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Huaqi Tang, Chunsheng Liu, Yanpeng Li, Xinyue Zhang, Guojie Xu
Fungi of the Alternaria genus are associated with allergic diseases, with Alternaria alternata being one of the most prevalent species. A. alternata has been frequently reported as the etiologic agent of hypersensitivity pneumonitis, allergic rhinosinusitis, bronchial asthma, and other diseases. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay and a real-time PCR assay to detect low levels of A. alternata in herbal tea samples. The LAMP assay can detect as little as 3 pg/μL of A...
October 18, 2016: Journal of AOAC International
Yohei Kurosaki, Sayaka Okada, Sayuri Nakamae, Jiro Yasuda
Bovine papular stomatitis virus (BPSV) causes pustular cutaneous disease in cattle worldwide. This paper describes the development of a specific loop-mediated isothermal amplification (LAMP) assay to detect BPSV which did not cross-react with other parapoxviruses. To assess analytical sensitivity of this LAMP assay, DNA was extracted from serially diluted BPSV from which the infectious titer was determined by a novel assay based on calf kidney epithelial cells. The LAMP assay had equivalent analytical sensitivity to quantitative PCR, and could detect as few as 86 copies of viral DNA per reaction...
October 14, 2016: Journal of Virological Methods
Xiangfeng He, Fei Xue, Shufa Xu, Wenhe Wang
Lily symptomless virus (LSV) is one of the most prevalent viruses that infect lily plants worldwide. A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of LSV, using two primer pairs that specifically amplified the conserved sequence of LSV coat protein. The optimum reaction conditions were as follows: 4mM MgCl2 and 0.8M betaine with incubation at 64°C for 30min. The limit of detection of LSV from infected lily leaves was 10-fold higher for RT-LAMP than for conventional RT-PCR...
October 11, 2016: Journal of Virological Methods
Yuka Okuwa, Michiko Miyamato-Hayashi, Takaichi Tanaka, Yuji Hayakawa, Yasuo Inoshima
Various techniques for screening and detection of bovine leukemia virus (BLV) were compared to ascertain a rapid and simple technique for routine examination. The performance of real-time PCR, nested PCR and loop-mediated isothermal amplification (LAMP) assays was compared using DNA extracted from whole blood instead of white blood cells (WBCs) of 23 cattle. Real-time PCR, LAMP and nested PCR detected 18, 16 and 11 BLV-positive cattle, respectively. These results suggest that LAMP using DNA from whole blood could enable rapid examination, as isolation of WBCs and electrophoresis is time-consuming and could be useful as a simple, and rapid method for routine screening of BLV...
October 3, 2016: Journal of Veterinary Medical Science
Tian-Min Qiao, Jing Zhang, Shu-Jiang Li, Shan Han, Tian-Hui Zhu
Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study...
October 2016: Plant Pathology Journal
Robert D Stedtfeld, Tiffany M Stedtfeld, Farag Samhan, Yogendra H Kanitkar, Paul B Hatzinger, Alison M Cupples, Syed A Hashsham
Nucleic acid amplification of biomarkers is increasingly used to monitor microbial activity and assess remedial performance in contaminated aquifers. Previous studies described the use of filtration, elution, and direct isothermal amplification (i.e. no DNA extraction and purification) as a field-able means to quantify Dehalococcoides spp. in groundwater. This study expands previous work with direct loop mediated isothermal amplification (LAMP) for the detection and quantification of Dehalobacter spp. in groundwater...
October 5, 2016: Journal of Microbiological Methods
Zeynep Koloren, Onuralp Seferoğlu, Panagiotis Karanis
A total of 420 environmental water samples and 120 drinking water samples from 45 different sampling sites of the Black Sea in Turkey were collected between 2012 and 2014. Genomic DNA was isolated from all the investigated water samples and comparativelly analyzed by Loop-mediated isothermal amplification (LAMP) of the elongation factor 1 Alfa (EF1α) gene, and by nested Polymerase Chain Reaction (nPCR) of the small subunit (SSU) rRNA and semi-nested PCR (snPCR) of the glutamate dehydrogenase gene (GDH). 141 (58...
September 30, 2016: Acta Tropica
Ilada Choopara, Narong Arunrut, Wansika Kiatpathomchai, Deborah Dean, Naraporn Somboonna
We developed an assay comprising crude DNA lysis by simple heat treatment coupled loop-mediated isothermal amplification with hydroxynaphthol blue (LAMP-HNB) for C. trachomatis detection, and evaluated the developed assay for its feasibility as one-step point-of-care detection on 284 endocervical swab specimens from clinically symptomatic C. trachomatis and healthy subjects. This assay is sensitive to 0.04 pg of ompA, specific with 6 primers targeting C. trachomatis ompA region, rapid (45 min total assay time), inexpensive (~3 USD/reaction), does not require sophisticated instrumentation, and has comparable assay effectiveness (95% specificity, 90-100% sensitivity) to the bacterial DNA isolation by commercial kit coupled with PCR and gel electrophoresis (98-100% specificity, 87-100% sensitivity) based on the clinical samples test...
September 30, 2016: Letters in Applied Microbiology
Romy E Verbeek, Peter D Siersema, Frank P Vleggaar, Fiebo J Ten Kate, George Posthuma, Rhonda F Souza, Judith de Haan, Jantine W P M van Baal
BACKGROUND AND AIMS: Inflammation plays an important role in the development of esophageal adenocarcinoma and its metaplastic precursor lesion, Barrett's esophagus. Toll-like receptor (TLR) 2 signalling and lysosomal function have been linked to inflammation-associated carcinogenesis. We examined the expression of TLR2 in the esophagus and the effect of long-term TLR2 activation on morphological changes and expression of factors involved in lysosomal function in a Barrett's esophagus epithelium cell line...
September 2016: Journal of Gastrointestinal and Liver Diseases: JGLD
Damien M O'Halloran
Overlapping PCR is commonly used in many molecular applications that include stitching PCR fragments together, generating fluorescent transcriptional and translational fusions, inserting mutations, making deletions, and PCR cloning. Overlapping PCR is also used for genotyping and in detection experiments using techniques such as loop-mediated isothermal amplification (LAMP). STITCHER is a web tool providing a central resource for researchers conducting all types of overlapping assembly PCR experiments with an intuitive interface for automated primer design that's fast, easy to use, and freely available online...
2017: Methods in Molecular Biology
S Y Youn, O M Jeong, B K Choi, S C Jung, M S Kang
Raw chicken products are major causes of human foodborne salmonellosis worldwide. In particular, there is a significant risk of human exposure to Salmonella originating from the chicken slaughtering process. Controlling the contamination of chicken carcasses by Salmonella has been a considerable challenge in chicken-slaughtering facilities and involves routine microbiological monitoring using reliable detection methods. Simple and rapid detection methods, particularly those capable of determining cell viability, will significantly facilitate routine monitoring of Salmonella Here, we report an invA-based loop-mediated isothermal amplification method coupled with a simple propidium monoazide treatment (PMA-LAMP) for simple and rapid detection and quantification of viable Salmonella in rinse water of chicken carcasses...
September 24, 2016: Poultry Science
Karin Johansson, Hanna Karlsson, Torbjörn Norén
Diagnostic testing for Clostridium difficile infection (CDI) has, in recent years, seen the introduction of rapid dual-EIA (enzyme immunoassay) tests combining species-specific glutamate dehydrogenase (GDH) with toxin A/B. In a prospective study, we compared the C. DIFF Quik Chek Complete test to a combination of selective culture (SC) and loop-mediated isothermal amplification (LAMP) of the toxin A gene. Of 419 specimens, 68 were positive in SC including 62 positive in LAMP (14.7%). The combined EIA yielded 82 GDH positives of which 47 were confirmed toxin A/B positive (11%) corresponding to a sensitivity and specificity of 94% for GDH EIA compared to SC and for toxin A/B EIA a sensitivity of 71% and a specificity of 99% compared to LAMP...
November 2016: APMIS: Acta Pathologica, Microbiologica, et Immunologica Scandinavica
Sukumar Biswas, Wei Fan, Rong Li, Sifan Li, Wenli Ping, Shujun Li, Alexandra Naumova, Tamara Peelen, Esther Kok, Zheng Yuan, Dabing Zhang, Jianxin Shi
Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-time PCR (qPCR) assay and the other loop-mediated isothermal amplification (LAMP) assay, were developed for the reliable and efficient detection of the presence of tobacco (Nicotiana tabacum) in various tobacco samples and commodities...
2016: International Journal of Analytical Chemistry
Yasuo Inoshima, Masaki Takasu, Naotaka Ishiguro
Orf virus infection has been prevalent continuously in the population of wild Japanese serows (Capricornis crispus), goat-like grazing cloven-hoofed mammal species that live mainly in mountainous areas of Japan. Currently, definitive diagnosis of infection requires time-consuming laboratory work. To diagnose rapidly on-site, we developed a field-friendly procedure for the detection of orf virus from oral cavity lesions. DNA was extracted from goat saliva spiked with orf virus as a proxy for Japanese serows by a commercial kit without the use of electricity, and the quality of the extracted DNA was evaluated by conventional polymerase chain reaction (PCR)...
September 15, 2016: Journal of Veterinary Medical Science
Xiefeng Yao, Pingfang Li, Jinghua Xu, Man Zhang, Runsheng Ren, Guang Liu, Xingping Yang
Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D...
2016: Frontiers in Microbiology
Mohamed E Ahmed, Mawahib H Eldigail, Fatima M Elamin, Ibtisam A Ali, Martin P Grobusch, Imadeldin E Aradaib
BACKGROUND: Cystic echinococcosis (CE) or hydatidosis, caused by the larval stage of Echinococcus granulosus (EG)-complex, is a neglected parasitic disease of public health importance. The disease is endemic in many African and Mediterranean countries including the Sudan. The objective of the present study was to develop and evaluate a real-time loop-mediated isothermal amplification (LAMP) assay for simple and rapid detection of CE in humans and domestic live stock in Sudan. METHODS: A set of six LAMP primers, designed from the mitochondrial NADH-1 gene of EG cattle strain of genotype 5 (G5), was used as a target for LAMP assay...
2016: BMC Veterinary Research
Bikash R Prusty, Pallab Chaudhuri, V K Chaturvedi, Mohini Saini, B P Mishra, Praveen K Gupta
Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B...
June 2016: Indian Journal of Microbiology
Gaolian Xu, Debbie Nolder, Julien Reboud, Mary C Oguike, Donelly A van Schalkwyk, Colin J Sutherland, Jonathan M Cooper
We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper-folding technique of origami to enable the sequential steps of DNA extraction, loop-mediated isothermal amplification (LAMP), and array-based fluorescence detection. A low-cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a "sample-to-answer" diagnosis from a finger-prick volume of human blood, within 45 min, with minimal user intervention...
August 24, 2016: Angewandte Chemie
Megha Sharma, Kusum Sharma, Aman Sharma, Nalini Gupta, Arvind Rajwanshi
INTRODUCTION: Tuberculous lymphadenitis (TBLA), the most common presentation of tuberculosis, poses a significant diagnostic challenge in the developing countries. Timely, accurate and cost-effective diagnosis can decrease the high morbidity associated with TBLA especially in resource-poor high-endemic regions. The loop-mediated isothermal amplification assay (LAMP), using two targets, was evaluated for the diagnosis of TBLA. MATERIAL AND METHODS: LAMP assay using 3 sets of primers (each for IS6110 and MPB64) was performed on 170 fine needle aspiration samples (85 confirmed, 35 suspected, 50 control cases of TBLA)...
September 2016: Tuberculosis
Ozlem Yaren, Kevin M Bradley, Patricia Moussatche, Shuichi Hoshika, Zunyi Yang, Shu Zhu, Stephanie M Karst, Steven A Benner
Noroviruses are the major cause of global viral gastroenteritis with short incubation times and small inoculums required for infection. This creates a need for a rapid molecular test for norovirus for early diagnosis, in the hope of preventing the spread of the disease. Non-chemists generally use off-the shelf reagents and natural DNA to create such tests, suffering from background noise that comes from adventitious DNA and RNA (collectively xNA) that is abundant in real biological samples, especially feces, a common location for norovirus...
November 2016: Journal of Virological Methods
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