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.

A microfluidic lab-on-a-disc integrated loop mediated isothermal amplification for foodborne pathogen detection

2016 ◽  
Vol 227 ◽  
pp. 600-609 ◽  
Author(s):  
Abkar Ahmed Sayad ◽  
Fatimah Ibrahim ◽  
Shah Mukim Uddin ◽  
Koh Xiu Pei ◽  
Mas S. Mohktar ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Foodborne Pathogen ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Foodborne Pathogen Detection

Retroreflection-based sandwich type affinity sensing of isothermal gene amplification product for foodborne pathogen detection

The Analyst ◽  
2022 ◽  
Author(s):  
Danbi Lee ◽  
Eunsuk Kim ◽  
Kyung Won Lee ◽  
Ka Ram Kim ◽  
Hyeong Jin Chun ◽  
...  
Keyword(s):  
Gene Amplification ◽  
Molecular Diagnostics ◽  
Pathogen Detection ◽  
Target Genes ◽  
Foodborne Pathogen ◽  
Isothermal Amplification ◽  
Amplification Product ◽  
Loop Mediated Isothermal Amplification ◽  
Sandwich Type ◽  
Foodborne Pathogen Detection

Loop-mediated isothermal amplification (LAMP) is an outstanding method for molecular diagnostics, as the rapid, specific, and sensitive amplification of target genes is possible. However, it is necessary to measure fluorescence...

scholarly journals Development of a Loop-mediated isothermal amplification (LAMP) technique for specific and early detection of Mycobacterium leprae in clinical samples

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nupur Garg ◽  
Upasana Sahu ◽  
Sudeshna Kar ◽  
Farhan J. Ahmad
Keyword(s):  
Early Detection ◽  
Mycobacterium Leprae ◽  
Negative Control ◽  
Rapid Diagnosis ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Loop Mediated Isothermal Amplification

AbstractLeprosy, a progressive, mutilating and highly stigmatized disease caused by Mycobacterium leprae (ML), continues to prevail in the developing world. This is due to the absence of rapid, specific and sensitive diagnostic tools for its early detection since the disease gets notified only with the advent of physical scarring in patients. This study reports the development of a Loop-mediated isothermal amplification (LAMP) technique for fast, sensitive and specific amplification of 16S rRNA gene of ML DNA for early detection of leprosy in resource-limited areas. Various parameters were optimized to obtain robust and reliable amplification of ML DNA. Blind clinical validation studies were performed which showed that this technique had complete concurrence with conventional techniques. Total absence of amplification of negative control DNA confirmed the specificity of this test. Various visual detection methods viz. colorimetric, turbidity differentiation and bridge flocculation were standardized to establish easy-to-read and rapid diagnosis. This technique eliminates the lack of accuracy and sensitivity in skin smear tests in patients and the requirement for expensive lab equipments and trained technicians. The technique holds promise for further expansion and has the potential to cater to the unmet needs of society for a cheap, highly-sensitive and robust rapid diagnosis of ML.

HIGHLY SENSITIVE H7N9 AVIAN INFLUENZA VIRUS DETECTION USING REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION AND OLIGONUCLEOTIDE MICROARRAY

2015 ◽  
Vol 41 (04) ◽  
pp. 251-255
Author(s):  
Lih-Chiann Wang ◽  
Dean Huang
Keyword(s):  
Avian Influenza ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
High Sensitivity ◽  
Oligonucleotide Microarray ◽  
Influenza Viruses ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Loop Mediated Isothermal Amplification ◽  
Viral Copy Number

H7N9 avian influenza viruses have circulated in the human population and poultry flocks in China since 2013. H7N9 virus monitoring is imperative in Taiwan due to the frequent contact between China and Taiwan. Traditional viral molecular detection methods using RT-PCR and sequencing are time- and labor-intensive, thus a simpler and cheaper method with high sensitivity and specificity is worth developing. We successfully detected human and wild bird H7N9 viruses in this study using reverse transcription loop-mediated isothermal amplification (RT-LAMP) and oligonucleotide microarray. The detection limit was as low as one viral copy number. The specific matching reaction between the templates and microarray probes during hybridization ensured the high detection effectiveness. The excellent sensitivity and specificity of the RT-LAMP-microarray makes it a powerful H7N9 surveillance approach in Taiwan.

scholarly journals Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Erwinia amylovora on Pear and Apple Fruit Flowers

Plant Disease ◽  
2011 ◽  
Vol 95 (4) ◽  
pp. 423-430 ◽  
Author(s):  
Todd N. Temple ◽  
Kenneth B. Johnson
Keyword(s):  
Rapid Detection ◽  
Pathogen Detection ◽  
Fire Blight ◽  
Management Practices ◽  
Erwinia Amylovora ◽  
Lamp Assay ◽  
Apple Fruit ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Forecasting Models

Fire blight of pear and apple is frequently an inoculum-limited disease but weather-based forecasting models commonly assume that the pathogen is omnipresent. To improve fire blight risk assessment during flowering, we developed a rapid pathogen detection protocol that uses loop-mediated isothermal amplification (LAMP) to detect DNA of epiphytic Erwinia amylovora on samples of pear and apple flowers. LAMP detected a single flower colonized epiphytically by E. amylovora in a sample of 100 flower clusters (approximately 600 flowers). Samples of 100 flower clusters from orchards (approximately one sample per hectare) were washed and subjected to LAMP, which was completed in 2 h. In three experimental orchards inoculated with E. amylovora, positive LAMP reactions were attained from nine of nine 100-flower cluster samples; pathogen populations in the floral washes averaged 5.2 × 103 CFU per flower as determined by dilution plating. Samples of pear and apple flowers obtained from 60 commercial orchards located in Oregon, Washington, California, and Utah resulted in detection of E. amylovora by LAMP assay from 34 sites, 20 of which developed fire blight. Of samples at early bloom, 10% were positive for epiphytic E. amylovora compared with 28% at petal fall; pathogen density in washes of positive samples averaged 3.2 × 102 CFU per flower. In another 26 orchards, all floral washes were negative for E. amylovora by LAMP and by dilution plating; a light severity of fire blight was observed in 8 of these orchards. Overall, positive detection of epiphytic E. amylovora in commercial orchards by LAMP-based scouting generally occurred at later stages of bloom after heat (risk) units had begun to accumulate, an indication that weather-based forecasting models may be an adequate measure of fire blight risk for many orchardists. Nonetheless, several orchardists communicated that information from the LAMP-based rapid detection protocol resulted in modification of their fire blight management practices.

scholarly journals Development of a rapid detection method for Trichomonas vaginalis based on loop-mediated isothermal amplification targeting adhesion protein 65

2020 ◽  
Author(s):  
Yuhua Li ◽  
Haoran Li ◽  
Xiaoxiao Song ◽  
Hao Zhang ◽  
Yujuan Duan ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Trichomonas Vaginalis ◽  
Detection Method ◽  
Reaction System ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Adhesion Protein ◽  
Loop Mediated Isothermal Amplification ◽  
Pcr Targeting

Abstract Background: Trichomoniasis resulting from Trichomonas vaginalis (T. vaginalis) has been considered as a commonly seen disease with the transmission way of sex. At present, the detection methods of T. vaginalis mainly include wet mount microscopy, culture, PCR, immunofluorescence and ELISA. However, all of these detection methods exist shortcomings.Methods: In this study, a loop-mediated isothermal amplification (LAMP) assay that targeted the species-specific sequence of adhesion protein 65 (AP65) gene had been conducted to detect T. vaginalis. The optimum reaction system and conditions were optimized in this rapid detection method.Results: The results of sensitivity analysis showed that the LAMP assay targeting the AP65 gene was 1000 times more sensitive than the nested PCR targeting the actin gene commonly used for detection of T. vaginalis, and the detecting limitation of the former was 10 trichomonad. Moreover, the amplification of the target gene AP65 by LAMP assay exhibited high specificity and the product was exclusively from T. vaginalis. The detection technique of LAMP did not exhibit cross-reactivity with the common pathogens of Trichinella spiralis, Toxoplasma gondii, Escherichia coli, Candida albicans, Staphylococcus aureus, Haemophilus.Conclusions: According to the present study, the LAMP assay with the target of AP65 gene, was suitable for the early diagnosis of T. vaginalis infections. Consequently, the LAMP assay was proposed by the current study as a point-of-care examination and an alternative molecular tool which exhibited the potential value in the treatment, control and prevention of trichomoniasis transmission and relevant complication.

scholarly journals Development of a convenient detection method for Trichomonas vaginalis based on loop-mediated isothermal amplification targeting adhesion protein 65

2020 ◽  
Author(s):  
Yuhua Li ◽  
Shuai Wang ◽  
Haoran Li ◽  
Xiaoxiao Song ◽  
Hao Zhang ◽  
...  
Keyword(s):  
Trichomonas Vaginalis ◽  
Detection Method ◽  
Trichinella Spiralis ◽  
Reaction System ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Adhesion Protein ◽  
Loop Mediated Isothermal Amplification ◽  
Pcr Targeting

Abstract Background: Trichomoniasis resulting from Trichomonas vaginalis (T. vaginalis) has been considered as a commonly seen disease with the transmission way of sex. At present, the detection methods of T. vaginalis mainly include wet mount microscopy, culture, PCR, immunofluorescence and ELISA. However, all of these detection methods exist shortcomings. Methods: In this study, a loop-mediated isothermal amplification (LAMP) assay that targeted the species-specific sequence of adhesion protein 65 (AP65) gene had been conducted to detect T. vaginalis. The optimum reaction system and conditions were optimized in this rapid detection method. Results: The results of sensitivity analysis showed that the LAMP assay targeting the AP65 gene was 1000 times more sensitive than the nested PCR targeting the actin gene commonly used for detection of T. vaginalis, and the detecting limitation of the former was 10 trichomonad. Moreover, the amplification of the target gene AP65 by LAMP assay exhibited high specificity and the product was exclusively from T. vaginalis. The detection technique of LAMP did not exhibit cross-reactivity with the common pathogens of Trichinella spiralis, Toxoplasma gondii, Escherichia coli, Candida albicans, Staphylococcus aureus, Haemophilus. Conclusions: According to the present study, the LAMP assay with the target of AP65 gene, was suitable for the early diagnosis of T. vaginalis infections. Consequently, the LAMP assay was proposed by the current study as a point-of-care examination and an alternative molecular tool which exhibited the potential value in the treatment, control and prevention of trichomoniasis transmission and relevant complication.

scholarly journals Development of a quantitative loop-mediated isothermal amplification assay for the field detection ofErysiphe necator

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4639 ◽  
Author(s):  
Lindsey D. Thiessen ◽  
Tara M. Neill ◽  
Walter F. Mahaffee
Keyword(s):  
Powdery Mildew ◽  
Pathogen Detection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Management Tool ◽  
Willamette Valley ◽  
Loop Mediated Isothermal Amplification ◽  
Grape Powdery Mildew

Plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. More recently, a loop-mediated isothermal amplification (LAMP) assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low DNA concentrations. A quantitative LAMP (qLAMP) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the Willamette Valley of Oregon. Custom impaction spore samplers were placed at a research vineyard and six commercial vineyard locations, and were tested bi-weekly by the lab and by growers. Grower-conducted qLAMP assays used a beta-version of the Smart-DART handheld LAMP reaction devices (Diagenetix, Inc., Honolulu, HI, USA), connected to Android 4.4 enabled, Bluetooth-capable Nexus 7 tablets for output. Quantification by a quantitative PCR assay was assumed correct to compare the lab and grower qLAMP assay quantification. Growers were able to conduct and interpret qLAMP results; however, theErysiphe necatorinoculum quantification was unreliable using the beta-Smart-DART devices. The qLAMP assay developed was sensitive to one spore in early testing of the assay, but decreased to >20 spores by the end of the trial. The qLAMP assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other, more reliable molecular tools.

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