparable to that of the PCR method, which requires less complex equipment and facilities for performance. Methods:The research design describes the laboratory experiment and evaluates a LAMP kit for the diagnosis of C. sinensis in the laboratory and the small-scale field assessment, using 150 human stool samples from fascioliasis endemic areas. Results: The LAMP primer sets have been designed and selected; the conditions and components of the LAMP reaction for C. sinensisdiagnosis have been optimised. Evaluation results of the LAMP kit in the laboratory showed a sensitivity of the test was 98.63% (95% CI: 91.92-99.99%); a specificity was 100% (95% CI: 85.25-100%); the limit of detection was 26 gene copies/μl. The LAMP test kit was found to be stable within 12 months at -20±5oC, and 6 months after opening the tubes. The field assessment of the LAMP kit in Ninh Binh province showed a C. sinensis rate of 6.66%, which was similar to the rate obtained from the studies using the real-time PCR method. Conclusions: The research has successfullyproduced a LAMP kit for the diagnosis of C. sinensis that is sensitive >95% and highly specific >95%. The LAMP kit is stable within 12 months at -20±5oC and for 6 months after opening the tubes

Dry Lamp: A Point of Care Diagnostics for Diagnosis of Bovine Tropical Theileriosis

ABSTRACT Background & objectives: Theileriosis is an important tick-borne hemoprotozoan disease of cattle which causes severe economic loss due to morbidity and mortality. A diagnostic test having high sensitivity, specificity and easy application at the field level is the need of the hour. In this regard Loop mediated isothermal amplification (LAMP) is proven to be a sensitive, easy and time efficient method. One of the major obstacles for the application of LAMP is the difficulty in maintaining the cold chain to preserve reagents. Thus, the challenge is to develop a LAMP kit in a ready-to-use format with dried reagents useful for quick and simple application in field conditions. Methods: The optimized reaction of wet LAMP was followed for the standardization of dry LAMP with certain modifications which are needful. The major modification is vitrification technology of enzyme using trehalose. Results: LAMP assay (dry and wet LAMP) was found to be more sensitive (100%) when compared to microscopy (69.5%) and PCR (86.9%). It was observed that the dry LAMP reaction tubes at room temperature as well as refrigeration temperature provided successful amplification till 7 weeks. Interpretation & conclusion: The drying conditions of LAMP reagents were optimized, and finally managed to dry them in a single reaction tube without reducing the sensitivity. This technology enables us to transport LAMP kits to areas where the cold chain is not easily available.

Head-to-head comparison of two loop-mediated isothermal amplification (LAMP) kits for diagnosis of malaria in a non-endemic setting

BackgroundLight microscopy and rapid diagnostic tests (RDT) have long been the recommended diagnostic methods for malaria. However, in recent years, loop-mediated isothermal amplification (LAMP) techniques have been shown to offer superior performance, in particular concerning low-grade parasitaemia, by delivering higher sensitivity and specificity with low laboratory capacity requirements in little more than an hour. In this study, the diagnostic performance of two LAMP kits were assessed head-to-head, compared to highly sensitive quantitative real time PCR (qPCR), in a non-endemic setting.MethodsIn this retrospective validation study two LAMP kits; Alethia® Illumigene Malaria kit and HumaTurb Loopamp™ Malaria Pan Detection (PDT) kit, were evaluated head-to-head for detection of Plasmodium-DNA in 133 biobanked blood samples from suspected malaria cases at the Clinical Microbiology Laboratory of Region Skåne, Sweden to determine their diagnostic performance compared to qPCR.ResultsOf the 133 samples tested, qPCR detected Plasmodium DNA in 41 samples (defined as true positives), and the two LAMP methods detected 41 and 37 of those, respectively. The results from the HumaTurb Loopamp™ Malaria PDT kit were in complete congruence with the qPCR, with a sensitivity of 100% (95% CI 91.40–100%) and specificity of 100% (95% CI 96.07–100%). The Alethia® Illumigene Malaria kit had a sensitivity of 90.24% (95% CI 76.87–97.28) and a specificity of 95.65% (95% CI 89.24–98.80) as compared to qPCR.ConclusionsThis head-to-head comparison showed higher performance indicators of the HumaTurb Loopamp™ Malaria PDT kit compared to the Alethia® illumigene Malaria kit for detection of malaria.

Development and Evaluation of Loop-Mediated Isothermal Amplification (LAMP) as a Preliminary Diagnostic Tool for Brown Root Rot Disease Caused by Phellinus noxius (Corner) G. H. Cunningham in Hong Kong Urban Tree Management

Brown Root Rot Disease (BRRD) is one of the most devastating urban tree diseases in tropical and subtropical areas, including Hong Kong. It can result in tree death in a few months and is difficult to detect in the early stages of development. Fungal isolation and PCR methods are currently the most widely adopted methods to diagnose the disease. However, they are both time and technically demanding. Loop-mediated isothermal amplification (LAMP) is a superior molecular-based diagnostic method with great specificity, accessibility, and effectivity. In this study, 15 BRRD-positive and 15 BRRD-negative trees were sampled from 19 roadside slopes in Hong Kong from the end of 2020 to the middle of 2021. The wood tissues were isolated and cultivated in PN3 and PDA agars for the disease diagnosis. The mycelium samples in PDA were directly conducted in LAMP kits (mLAMP) to substitute the purified DNA materials. Wood tissues were also used in LAMP kits (wLAMP) as impurified and highly contaminated samples. The results of mLAMP and wLAMP were compared with the results of isolation to evaluate the specificity and sensitivity of LAMP method. The results showed that mLAMP had 100% sensitivity and 73.3% specificity. For wLAMP, both the sensitivity and specificity were 73.3%. For symptomless trees, 85.7% and 64.3% congruencies were found in mLAMP and wLAMP, respectively. Based on the results of this study, the co-application of LAMP in the current tree management work was also discussed. We envisaged LAMP is a sensitive, prompt, and user-friendly method to diagnose BRRD and it could favor the BRRD diagnosis in fields by accelerating and promoting large-scale screening.

Plasmonic LAMP: Improving the Detection Specificity and Sensitivity for SARS-CoV-2 by Plasmonic Sensing of Isothermally Amplified Nucleic Acids.

The ability to detect pathogens specifically and sensitively is critical to combat infectious diseases outbreaks and pandemics. Colorimetric assays involving loop‐mediated isothermal amplification (LAMP) provide simple readouts yet suffer from the intrinsic non‐template amplification. Herein, a highly specific and sensitive assay relying on plasmonic sensing of LAMP amplicons via DNA hybridization, termed as plasmonic LAMP, is developed for the severe acute respiratory syndrome‐related coronavirus 2 (SARS‐CoV‐2) RNA detection. This work has two important advances. First, gold and silver (Au–Ag) alloy nanoshells are developed as plasmonic sensors that have 4‐times stronger extinction in the visible wavelengths and give a 20‐times lower detection limit for oligonucleotides over Au counterparts. Second, the integrated method allows cutting the complex LAMP amplicons into short repeats that are amendable for hybridization with oligonucleotide‐functionalized Au–Ag nanoshells. In the SARS‐CoV‐2 RNA detection, plasmonic LAMP takes ≈75 min assay time, achieves a detection limit of 10 copies per reaction, and eliminates the contamination from non‐template amplification. It also shows better detection specificity and sensitivity over commercially available LAMP kits due to the additional sequence identification. This work opens a new route for LAMP amplicon detection and provides a method for virus testing at its early representation.

Validation of a Commercial Loop-Mediated Isothermal Amplification (LAMP)-Based Kit for the Detection of Salmonella spp. According to ISO 16140:2016

The traditional cultural method (PCR and Real-Time PCR) for Salmonella spp. detection and identification is laborious and time-consuming. A qualitative LAMP method detecting Salmonella spp. was validated in compliance with ISO 16140:2016. The results show a relative accuracy, sensitivity, and specificity of 100% in comparison with the reference method ISO 6579-1:2017; the LOD50 was set as 0.4 CFU/g. Additionally, a field study was carried out comparing the LAMP kit, a commercially available Real-Time PCR kit (FoodProof Salmonella, Biotecon Diagnostics), and the reference cultural method. The Salmonella spp. LAMP kit was suitable for reliable detection of Salmonella spp., simplifying and reducing the extent and the steps of the analytical process. A total of 105 samples of raw poultry meat were screened for the presence of Salmonella spp. according to three methods: the LAMP kit Salmonella spp. (Enbiotech), the Real-Time PCR kit FoodProof Salmonella (Biotecon), and the reference cultural method. Using these three methods, only one sample out of the 105 (0.95%) tested was positive for Salmonella spp. This sample was further investigated using the reference method described in ISO 6579-3:2014, in order to characterise the Salmonella strain. Following this further biochemical identification and serological typing, the isolate was characterised as Salmonella Infantis.

Performance and Application of Commercially Available Loop-Mediated Isothermal Amplification (LAMP) Kits in Malaria Endemic and Non-Endemic Settings.

Loop-mediated isothermal amplification (LAMP) is a sensitive molecular tool suitable for use as a near point-of-care test for the diagnosis of malaria. Recent meta-analyses have detailed high sensitivity and specificity of malaria LAMP when compared to microscopy, rapid diagnostic tests, and polymerase chain reaction in both endemic and non-endemic settings. Despite this, the use of malaria LAMP has primarily been limited to research settings to date. In this review, we aim to assess to what extent commercially available malaria LAMP kits have been applied in different settings, and to identify possible obstacles that may have hindered their use from being adopted further. In order to address this, we conducted a literature search in PubMed.gov using the search terms (((LAMP) OR (Loop-mediated isothermal amplification)) AND ((Malaria) OR (Plasmodium))). Focusing primarily on studies employing one of the commercially available kits, we then selected three key areas of LAMP application for further review: the performance and application of LAMP in malaria endemic settings including low transmission areas; LAMP for malaria screening during pregnancy; and malaria LAMP in returning travelers in non-endemic settings.

Improvement of a new selective enrichment broth for culturing Salmonella in ready‐to‐eat fruits and vegetables

AbstractFruits and vegetables are consumed raw, which is a potential health hazard toward consumers. Consequently, a novel selective enrichment broth (selective Luria‐Bertani broth, SLB) combined with a loop‐mediated isothermal amplification (LAMP) kit was developed for inspection of Salmonella in ready‐to‐eat fruits and vegetables. SLB was formulated by improved Luria‐Bertani (LB), the recovery of acid‐injured Salmonella in SLB was superior to Selenite Cystine Broth (SC) (p < 0.05). In addition, the growth speed of Salmonella in SLB was exceeding among LB and SC, and growth inhibition of non‐target bacteria without compromising Salmonella. Fruits and vegetables, inoculated with Salmonella, were inspected using a LAMP kit and by spreading on selective agar plates after incubation in SLB (10 hr) and Buffered Peptone Water (BPW)‐SC (34 hr). The LAMP kit had a high detection rate (100%) for both the culture methods, whereas detection rates after incubation in BPW‐SC were lower than those in SLB using the coating detection method. In general, SLB had suitable solubility and needed less incubation times, and the combination of SLB and LAMP method could enhance the accuracy of test results.

Comparison of colorimetric loop-mediated isothermal amplification kit and reverse transcription-polymerase chain reaction in the diagnosis of peste des petits ruminants in sheep and goats in Southeast Nigeria

Background and Aim:Peste des petits ruminants (PPR) is an acute, extremely contagious transboundary viral disease of small ruminants with severe economic consequences, caused by PPR virus. Cost-effective and rapid diagnosis of the disease is essential for prompt management and control. This study aimed to compare the application of a commercial colorimetric loop-mediated isothermal amplification (cLAMP) kit and reverse transcriptase-polymerase chain reaction (RT-PCR) in the diagnosis of PPR in sheep and goats in Southeast Nigeria.Materials and Methods:Nasal swab samples were collected from West African Dwarf sheep and goats showing clinical signs suggestive of PPR (n=80) and those without any clinical signs (n=140) of the disease. The diagnosis was achieved through detection of PPR viral genome in the samples using a cLAMP kit and RT-PCR. cLAMP assay was done directly on nasal swab samples without ribosomal nucleic acid extraction. A set of six primers targeting the matrix gene protein was used for the cLAMP assay.Results:PPR viral genome was detected by both cLAMP and RT-PCR in 51 (63.8%) of the 80 samples from sheep and goats with signs suggestive of PPR while 14 (10%) of those without signs tested positive for PPR by both assay methods. There was a 100% agreement in the cLAMP and RT-PCR results. However, cLAMP was a faster, easier, and less expensive method compared to RT-PCR.Conclusion:The cLAMP assay demonstrates the potential for a point of care diagnosis in the field and a valuable diagnostic tool in areas with poor electricity supply as well as in a less equipped diagnostic laboratory. Since the reagents are affordable, cLAMP can be a diagnostic tool of choice in the detection and surveillance of PPR virus in countries with limited resources.

Progress in loop-mediated isothermal amplification assay for detection of Schistosoma mansoni DNA: towards a ready-to-use test

Schistosomiasis is one of the most prevalent Neglected Tropical Disease, affecting approximately 250 million people worldwide. Schistosoma mansoni is the most important species causing human intestinal schistosomiasis. Despite significant efforts in recent decades, the global disease burden of schistosomiasis remains extremely high. This could partly be attributed to the absence of accurate diagnostic tools, primarily in endemic areas. Loop-mediated isothermal amplification (LAMP) is increasingly used in molecular diagnostics as a field-friendly alternative to many other complex molecular methods and it has been proposed as an ideal candidate for revolutionizing point-of-care molecular diagnostics. In a previous work, a LAMP-based method to detect S. mansoni DNA (SmMIT-LAMP) was developed by our research group for early diagnosis of active schistosomiasis in an experimental infection murine model. The SmMIT-LAMP has been further successfully evaluated in both human stool and snail samples and, recently, in human urine samples. In this study, we developed an important improvement for SmMIT-LAMP molecular assay, transforming it into a cold maintenance dry format suitable for potentially manufacturing as kit for ready-to-use for schistosomiasis diagnosis. This procedure could be applied to create dry LAMP kits for a laboratory setting and for diagnostic applications for other neglected tropical diseases.

Evaluation of fluorimetry and direct visualization to interpret results of a loop-mediated isothermal amplification kit to detect Leishmania DNA

BackgroundNucleic acid amplification tests (NAATs) have proven to be advantageous in the diagnosis of leishmaniases, allowing sensitive diagnosis of: (i) cutaneous leishmaniasis in long duration lesions and (ii) visceral leishmaniasis using a less-invasive sample like peripheral blood, in opposition to tissue aspiration required for parasite demonstration by microscopy. Despite their benefits, the implementation of NAATs for leishmaniasis diagnosis at the point-of-care has not been achieved yet, mostly due to the complexity and logistical issues associated with PCR-based methods.MethodsIn this work, we have evaluated the performance of a ready-to-use loop-mediated isothermal amplification (LAMP) kit using two real time fluorimeters to amplify leishmanial DNA obtained by silica column-based and Boil & Spin protocols.ResultsThe different approaches used to run and interpret the LAMP reactions showed a performance equivalent to PCR and real-time PCR, using spiked and clinical samples. The time to positivity obtained with real-time fluorimetry showed an excellent correlation with both Ct values and parasite load from real-time quantitative PCR.ConclusionsThe results obtained open the possibility of using a highly stable, ready-to-use LAMP kit for the accurate diagnosis of leishmaniasis at the point-of-care. Furthermore, the feasibility of relating time to positivity, determined with a portable real-time fluorimeter, with the parasite burden could have a wider application in the management of leishmaniasis, such as in treatment efficacy monitoring or as a pharmacodynamics tool in clinical trials.

Evaluation of loop mediated isothermal amplification (LAMP) assay in the diagnosis of tubercular lymphadenitis: A pilot study

Tubercular lymphadenitis (TBLA) contributes to 30–40% of extrapulmonary TB cases in the immunocompetent individuals and 40–50% in people with HIV. Current diagnostic methods for TBLA like Gene-Xpert or PCR are costly and conventional methods like fine needle aspiration cytology, histopathology lack sensitivity and specificity. Culture which is considered as gold standard require high turnaround time. Loop mediated isothermal amplification (LAMP) assay has been developed as a novel technique for nucleic acid amplification and has shown promising results in the diagnosis of pulmonary tuberculosis. Present study evaluated the Nu-LAMPTM TB Kit (RAS Life Sciences Pvt. Ltd, a bioMerieux group company) for diagnosis of TBLA comparing with conventional tests (cytology, ZN smear, culture). The sensitivity, specificity, PPV and NPV of LAMP assay was found to be 33.3%, 91.2%, 40% and 88.57% as compared to 100%, 76.5%, 42.9% and 100% of ZN staining and 100%, 73.5%, 40% and 100% of cytopathology. The low sensitivity of LAMP assay in the present study addresses the need for comparison and validation of the commercially available LAMP kits before used for patient diagnosis.

Evaluation of in-house loop-mediated isothermal amplification for tuberculosis diagnosis compared with Xpert MTB/RIF.

To evaluate the diagnostic performances of an in-house loop-mediated isothermal amplification (LAMP) kit and the Xpert MTB/RIF test for the diagnosis of pulmonary tuberculosis in a resource-limited setting, this study was performed at the University Teaching Hospital, Ministry of Health, the Republic of Zambia. Two hundred sputum specimens obtained from new tuberculosis (TB) suspects were used for the evaluation of the diagnostic performance of an in-house LAMP kit in comparison with the Xpert MTB/RIF kit. The sensitivity of in-house LAMP and Xpert MTB/RIF was 96.9% and 95.4% in smear-positive samples, 96.8% and 100% in smear-positive/culture-positive samples, and 39.1% and 73.9% in smear-negative/culture-positive samples, respectively. The specificity of in-house LAMP and MTB/RIF kits with culture was 96.5% and 94.5%, respectively. This indicated the superiority of the Xpert MTB/RIF kit; however, mechanical errors during sample processing and the insufficient quantity of samples by Xpert MTB/RIF kit occurred at 2.0% and 19.7%, respectively, comparing to the 100% accessibility of in-house LAMP. Considering the results obtained in this study together with the easy setup with much simpler equipment, such as an aluminum heat block or water bath, in in-house LAMP compared with real-time polymerase chain reaction equipment in Xpert MTB/RIF kit, the applicability of in-house LAMP for the screening of tuberculosis directly from sputum in resource-limited setting seemed to be high.

Evaluation and validation of a loop-mediated isothermal amplification test kit for detection of Hymenoscyphus fraxineus

Hymenoscyphus fraxineus (anamorph Chalara fraxinea) is the ascomycete fungus which causes ash dieback, a potentially lethal disease of Fraxinus excelsior in Europe. Isolation and culturing of H. fraxineus is time consuming and there is a need for rapid, specific diagnostic tools to assist in the deployment of appropriate phytosanitary measures. Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method which can be used for on-site diagnosis. OptiGene have recently released a kit for rapid in planta detection of H. fraxineus using LAMP. The performance of the kit was evaluated for use in the laboratory and in the field in a comparison with real-time PCR, and the kit was also validated according to EPPO standard 7/98. The analytical sensitivity of the kit was found to be 7 pg of DNA extracted from pure culture. The kit was rapid, with an average time to positive of 15.5 min for field samples. A comparison of real-time PCR and LAMP was carried out in the laboratory and in the field. The diagnostic sensitivity and specificity of the LAMP kit in the laboratory were 90.2% and 98.0% respectively, and 98.3% and 88.6% in the field. Positive and negative predictive values and likelihood ratios were also calculated and considered in relation to the prevalence of the disease. The kit was found to be a useful diagnostic tool which can be applied in the field.

肺結核症例における結核菌群核酸検出検査のためのLAMP法（Direct TB-LAMP）の有用性の検討

To evaluate the efficiency of the direct tuberculosis-loop-mediated isothermal amplification (TB-LAMP) assay by using non-centrifuged sputum samples. STUDY PERIOD AND METHODS: The study was conducted between June 2013 and February 2014. We collected 111 sputum samples from patients who had been radiographically diagnosed with tuberculosis and had not received any treatments for longer than 5 days. In the direct TB-LAMP assay, a loop-mediated isothermal amplification kit and 60-μL sputum samples were used. A direct smear microscopy test was used as the smear test. Then, the same sputum samples were processed with a CCE pretreatment reagent, and 100 μL of the solution samples were cultured by using the mycobacterial growth indicator tube (MGIT) culture method. Forty-six of the 111 samples were positive in the smear microscopy tests. All the smear-positive samples were positive in both the MGIT and direct TB-LAMP assay (100%). The mean positive detection time with the direct TB-LAMP assay was 13 minutes 55 seconds. Of 56 smear-negative and MGIT positive samples, 44 (78.6%) were judged to be positive using the direct TB-LAMP assay, with a mean positive detection time of 15 minutes 59 seconds. The direct TB-LAMP assay using non-centrifuged sputum samples was demonstrated to have a high detection rate and thus may be considered useful for rapid and effective tuberculosis diagnosis.

Loop-Mediated Isothermal Amplification (LAMP), An Innovation in Gene Amplification: Bridging the Gap in Molecular Diagnostics; A Review

Loop-Mediated Isothermal Amplification (LAMP), through its uniqueness has provided the world with a powerful molecular diagnostic tool. This innovative gene amplification technique, amplifies nucleic acid at a very rapid pace, maintaining high sensitivity, specificity and efficiency. The most innovative features of LAMP, is the simplicity of its protocol, and overall low cost of application. Other features of LAMP are highlighted in this review, though with few limitations. The demerits include highly technical and cumbersome primer design protocol, and relative scarcity of some materials needed for the technique. The standout features however, ranges from the strand displacement activity of the Bst and Bsm polymerase enzymes, to its ability to amplify gene in an unprocessed samples. The PCR and other several pre-existing techniques, has contributed immensely in disease diagnosis and molecular biology at large. However, their use in molecular diagnostics is mostly limited to highly resourced establishment, leaving the low resourced settings with little or no hope. The cheapness and user-friendliness of LAMP amongst other advantages made it an ideal diagnostic tool that provides solution to the odds of PCR and its likes. The outstanding qualities displayed by LAMP in various researches, has led to several modifications in the technique. Presently several forms of LAMP kits, such as Lyophilized LAMP Kit, electric LAMP device (eLAMP), and combined use of LAMP and Lateral Flow Assay (LFA), are been adopted for diagnosis of many pathogens.

Evaluation of Commercial Kit Based on Loop-Mediated Isothermal Amplification for Rapid Detection of Low Levels of Uninjured and Injured Salmonella on Duck Meat, Bean Sprouts, and Fishballs in Singapore

The objective of this study was to evaluate performance of the commercial kit based on loop-mediated isothermal amplification (LAMP) in comparison with the International Organization for Standardization method for detecting uninjured and sublethally injured Salmonella cells artificially inoculated at levels of 100 and 101 CFU/25 g on raw duck wing, raw mung bean sprouts, and processed fishballs. Injured cells were prepared by a heat treatment for duck wings and fishball samples and a chlorine treatment for bean sprout samples. Additionally, a validation study was performed on naturally contaminated food samples sold in Singapore. A total of 110 samples of each commodity were analyzed in this study. Regardless of inoculum levels, the detection by the commercial LAMP kit showed 100% sensitivity and specificity for both inoculated and uninoculated samples compared with the International Organization for Standardization method, with the exception of bean sprout samples. Only 20% of bean sprout samples inoculated with 100 CFU/25 g injured Salmonella cells were positive by using the commercial LAMPbased kit. However, all negative samples became positive following a secondary enrichment in Rappaport-Vassiliadis medium with soy broth or after concentration by centrifugation. These results suggest that secondary enrichment or centrifugation should be considered as an additional step to increase the sensitivity of the commercial LAMP-based kit with low numbers of injured target cells in samples with high background microflora (such as mung bean sprouts). The validation study also showed that the commercial LAMP-based kit provided 91% sensitivity and 95% specificity for naturally contaminated samples. Thus, this study demonstrates that the commercial LAMP-based kit might be a cost-effective method, as this system could provide rapid, accurate detection of both uninjured and injured Salmonella cells on raw duck wings, raw mung bean sprouts, and processed fishballs in less than 26 h.

Field deployment of loop-mediated isothermal amplification for centralized mass-screening of asymptomatic malaria in Zanzibar: a pre-elimination setting.

BackgroundMolecular tools for detection of low-density asymptomatic Plasmodium infections are needed in malaria elimination efforts. This study reports results from the hitherto largest implementation of loop-mediated isothermal amplification (LAMP) for centralized mass screening of asymptomatic malaria in Zanzibar.MethodsHealthy individuals present and willing to participate in randomly selected households in 60 villages throughout Zanzibar were screened for malaria by rapid diagnostic tests (RDT). In 50 % of the study households, participants were asked to provide 60 μL of finger-prick blood for additional LAMP screening. LAMP was conducted in two centralized laboratories in Zanzibar, by trained technicians with limited or no previous experience of molecular methods. The LAMP assay was performed with LoopampTM MALARIA Pan/Pf Detection Kit (Eiken Chemical Company, Japan). Samples positive for Plasmodium genus (Pan)-LAMP were re-tested using Plasmodium falciparum-specific LAMP kits.ResultsPaired RDT and LAMP samples were available from 3983 individuals. The prevalence of asymptomatic malaria was 0.5 % (CI 95 % 0.1-0.8) and 1.6 % (CI 95 % 1.1-2.2) by RDT and Pan-LAMP, respectively. LAMP detected 3.4 (CI 95 % 2.2-5.2) times more Plasmodium positive samples than RDT. DNA contamination was experienced, but solved by repetitive decontamination of all equipment and reagents.ConclusionsLAMP is a simple and sensitive molecular tool, and has potential in active surveillance and mass-screening programmes for detection of low-density asymptomatic malaria in pre-elimination settings. However, in order to deploy LAMP more effectively in field settings, protocols may need to be adapted for processing larger numbers of samples. A higher throughput, affordable closed system would be ideal to avoid contamination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0731-2) contains supplementary material, which is available to authorized users.

Loop-mediated isothermal amplification of DNA (LAMP): a new diagnostic tool lights the world of diagnosis of animal and human pathogens: a review.

Diagnosis is an important part in case of animal husbandry as treatment of a disease depends on it. Advancement in molecular biology has generated various sophisticated tools like Polymerase Chain Reaction (PCR), its versions along with pen-side diagnostic techniques. Every diagnostic test however has both advantages and disadvantages; PCR is not an exception to this statement. To ease the odds faced by PCR several non-PCR techniques which can amplify DNA at a constant temperature has become the need of hour, thus generating a variety of isothermal amplification techniques including Nucleic Acid Sequence-Based Amplification (NASBA) along with Self-Sustained Sequence Replication (3SR) and Strand Displacement Amplification (SDA) and Loop mediated isothermal amplification (LAMP) test. LAMP stands out to be a good and effective diagnostic test for empowering in developing countries as it does not require sophisticated equipments and skilled personnel and proves to be cost-effective. Performance of LAMP mainly relies on crafting of six primers (including 2 loop primers) ultimately accelerating the reaction. LAMP amplifies DNA in the process pyrophosphates are formed causing turbidity that facilitates visualisation in a more effective way than PCR. The Bst and Bsm polymerase are the required enzymes for LAMP that does not possess 5'-3' exonuclease activity. Results can be visualized by adding DNA binding dye, SYBR green. LAMP is more stable than PCR and real-time PCR. Non-involvement of template DNA preparation and ability to generate 10(9) copies of DNA are added benefits that make it more effective than NASBA or 3SR and SDA. Thus, it fetches researcher's interest in developing various versions of LAMP viz., its combination with lateral flow assay or micro LAMP and more recently lyophilized and electric (e) LAMP. Availability of ready to use LAMP kits has helped diagnosis of almost all pathogens. LAMP associated technologies however needs to be developed as a part of LAMP platform rather than developing them as separate entities. This review deals with all these salient features of this newly developed tool that has enlightened the world of diagnosis.

Highly Sensitive Detection of Malaria Parasitemia in a Malaria-Endemic Setting: Performance of a New Loop-Mediated Isothermal Amplification Kit in a Remote Clinic in Uganda

Background. Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too complex for field deployment. A new commercial molecular assay based on loop-mediated isothermal amplification (LAMP) was assessed for field use.Methods. Malaria LAMP (Eiken Chemical, Japan) was evaluated for samples from 272 outpatients at a rural Ugandan clinic and compared with expert microscopy, nested PCR, and quantitative PCR (qPCR). Two technicians performed the assay after 3 days of training, using 2 alternative blood sample–preparation methods and visual interpretation of results by fluorescence assay.Results. Compared with 3-well nested PCR, the sensitivity of both LAMP and single-well nested PCR was 90%; the microscopy sensitivity was 51%. For samples with a Plasmodium falciparum qPCR titer of ≥2 parasites/µL, LAMP sensitivity was 97.8% (95% confidence interval, 93.7%–99.5%). Most false-negative LAMP results involved samples with parasitemia levels detectable by 3-well nested PCR but very low or undetectable by qPCR.Conclusions. Malaria LAMP in a remote Ugandan clinic achieved sensitivity similar to that of single-well nested PCR in a United Kingdom reference laboratory. LAMP dramatically lowers the detection threshold achievable in malaria-endemic settings, providing a new tool for diagnosis, surveillance, and screening in elimination strategies.