Simple DNA Extraction Research Articles

Simplified, rapid DNA extraction protocol for STR typing from bones

Bones are one of the body parts most tolerant towards various environmental conditions, especially as unidentified remains found in several circumstances: mass disaster, terrorism, war, and plague. Typically, the most complex and time-consuming part of the STR typing process is DNA extraction, with many different methods developed, such as organic extraction, total demineralization (TD), and silica-based column extraction. These protocols may take more than a day to complete. In such events where time is of the essence for human identification of the remains, a simple and fast but effective method is required. This study aimed to develop a simplified and rapid DNA extraction protocol from bone samples. Two bone types (fresh femur and tibia) were incubated in a buffer of SLS, EDTA, proteinase K, and DTT. We also evaluated extraction from different amounts of bone powder. Comparison was done for the developed method against standard total demineralisation protocol. Quantity of DNA extracted was slightly lower for the developed protocol compared to TD, however this did not affect STR typing and profile interpretability. DNA extraction was also possible from merely 0.1 g of bone. The developed method used significantly shorter incubation time and processing steps. Our final protocol only required two hours of incubation and 0.1 g of bone powder. STR typing results gave full profiles. The rapid protocol could be used when there is an urgent need of STR typing for human identification or when the sample amount is limited with a significant reduction in cost and time for investigation.

Simple, rapid and cost-effective DNA extraction method for high quality DNA suitable for PCR based downstream application in mungbean [Vigna radiata (L.) Wilczek

Mungbean is a widely cultivated pulse crop. It is a source of high-quality protein for human consumption. Several new mungbean varieties have been developed employing molecular breeding tools. The quality and quantity of extracted DNA are very important for PCR amplification, particularly for the inter-simple sequence repeat-anchored resistance gene analog (ISSR-RGA) marker. In addition, reducing the time required for DNA extraction is essential in cases when large number of genotypes are analysed. Thus, the aim of this study was to compare eight combinations of two homogenization methods (manual grinding and Bullet blender® homogenizer) and four modified DNA extraction protocols. The effectiveness of DNA extraction for PCR amplification was evaluated using 3 PCR based markers, simple sequence repeat (SSR), ISSR and ISSR-RGA, on 6% polyacrylamide gel. Using homogenizer with modified protocol 2 (T6) resulted in a high DNA concentration (1032.60 ng/μl) and an A260/A280 ratio of 1.80, indicating high DNA purity. The PCR amplification of the resulting DNA with three types of molecular markers showed clear DNA bands, suggesting that DNA quality was appropriate for various molecular studies. In addition, homogenizers allowed processing of large number of samples in one go with minimal cost. These results suggest that this simple, rapid and cost-effective DNA extraction method is useful for marker-assisted breeding.

An innovative DNA extraction method: Water versus commercial buffers

The main objective of DNA extraction is to obtain good quality genetic material in order to carry out its amplification, and corresponding analysis. Most laboratories tend to resort to commercial extraction buffers, which allow a simple and rigorous DNA extraction, with limited handling of the sample, but with high financial cost.As an alternative, we proposed to use water as reagent to extract DNA from blood spots, collected on Whatman® FTA® Cards (Sigma-Aldrich®). We propose to take advantage of hypotonic solutions, in order to obtain a better efficiency in the extraction of genetic material (DNA).On this sense, 219 extractions were performed with Whatman® FTA® Cards (SigmaAldrich®) stained with blood, based on Garcia-Palacios et al., extraction method, with some modifications. Extractions were performed with two types of water (sterile (Lonza™) and distilled (Milli-Q, MilliporeSigma®), as well as, with a commercial extraction kit (Prep-n-Go™, ThermoFisher™ Scientific, Foster City, USA). To evaluate and compare each reagent effectiveness, we amplified DNA extract with mitochondrial DNA Hypervariable regions I and II, and with nuclear DNA markers (autosomal STRs and X-InDels).Our preliminary results suggest that distilled water allows an extraction as effective, or even better, as that obtained with the commercial buffer. These preliminary results offer the possibility for laboratories to choose an inexpensive protocol for DNA extraction, avoiding expensive commercial buffers. Further research is needed to evaluate the performance of each method in blood samples, but also in other biological samples.

Whole-Genome Bisulfite Sequencing and Epigenetic Variation in Cereal Methylomes.

Whole-genome bisulfite sequencing (WGBS) is a technique used for the analysis of genome-wide DNA methylation patterns (DNA methylomes) at a single-base resolution. Here, I describe a simple DNA extraction method from rice endosperm and the universal protocol of WGBS, MethylC-sequencing library preparation. The use of benzyl chloride allows for the extraction of high-quality genomic DNA from starchy endosperm, while sodium bisulfite converts unmethylated cytosine to uracil, whereas methylated cytosine is unchanged. The bisulfite conversion of whole genome sequencing libraries before the final amplification step allows for the discrimination of methylated from unmethylated cytosines in a genome-wide manner.

Experimentation on artificial inoculation studies for persistence of shiga-like toxin-producing Escherichia coli (E. coli O157) in agricultural soils and vegetables using real-time PCR.

Diarrheagenic Escherichia coli O157 is an important reason for largest food borne inflectional outbreaks. E. coli O157 invades into the food chain through contaminated irrigation water and soil causing infectious diseases to humans. In our previous study, we have evaluated the persistence of E. coli O157 through plate count methods. However, conventional cultural procedures are less sensitive to discriminate the pathogenic strain and are time consuming. Therefore, in the present study we have enumerated the persistence of E. coli O157 in soil and vegetables using specific shiga toxin genes (stx1, stx2) through quantitative PCR. Initially, we have standardized a simple Sephadex-based DNA extraction protocol that could detect 2-3 cells/25g of vegetables. Further, quantitative PCR analysis showed a 103 fold difference in the enumeration of persistence as compared to simple plating techniques. Thus, qPCR-based persistence study can be used for rapid and accurate detection techniques for analyzing E. coli O157 contamination. PRACTICAL APPLICATIONS: Our experiment on E. coli O157 expression could be used as a scale for further studies on E. coli O157 pollution in the cropped soils, additionally the DNA extraction protocol experimented by us could be used in all sensitive quantitative assays, as it could detect the expression in lowest cell loads. However, our methodology is a more reliable and sensitive assay compared to normal cultural methods. Our experiment provides a strong evidence of persistence of E. coli O157 prevailing up to half or full cropping season.

A Simple DNA Extraction Method for Begomovirus Detection and Genotyping of Host Plants

A Simple DNA Extraction Method for Begomovirus Detection and Genotyping of Host Plants

Utilization of recombinase polymerase amplification combined with a lateral flow strip for detection of Perkinsus beihaiensis in the oyster Crassostrea hongkongensis

BackgroundPerkinsosis, a disease caused by the protist Perkinsus, is responsible for mass mortalities of many molluscan species worldwide. The rapid, early and accurate detection of Perkinsus infection is necessary to react to outbreaks, and manage disease transmission. Current methods for diagnosis of Perkinsus spp. are time-consuming or require professional equipment and experienced personnel, rendering them unsuitable for field application. Recombinase polymerase amplification (RPA) assay is a highly sensitive and selective isothermal amplification technique that operates at temperatures of 37–42 °C, requires minimal sample preparation, and is capable of amplifying as low as 1–10 target DNA copies in less than 20 minutes.MethodsWe report a novel RPA assay that amplifies the internal transcriber spacer (ITS) region of P. beihaiensis, which, followed by rapid detection of amplicons using a lateral flow (LF) strip, enables easy visualization of results by the naked eye.ResultsThe LF-RPA assay successfully amplified P. beihaiensis DNA using a set of primers of 20–25 bp in length. After incubation at 37 °C for 25 min, results were read within 5 min by the naked eye on a lateral flow strip. Our LF-RPA assay was comparably sensitive to qPCR assay, and capable of detecting as few as 26 copies of P. beihaiensis DNA. Cross-amplification occurred with other two Perkinsus species, P. olseni and P. chesapeaki, but not with other potential pathogen taxa in culture environments. We compared the performance of LF-RPA, conventional PCR and qPCR assays on 60 oyster samples. While LF-RPA assay results were 86.2% as sensitive, 77.4% as specific, and generally in agreement with those of conventional PCR results, they were more (93.3%) sensitive, (86.7%) specific, and agreed better with qPCR assay results. Future research should focus on developing simple DNA extraction methods that do not require professional laboratories and complicated extraction procedures, to facilitate application of this LF-RPA assay in the field.ConclusionsOur LF-RPA assay provides a rapid and efficient method for detecting species of Perkinsus. This novel assay has potential to be used in field applications.

Whole-genome sequencing for rapid, reliable and routine investigation of Mycobacterium tuberculosis transmission in local communities.

Contact investigations following the diagnosis of active tuberculosis (TB) are paramount for the control of the disease. Epidemiological data are very powerful for contact tracing but might be delayed and/or difficult to integrate, especially in the setting of multiple contact-tracing investigations. The aim of this study was to address the added-value of whole-genome sequencing (WGS) to routine local TB surveillance systems. From November 2016 to July 2017, the local TB surveillance system identified three clusters that could constitute a unique larger outbreak. Epidemiological and clinical information were integrated with WGS genotyping data of Mycobacterium tuberculosis strains obtained using a simple DNA extraction method coupled with sequencing using an Illumina MiSeq platform and an in-house bioinformatics pipeline for single nucleotide polymorphism (SNP) analysis. Epidemiological investigations identified three putative TB clusters potentially interrelated including eight patients with active TB. Seven M. tuberculosis isolates were available and analysed by WGS. Using a 5-SNP threshold to define recent transmission, WGS-based genotyping supported the occurrence of the three clusters as well as a link between clusters 1 and 2 (SNP ≤1), constituting a larger outbreak. This outbreak was clearly delineated by refuting a potential link with the third cluster (SNP >500). Genotyping data did not support the belonging of patient 7 to any studied cluster. This study illustrates the usefulness of WGS genotyping for routine TB surveillance systems in local communities to rapidly confirm or disprove epidemiological hypotheses and delineate TB clusters, especially in the context of multiple contact-tracing investigations.

Extraction of Plant DNA by Microneedle Patch for Rapid Detection of Plant Diseases.

In-field molecular diagnosis of plant diseases via nucleic acid amplification is currently limited by cumbersome protocols for extracting and isolating pathogenic DNA from plant tissues. To address this challenge, a rapid plant DNA extraction method was developed using a disposable polymeric microneedle (MN) patch. By applying MN patches on plant leaves, amplification-assay-ready DNA can be extracted within a minute from different plant species. MN-extracted DNA was used for direct polymerase chain reaction amplification of plant plastid DNA without purification. Furthermore, using this patch device, extraction of plant pathogen DNA ( Phytophthora infestans) from both laboratory-inoculated and field-infected leaf samples was performed for detection of late blight disease in tomato. MN extraction achieved 100% detection rate of late blight infections for samples after 3 days of inoculation when compared to the conventional gold standard cetyltrimethylammonium bromide (CTAB)-based DNA extraction method and 100% detection rate for all blind field samples tested. This simple, cell-lysis-free, and purification-free DNA extraction method could be a transformative approach to facilitate rapid sample preparation for molecular diagnosis of various plant diseases directly in the field.

A simplified DNA extraction protocol for unsorted bulk arthropod samples that maintains exoskeletal integrity

AbstractHigh‐throughput DNA sequencing offers an efficient tool for assessing the taxonomic content of bulk arthropod samples. Many current DNA extraction protocols however require extensive handling of samples, like specimen‐based DNA extractions, or sorting of samples and are thus unsuitable for large scale studies. Furthermore, protocols often include homogenization and thus imply partial or complete destruction of the sample constituents. The aim of this study was therefore to investigate steps related both to sample pre‐processing and DNA extraction of unsorted bulk arthropod samples and explore possibilities for simplifying sample processing and thus increase sample handling efficiency without losing taxonomic information. Using mock bulk arthropod samples, we compare laboratory steps related to DNA extraction and semi‐automatic handling. Specifically, we (a) assess whether aliquots of digest buffer from bulk arthropod samples adequately describe the community composition; (b) compare a non‐destructive and a destructive DNA extraction method; (c) compare a phenol/chloroform inhibitor removal method with the exclusion of the same; and (d) compare manual purification to automated DNA purification on a QIAcube laboratory robot. Using DNA metabarcoding and mock bulk arthropod samples, we show that it is possible to efficiently process unsorted arthropod bulk samples with a non‐destructive DNA extraction approach. We found that homogenizing samples yielded more DNA but also generally produced more inconsistent results when compared to non‐destructive extraction. When assessing the recovered taxonomic content of samples (operational taxonomic units, OTUs), intact samples performed at least comparable to, if not better, than homogenized samples. Additionally, we show that sample processing can be further simplified from using a defined volume of digest, no phenol/chloroform purification and automated DNA purification. This approach can be a way to process hundreds of unsorted bulk samples effectively, consistently and with a minimum loss of valuable morphologic information.

Rapid and in-situ detection of fecal indicator bacteria in water using simple DNA extraction and portable loop-mediated isothermal amplification (LAMP) PCR methods

Contamination of water by fecal matter and potential human enteric pathogens is a serious health concern. Microbiological water quality has been assessed by conventional culture-based methods of fecal indicator bacteria (FIB). Recently, molecular techniques for FIB have been introduced as alternative tools for rapid detection. However, such molecular techniques require a modern laboratory setting, expensive equipment, and skilled personnel. In this study, we developed a simple and rapid DNA extraction method based on a syringe filter without any specialized equipment. Furthermore, loop-mediated isothermal amplification (LAMP) PCR for fecal indicator bacteria (FIB) (i.e. E. coli and E. faecalis) was carried out using the DNA extracts from the syringe-filter based DNA extraction method. The efficiency of the extracted DNA from the syringe-filter based method was comparable to the results of the commercial kit method. We also tested fresh and marine-water collected directly from different locations in Singapore that were spiked with E. coli or E. faecalis. The LAMP assays combined with our DNA extraction method showed higher sensitivity and more tolerance to PCR inhibitors than that of conventional PCR methods. We further developed a portable LAMP device to conduct isothermal PCR reactions for rapid on-site measurement of FIB. As the color changes in the end point of the LAMP reaction can be observed with the naked eye, the portable LAMP device was easily operated and quick, obtaining results in 30 min. The simple, portable and user-friendly platform can be used as an initial screening for the rapid detection of the presence FIB in lower-resource settings. In conclusion, the portable LAMP device coupled with a syringe-filter based DNA extraction method enables us to detect the presence of FIB for assessing microbial water quality within 1 h without any sophisticated laboratory equipment or highly trained personnel.

Rapid and equipment-free detection of Phytophthora capsici using lateral flow strip-based recombinase polymerase amplification assay.

Phytophthora capsici is an important oomycete pathogen that causes devastating diseases in various crops. Methods for the rapid detection of P. capsici are important for disease control and eradication programmes. Here, we developed an assay based on lateral flow strip-based recombinase polymerase amplification (LF-RPA) for the rapid and equipment-free detection of P. capsici. The specific primers and a probe were designed using the sequence of Ypt1, and the optimal assay conditions were 40°C for 20min. The specificity of the assay was verified using closely related oomycetes and fungal species, and its detection limit was 10pg of genomic DNA. In combination with a simple DNA extraction method, the LF-RPA assay enabled detection of P. capsici in diseased pepper samples without specialized equipment within 30min. Consequently, the LF-RPA assay developed in this study enables rapid and equipment-free detection of P. capsici and has potential for further development as a diagnostic kit for application in the field or in resource-limited laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: We developed a novel assay based on lateral flow strip-based recombinase polymerase amplification (LF-RPA) for the rapid and equipment-free detection of Phytophthora capsici. In combination with a simple DNA extraction method, the LF-RPA assay detected P. capsici in field samples without specialized equipment within 30min. The assay has potential for further development as a diagnostic kit for application in the field or in resource-limited laboratories.

Quantitative real-time PCR with high-throughput automatable DNA preparation for molecular screening of Nosema spp. in Antheraea pernyi.

Accurate diagnosis of pathogenic Nosema spp. in Antheraea pernyi samples is considered especially useful for reducing economic losses in sericulture and improving food safety by maintaining pathogen-free pupae. However, microscopy and immunologic methods have poor diagnostic sensitivity, while the more sensitive PCR methods remain costly and time-consuming for template preparation. To address this issue, we introduce a sensitive ALMS-qPCR method that combines fast, simple DNA extraction using Alkali Lysis followed by Magnetic bead Separation (ALMS) and quantitative real-time PCR (qPCR). This approach is especially fit for large-scale pathogen molecular screening, because the DNA preparation procedure is fast (<0.94 min per sample) and is high-throughput (performs on a 96-well plate). It is cost-effective, since the most expensive materials can be made in the lab and can be recycled, while the automated procedure can help to minimize labor cost. Though the DNA preparation procedure was substantially simplified, common PCR inhibitory factors were not observed. The sensitivity of ALMS-qPCR is high and the limit of detection is 0.045 parasites/μL. Large-scale screening of Nosema spp. in 3000 Antheraea pernyi samples confirmed the efficacy of the ALMS-qPCR method. Sensitivity is much higher than clinical microscopy, especially for host groups with low infection prevalence and levels. High-throughput ALMS-qPCR, combining automated DNA preparation and sensitive qPCR, provides an enhanced approach for pébrine screening and epidemiological studies. The application of ALMS-qPCR in the sericulture industry will help to strengthen pébrine control and breed pathogen-free species, which means much safer food provision and better genetic resource conservation.

Sex identification of pigeons using polymerase chain reaction analysis with simple DNA extraction

Sex identification plays an important role in avian production. Hitherto, it is difficult to distinguish the sexes of monomorphic birds based on their external features. The chromo-helicase-DNA-binding genes contain CHD-W gene and CHD-Z gene, which are located on the W chromosome and Z chromosome, respectively. Since CHD-W gene is unique to females, the polymerase chain reaction can be used for sex identification. However, extracting DNA procedures for verifying the sex is tedious and expensive. To address these disadvantages, the objective of this study was to develop a simple DNA extraction assay to efficiently process blood, liver, and feather samples. The results showed that 2% dimethylsulfoxide was suitable for processing blood, and phosphate-buffered saline was suitable for processing liver and feather samples. The specific primers were designed, and the length of the targets is 474 bp on Z chromosome and 319 bp on W chromosome. The pigeons were identified as females based on the presence of two bands on the gel, and as males based on the presence of one band. Taken together, our results suggested that feather samples were more appropriate than blood or liver for sex identification of pigeons. Compared to the traditional DNA extraction, this method shortened the assay time and reduced the cost.

Using loop-mediated isothermal amplification for fast species delimitation in eels (genus Anguilla), with special reference to the European eel (Anguilla anguilla)

Fast, reliable and easy to handle methods are required to survey the introduction of foreign eel species into German waters, to detect incorrect labelling of food as well as for ecological field studies e.g. on board a ship. Several DNA-based methods are available for identification of the European eel (Anguilla anguilla). However, the need for expensive and bulky equipment is not convenient for all applications, particularly those related to field work. Therefore, loop-mediated isothermal amplification (LAMP) assays were developed for identification of A. anguilla and the genus Anguilla, respectively. The Anguilla genus LAMP assay was able to detect at least 500 pg DNA per reaction of all four tested Anguilla species, the European eel (A. anguilla), its closest relatives (A. rostrata and A. australis), and the Japanese eel (A. japonica). The detection limit for the A. anguilla specific assay was also 500 pg DNA per reaction and, in combination with a rapid simplified DNA extraction method, both assays were able to detect one single egg of the species A. anguilla per LAMP reaction. None of the two LAMP assays showed false positive results among 112 non-Anguilla species tested. The Anguilla genus-specific LAMP system detected all 34 Anguilla specimens examined, while the second LAMP only detected the 18 A. anguilla specimens, but not the 16 specimens classified as other Anguilla species. The total analysis time, including DNA extraction and data evaluation, was no more than 90 min per experiment. A further time reduction to 70 min is possible by shortening the amplification time and omitting the melting curve analysis without lose of critical information.

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

AbstractPhytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.

Efficient coralline algal psbA mini barcoding and High Resolution Melt (HRM) analysis using a simple custom DNA preparation

Coralline algae form extensive maerl and rhodolith habitats that support a rich biodiversity. Calcium carbonate harvesting as well as trawling activities threatens this ecosystem. Eleven species were recorded so far as maerl-forming in NE Atlantic, but identification based on morphological characters is unreliable. As for most red algae, we now use molecular characters to resolve identification of these taxa. However, obtaining DNA sequences requires time and resource demanding methods. The purpose of our study was to improve methods for achieving simple DNA extraction, amplification, sequencing and sequence analysis to allow robust identification of maerl species and other coralline algae. Our novel and easy DNA preparation method for coralline algae was of sufficient quality for qPCR amplification and sequencing of all 47 tested samples. The new psbA qPCR assay successfully amplified a 350 bp fragment identifying six species and uncovering two new Operational Taxonomic Units. Molecular results were corroborated with anatomical examination using i.e. scanning electron microscopy. Finally, the qPCR assay was coupled with High Resolution Melt analysis that successfully differentiated the closely related species Lithothamnion erinaceum and L. cf. glaciale. This DNA preparation and qPCR technique should vitalize coralline research by reducing time and cost associated with molecular systematics.

Popularização da Ciência: Desmistificando o Dogma Central da Biologia Molecular

Esse projeto de extensão foi apresentado durante a XII Edição do Movimento Científico e Cultural de Aparecida de Goiânia, Goiás, Brasil (MOCCA). O objetivo foi promover o conhecimento científico básico sobre o fluxo da informação genética a partir da experimentação por métodos ilustrativos. Cada participante respondeu um questionário para avaliar seu conhecimento prévio sobre o tema. Em seguida, foi direcionado a um monitor que o acompanhava, no ambiente interativo, apresentando ilustrações com maquetes, painéis, modelos tridimensionais, bem como, na realização da extração simples de DNA de uma fruta. Ao final, o mesmo questionário foi reaplicado. A análise comparativa do desempenho dos participantes no questionário antes e depois da realização das atividades mostrou que as abordagens interativas e as práticas ilustrativas contribuíram, positivamente, para a melhor compreensão do conteúdo sugerindo que essa metodologia possa ser incorporada ao processo de ensino-aprendizagem.

A modified protocol for rapid DNA isolation from cotton (Gossypium spp.)

Extraction of high-quality DNA from Gossypium (Cotton) species is notoriously difficult due to high contents of polysaccharides, quinones and polyphenols other secondary metabolites. Here, we describe a simple, rapid and modified procedure for high-quality DNA extraction from cotton, which is amenable for downstream analyses. In contrast to other CTAB methods, the described procedure is rapid, omits the use of liquid nitrogen, phenol, CsCl gradient ultracentrifugation, uses inexpensive and less hazardous reagents, and requires only ordinary laboratory equipment. The procedure employed the high concentration of NaCl and use of PVP-10 to rid the problems associated with polysaccharides and polyphenols, respectively. The average yield was approximately 10–15 μg of good quality DNA from 100 mg of tissue weight, which is adequate for projects, like genetic mapping or marker-assisted plant breeding. This protocol can be performed in as little as 3 h and may be adapted to high-throughput DNA isolation.•Buffers A and B were redesigned from Paterson et al. (1993) and Porebski et al. (1997), respectively.•Ribonuclease A was added before chloroform extraction.•A simple, rapid and inexpensive DNA extraction method is described.

Combination of PURE-DNA extraction and LAMP-DNA amplification methods for accurate malaria diagnosis on dried blood spots

BackgroundMalaria is one of the most important parasitic infectious diseases for which almost half of the world’s population is at risk. Although several diagnostic methods are now available to detect the infection, more sensitive and applicable tests are still required in the field. The loop-mediated isothermal amplification (LAMP) method is a DNA amplification tool in which the DNA amplification can be achieved by incubation at a stable temperature. A malaria detection kit based on this methodology has already been commercialized and is being used in some countries. The kit includes two reaction tubes: one targeting the common Plasmodium genus (Pan tube) and the other specifically targeting Plasmodium falciparum (Pf tube). In parallel, a simple DNA extraction method, the procedure for ultra rapid extraction (PURE), which can produce a DNA solution suitable for the LAMP reaction without the use of a centrifuge, has also become available. In this study, the sensitivity of the combination of the PURE and LAMP methods (PURE–LAMP) was evaluated with archived dried clinical blood samples of imported malaria cases, including P. falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae.ResultsUsing a nested PCR as the reference, 117 samples including 46 P. falciparum, 7 P. vivax, 9 P. ovale, 4 P. malariae, and 51 negative cases were tested. The PURE–LAMP Pan correctly identified 64 of the 66 positives and the 51 negatives. Among the Pan-positive samples 45 P. falciparum were also detected with the PURE–LAMP Pf. The PURE–LAMP Pan and PURE–LAMP Pf had respective sensitivities of 96.96% (95% CI 89.47–99.63) and 97.82% (95% CI 88.47–99.94) and common specificity of 1.ConclusionThe PURE–LAMP system is accurate when used with dried blood spots and extendable to the field.