Optimization of the Amplicons Detection System of Loop-Mediated Isothermal Amplification on Microfluidic Compact Disk

2018 ◽  
pp. 7-12
Author(s):  
Shah Mukim Uddin ◽  
Fatimah Ibrahim ◽  
Jongman Cho ◽  
Kwai Lin Thong
Keyword(s):  
Detection System ◽  
Isothermal Amplification ◽  
Compact Disk ◽  
Loop Mediated Isothermal Amplification

scholarly journals A Portable Automatic Endpoint Detection System for Amplicons of Loop Mediated Isothermal Amplification on Microfluidic Compact Disk Platform

Sensors ◽  
2015 ◽  
Vol 15 (3) ◽  
pp. 5376-5389 ◽  
Author(s):  
Shah Uddin ◽  
Fatimah Ibrahim ◽  
Abkar Sayad ◽  
Aung Thiha ◽  
Koh Pei ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Detection System ◽  
Liquid Crystal Display ◽  
Foodborne Pathogen ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Endpoint Detection ◽  
Compact Disk ◽  
Loop Mediated Isothermal Amplification ◽  
The Impact

In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10−3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment.

scholarly journals Portable and label-free quantitative Loop-mediated Isothermal Amplification (qLAMP) for reliable COVID-19 diagnostics in 3 minutes: An Arduino-based detection system assisted by a pH microelectrode

2021 ◽  
Author(s):  
Mario Moisés Alvarez ◽  
Sergio Bravo-González ◽  
Everardo González-González ◽  
Grissel Trujillo-de Santiago
Keyword(s):  
Real Time ◽  
Detection System ◽  
Genetic Material ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Label Free ◽  
Viral Pathogens ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification System

Loop-mediated isothermal amplification (LAMP) has been recently studied as an alternative method for cost-effective diagnostics in the context of the current COVID-19 pandemic. Recent reports document that LAMP-based diagnostic methods have a comparable sensitivity and specificity to that of RT-qPCR. We report the use of a portable Arduino-based LAMP-based amplification system assisted by pH microelectrodes for the accurate and reliable diagnosis of SARS-CoV-2 during the first 3 minutes of the amplification reaction. We show that this simple system enables a straightforward discrimination between samples containing or not containing artificial SARS-CoV-2 genetic material in the range of 10 to 10,000 copies per 50 μL of reaction mix. We also spiked saliva samples with SARS-CoV-2 synthetic material and corroborated that the LAMP reaction can be successfully monitored in real time using microelectrodes in saliva samples as well. These results may have profound implications for the design of real-time and portable quantitative systems for the reliable detection of viral pathogens including SARS-CoV-2.

scholarly journals Establishment of a Rapid Detection Method for Rice Blast Fungus Based on One-Step Loop-Mediated Isothermal Amplification (LAMP)

Plant Disease ◽  
2019 ◽  
Vol 103 (8) ◽  
pp. 1967-1973 ◽  
Author(s):  
Ling Li ◽  
Shu Ya Zhang ◽  
Chuan-Qing Zhang
Keyword(s):  
Rapid Detection ◽  
Rice Blast ◽  
Detection System ◽  
Fungal Spores ◽  
Lamp Assay ◽  
Rice Blast Fungus ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Blast Fungus

Rice blast is one of the most serious diseases for rice, and controlling the filamentous fungus Magnaporthe oryzae that causes rice blast is crucial for global food security. Typically, early infected rice does not show symptoms. Therefore, the early diagnosis of rice blast is particularly important to avoid uncontrollable propagation of rice blast fungus. In the present work, a rapid and efficient loop-mediated isothermal amplification (LAMP) method was developed to detect the pathogen at the early infected stage of rice. The Alb1 superfamily hypothetical protein MGG_04322, a nuclear shuttling factor involved in ribosome and melanin biogenesis, was chosen as the target for designing the LAMP primers. The LAMP assay enabled rapid detection of as little as 10 pg of pure genomic DNA of M. oryzae. In addition, we established the quantitative LAMP (q-LAMP) detection system to quantify the conidia of rice blast fungus. The q-LAMP assay enabled rapid detection (within 35 min) of the fungal spores at a sensitivity of 3.2 spores/ml. In addition, the assay sets up the linearization formula of the standard curve as y = 0.3066 + 15.33x (where x = amplification of time), inferring that spore number = 100.60y. In addition, the q-LAMP assay was successfully used to detect the presence of the virulence strains of M. oryzae (wild type) in comparison with that of the two mutant strains by quantifying the biomass within host tissue. These results provide a useful and convenient tool for detecting M. oryzae that could be applied in the incubation period of rice blast before symptoms appear.

scholarly journals Development of a Multiplex Loop-Mediated Isothermal Amplification (LAMP) Method for Simultaneous Detection of Spotted Fever Group Rickettsiae and Malaria Parasites by Dipstick DNA Chromatography

Diagnostics ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 897
Author(s):  
Lavel Chinyama Moonga ◽  
Kyoko Hayashida ◽  
Naoko Kawai ◽  
Ryo Nakao ◽  
Chihiro Sugimoto ◽  
...  
Keyword(s):  
Detection System ◽  
Spotted Fever ◽  
Simultaneous Detection ◽  
Febrile Illness ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Spotted Fever Group ◽  
Loop Mediated Isothermal Amplification ◽  
Rickettsia Monacensis ◽  
Sfg Rickettsia

Spotted fever group (SFG) rickettsiae causes febrile illness in humans worldwide. Since SFG rickettsiosis’s clinical presentation is nonspecific, it is frequently misdiagnosed as other febrile diseases, especially malaria, and complicates proper treatment. Aiming at rapid, simple, and simultaneous detection of SFG Rickettsia spp. and Plasmodium spp., we developed a novel multiple pathogen detection system by combining a loop-mediated isothermal amplification (LAMP) method and dipstick DNA chromatography technology. Two primer sets detecting SFG Rickettsia spp. and Plasmodium spp. were mixed, and amplified products were visualized by hybridizing to dipstick DNA chromatography. The multiplex LAMP with dipstick DNA chromatography distinguished amplified Rickettsia and Plasmodium targeted genes simultaneously. The determined sensitivity using synthetic nucleotides was 1000 copies per reaction for mixed Rickettsia and Plasmodium genes. When genomic DNA from in vitro cultured organisms was used, the sensitivity was 100 and 10 genome equivalents per reaction for Rickettsia monacensis and Plasmodium falciparum, respectively. Although further improvement will be required for more sensitive detection, our developed simultaneous diagnosis technique will contribute to the differential diagnosis of undifferentiated febrile illness caused by either SFG Rickettsia spp. or Plasmodium spp. in resource-limited endemic areas. Importantly, this scheme is potentially versatile for the simultaneous detection of diverse infectious diseases.

scholarly journals Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Su Jeong Ahn ◽  
Yun Hee Baek ◽  
Khristine Kaith S. Lloren ◽  
Won-Suk Choi ◽  
Ju Hwan Jeong ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Seasonal Influenza ◽  
Detection System ◽  
Influenza Viruses ◽  
Isothermal Amplification ◽  
Type B ◽  
Multiplex Detection ◽  
Rt Pcr ◽  
Avian Influenza Viruses ◽  
Loop Mediated Isothermal Amplification

Abstract Background In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans. Methods We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses). Results We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%). Conclusions Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.

Loop-Mediated Isothermal Amplification (LAMP)-Based Turn on Fluorescent Paper (ToFP) Device for Detecting Rosellinia necatrix

2020 ◽  
Vol 16 (2) ◽  
pp. 166-178
Author(s):  
Se Hee Lee ◽  
Sang-Hee Lee ◽  
Kyungho Won ◽  
Myung-Su Kim ◽  
Hojin Ryu ◽  
...  
Keyword(s):  
Detection System ◽  
Fruit Trees ◽  
Isothermal Amplification ◽  
Rosellinia Necatrix ◽  
Loop Mediated Isothermal Amplification ◽  
Turn On ◽  
Highly Sensitive ◽  
Field Samples ◽  
Long Time ◽  
Time Loop

White root rot (WRR) disease caused by Rosellinia necatrix, a fungal pathogen, results in severe damage to various fruit trees, decreasing their marketability. Regular monitoring is a major process because the pathogen can remain in the soil around the host for a long time. Loop-mediated isothermal amplification (LAMP) is a highly sensitive and efficient amplification technology of nucleic acids (DNA or RNA) that can be performed at constant temperatures. Thus, it has been spotlighted as a useful tool for detecting several infectious agents. In the present study, LAMP-based Turn-on Fluorescent Paper (ToFP) devices were designed and applied to detect R. necatrix. LAMP conditions were optimized and found to be optimal at a reaction temperature (62 °C) and a reaction time (30 minutes). These reaction conditions were confirmed by applying them to infectious soil samples collected from the field. The limitation of detection was identified as 10 fg of genomic DNA under optimized LAMP conditions. These LAMP-based ToFP devices were generated with easily available stationery materials and the utility of these devices to analyze the LAMP results were confirmed through several experiments on a total of 14 field samples. The results showed that the developed LAMP-based detection system was very sensitive and had the advantages of rapid detection and high availability in the field.

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