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loop-mediated isothermal amplification

Kim A Piera, Ammar Aziz, Timothy William, David Bell, Iveth J González, Bridget E Barber, Nicholas M Anstey, Matthew J Grigg
BACKGROUND: Plasmodium knowlesi is the most common cause of malaria in Malaysia. However, microscopic diagnosis is inaccurate and rapid diagnostic tests (RDTs) are insufficiently sensitive. PCR is sensitive and specific but not feasible at a district level. Loop-mediated isothermal amplification (LAMP) shows potential with only basic requirements. A commercially available LAMP assay, the Eiken Loopamp™ MALARIA Pan Detection kit, is sensitive for Plasmodium falciparum and Plasmodium vivax, but has not previously been evaluated for P...
January 13, 2017: Malaria Journal
Fabio Gentilini, Renato Giulio Zanoni, Elisa Zambon, Maria Elena Turba
We compared 2 novel loop-mediated isothermal amplification (LAMP) assays that target either the 16S ribosomal RNA ( rrs) gene or the gene encoding a 32-kDa leptospiral lipoprotein ( lipL32) in order to assess the effect of the target on the accuracy of the LAMP assays. The most sensitive assay was the rrs assay with a limit of detection (LOD) of 1.2 × 10(1) genome equivalents per reaction. The novel lipL32 assay showed an LOD of 1.2 × 10(2) genome equivalents per reaction. Both assays showed adequate specificity when tested against a collection of bacteria commonly found in voided canine urine...
January 2017: Journal of Veterinary Diagnostic Investigation
Jinzhao Song, Changchun Liu, Michael G Mauk, Shelley C Rankin, James B Lok, Robert M Greenberg, Haim H Bau
BACKGROUND: The wide array of pathogens responsible for infectious diseases makes it difficult to identify causative pathogens with single-plex tests. Although multiplex PCR detects multiple targets, it is restricted to centralized laboratories, which delays test results or makes multiplexing unavailable, depriving healthcare providers of critical, real-time information. METHODS: To address the need for point-of-care (POC) highly multiplexed tests, we propose the 2-stage, nested-like, rapid (<40 min) isothermal amplification assay, dubbed rapid amplification (RAMP)...
January 10, 2017: Clinical Chemistry
Yuki Hashimoto, Yuki Hatayama, Nao Kojima, Shota Morishita, Satoko Matsumoto, Yuzuru Hosoda, Ayako Hara, Toru Motokura
BACKGROUND: Acute promyelocytic leukemia (APL) is a disease characterized by expression of Promyelocytic Leukemia-Retinoic Acid Receptor α (PML-RARα) chimeric mRNA. Although APL is curable, early death due to hemorrhage is a major problem. Here, we report the development of a simple and rapid diagnostic method for APL based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). METHODS: An RT-LAMP primer set was designed to detect three types of PML-RARα mRNA in a single reaction...
December 2016: Yonago Acta Medica
J Rödel, J A Bohnert, S Stoll, L Wassill, B Edel, M Karrasch, B Löffler, W Pfister
The use of molecular assays to rapidly identify pathogens and resistance genes directly from positive blood cultures (BCs) contribute to shortening the time required for the diagnosis of bloodstream infections. In this work, loop-mediated isothermal amplification (LAMP) assays have been examined for their potential use in BC diagnosis. Three different assays were applied. The commercially available eazyplex® MRSA test detects Staphylococcus aureus, S. epidermidis, mecA, and mecC. Two in-house assays [Gram-positive (GP) and Gram-negative (GN)] have been developed for the detection of streptococci, enterococci, vanA, vanB, Pseudomonas spp...
January 6, 2017: European Journal of Clinical Microbiology & Infectious Diseases
Cathrin P Stiedl, Karin Weber
Dogs with a 4-bp deletion in the MDR1 (or ABCB1) gene show intolerance to certain drugs routinely used in veterinary medicine, such as ivermectin, vincristine, and doxorubicin. The mutation leads to a dysfunctional P-glycoprotein drug transporter, which results in drug accumulation in the brain and severe neurotoxicity. A rapid and accurate in-house test to determine the genotype of patients in cases of acute neurotoxic signs or in tumor patients is desirable. We describe a cost-effective detection method with simple technical equipment for veterinary practice...
January 1, 2017: Journal of Veterinary Diagnostic Investigation
Wei Liu, Simo Huang, Ningwei Liu, Derong Dong, Zhan Yang, Yue Tang, Wen Ma, Xiaoming He, Da Ao, Yaqing Xu, Dayang Zou, Liuyu Huang
This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn(2+) dye, and the composite probe method), which cannot distinguish between the desired products and products of nonspecific amplification, thereby leading to false positives...
January 6, 2017: Scientific Reports
Patrick Bird, Jonathan Flannery, Erin Crowley, James Agin, David Goins, Lisa Monteroso
The 3M<sup>™</sup> Molecular Detection Assay (MDA) 2 <em>- Listeria </em>uses loop-mediated isothermal amplification and bioluminescence detection to rapidly detect <em>Listeria </em>spp. in a broad range of food types and environmental surfaces. Using an unpaired study design, the MDA 2 - <em>Listeria </em>was compared to the U.S. Department of Agriculture Food Safety and Inspection Service <em>Microbiology Laboratory Guidebook </em>Chapter 8.09 "<em>Isolation and Identification of Listeria monocytogenes from Red Meat, Poultry, and Egg Products, and Environmental Samples</em>" reference method for the detection of <em>Listeria </em>in deli turkey and raw chicken breast fillet...
November 17, 2016: Journal of AOAC International
Xi-Meng Sun, Yong-Sheng Ji, Xian-Yong Liu, Mei Xiang, Guang He, Li Xie, Jing-Xia Suo, Xun Suo
Loop-mediated isothermal amplification (LAMP), an attractive DNA amplification method, was developed as a valuable tool for the rapid detection of Toxoplasma gondii. In this study, species-specific LAMP primers were designed by targeting the AF146527 sequence, which was a conserved sequence of 200- to 300-fold repetitive 529 bp fragment of T.gondii. LAMP reaction system was optimized so that it could detect the minimal DNA sample such as a single tachyzoite or 10 copies of recombinant plasmid. No cross-reactivity was found when using DNA from other parasites as templates...
2017: PloS One
Byung Hyun Park, Seung Jun Oh, Jae Hwan Jung, Goro Choi, Ji Hyun Seo, Do Hyun Kim, Eun Yeol Lee, Tae Seok Seo
Point-of-care (POC) molecular diagnostics plays a pivotal role for the prevention and treatment of infectious diseases. In spite of recent advancement in microfluidic based POC devices, there are still rooms for development to realize rapid, automatic and cost-effective sample-to-result genetic analysis. In this study, we propose an integrated rotary microfluidic system that is capable of performing glass microbead based DNA extraction, loop mediated isothermal amplification (LAMP), and colorimetric lateral flow strip based detection in a sequential manner with an optimized microfluidic design and a rotational speed control...
December 14, 2016: Biosensors & Bioelectronics
Jolanta Sarowska, Magdalena Frej-Mądrzak, Agnieszka Jama-Kmiecik, Anna Kilian, Dorota Teryks-Wołyniec, Irena Choroszy-Król
BACKGROUND: Salmonella, one of the primary indicators of food safety, is a common cause of food poisoning of an epidemic nature around the world. These microorganisms can colonize the gastrointestinal tract of both people and animals, and next contaminate not only eggs, milk, meat and dairy products, but also vegetables, fruit, grains and even spices. OBJECTIVES: The aim of this study was to analyze the frequency of detection of Salmonella spp. in food samples using a reference PN-ISO method and an alternative method based on loop-mediated isothermal amplification (LAMP) coupled with bioluminescence...
September 2016: Advances in Clinical and Experimental Medicine: Official Organ Wroclaw Medical University
D Kumar, T K S Chauhan, R K Agarwal, K Dhama, P P Goswami, A K Mariappan, A K Tiwari, B P Mishra
We applied a probe-based real-time loop-mediated isothermal amplification (Cy5-RTqLAMP) technique targeting the avian reovirus (ARV) S3 gene to develop a rapid, sensitive, and specific method for virus detection and quantification. This test specifically detected the presence of ARV, but not other viruses or bacteria present in clinical or artificially spiked samples, including Newcastle disease virus, infectious bursal disease virus, fowl adenovirus, Marek's disease virus, Escherichia coli, and Salmonella spp...
December 24, 2016: Archives of Virology
Jacinto Gomes, Marcos Santos, Ana Amaro, Isabel Pereira da Fonseca, Gabriela Santos-Gomes, João Inácio
A loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Theileria annulata infection in cattle. The results were compared with a real-time PCR used for the quantification of T. annulata parasitaemia. One hundred bovine blood samples from 16 cattle farms were tested with LAMP and real-time PCR, with T. annulata DNA being detected in 66% and 67% of the samples, respectively. The results showed that the LAMP assay detects a parasitaemia as low as 0.00025%, indicating a high analytical sensitivity of LAMP for clinical diagnosis of bovine theileriosis...
December 22, 2016: Molecular and Cellular Probes
A Ch Stratakos, M Linton, S Millington, I R Grant
AIMS: To develop a multiplex loop-mediated isothermal amplification (LAMP) assay capable of quantifying Escherichia coli and differentiating verocytotoxigenic E. coli (VTEC). METHODS AND RESULTS: Primer sets were selected to amplify the phoA gene (all E. coli strains) and stx1 and/or stx2 genes (VTEC strains only). LAMP calibration curves demonstrated good quantification capability compared to conventional culture. The limits of detection 50% (LOD50 ) of the multiplex LAMP assay were 2...
December 19, 2016: Journal of Applied Microbiology
Yan Du, Arti Pothukuchy, Jimmy D Gollihar, Armin Nourani, Bingling Li, Andrew D Ellington
The detection of nucleic acid biomarkers for point-of-care (POC) diagnostics is currently limited by technical complexity, cost, and time constraints. To overcome these shortcomings, we have combined loop-mediated isothermal amplification (LAMP), programmable toehold-mediated strand-exchange signal transduction, and standard pregnancy test strips. The incorporation of an engineered hCG-SNAP fusion reporter protein (human chorionic gonadotropin-O(6) -alkylguanine-DNA alkyltransferase) led to LAMP-to-hCG signal transduction on low-cost, commercially available pregnancy test strips...
December 19, 2016: Angewandte Chemie
Kazuya Tone, Ryuichi Fujisaki, Takashi Yamazaki, Koichi Makimura
Loop-mediated isothermal amplification (LAMP) is widely used for differentiating causative agents in infectious diseases. Melting curve analysis (MCA) in conjunction with the LAMP method reduces both the labor required to conduct an assay and contamination of the products. However, two factors influence the melting temperature (Tm) of LAMP products: an inconsistent concentration of Mg(2+) ion due to the precipitation of Mg2P2O7, and the guanine-cytosine (GC) content of the starting dumbbell-like structure. In this study, we investigated the influence of inorganic pyrophosphatase (PPase), an enzyme that inhibits the production of Mg2P2O7, on the Tm of LAMP products, and examined the correlation between the above factors and the Tm value using MCA...
January 2017: Journal of Microbiological Methods
Kazunari Kamachi, Takumi Moriuchi, Yukihiro Hiramatsu, Nao Otsuka, Keigo Shibayama
We evaluated a commercial loop-mediated isothermal amplification (LAMP) assay kit for Bordetella pertussis detection. The LAMP primers were designed to target the ptxP1 allele of the pertussis toxin promoter, but the assay could detect B. pertussis ptxP3 and ptxP8 strains in addition to ptxP1 strains, with high analytical sensitivity.
December 11, 2016: Journal of Microbiological Methods
L Elvira-González, A V Puchades, C Carpino, A Alfaro-Fernandez, M I Font-San-Ambrosio, L Rubio, L Galipienso
Southern tomato virus (STV) is a double stranded RNA (dsRNA) virus belonging to genus Amalgavirus (family Amalgamaviridae) which has been detected in tomato plants showing stunting, fruit discoloration and size reduction. A one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of STV in total RNA or sap extracts (obtained just by grinding in buffer) from STV-infected tomato plants by using a set of three primers pairs which were designed to the sequence of the STV putative coat protein...
December 10, 2016: Journal of Virological Methods
Tiantian Wang, Sanghyo Kim, Jeong Ho An
Loop-mediated isothermal amplification (LAMP) is considered as one of the alternatives to the conventional PCR and it is an inexpensive portable diagnostic system with minimal power consumption. The present work describes the application of LAMP in real-time photon detection and quantitative analysis of nucleic acids integrated with a disposable complementary-metal-oxide semiconductor (CMOS) image sensor. This novel system works as an amplification-coupled detection platform, relying on a CMOS image sensor, with the aid of a computerized circuitry controller for the temperature and light sources...
December 10, 2016: Journal of Microbiological Methods
Yan Lu, Xueping Ma, Jianping Wang, Nan Sheng, Tianhui Dong, Qinxin Song, Jianzhong Rui, Bingjie Zou, Guohua Zhou
Loop-mediated isothermal amplification (LAMP) is a well-developed DNA amplification method with an ultra-high sensitivity, but it is difficult to recognize a single-base difference (like genotyping) in target-specific amplicons by conventional detection ways, such as the intercalation of dyes into dsDNA amplicons or the increase of solution turbidity along with the polymerization process. To allow genotyping based on LAMP suitable for POCT (point-of-care testing) or on-site testing, here we proposed a highly specific and cost-effective method for detecting a single-base difference in LAMP amplicons...
April 15, 2017: Biosensors & Bioelectronics
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