Point-of-Care Multiplexed Assays of Nucleic Acids Using Microcapillary-based Loop-Mediated Isothermal Amplification

2014 ◽  
Vol 86 (14) ◽  
pp. 7057-7062 ◽  
Author(s):  
Yi Zhang ◽  
Lu Zhang ◽  
Jiashu Sun ◽  
Yulei Liu ◽  
Xingjie Ma ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Point Of Care ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification

scholarly journals Scaling diagnostics in times of COVID-19: Colorimetric Loop-mediated Isothermal Amplification (LAMP) assisted by a 3D-printed incubator for cost-effective and scalable detection of SARS-CoV-2

2020 ◽  
Author(s):  
Everardo González-González ◽  
Itzel Montserrat Lara-Mayorga ◽  
Iram Pablo Rodríguez-Sánchez ◽  
Felipe Yee-de León ◽  
Andrés García-Rubio ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Point Of Care ◽  
Three Dimensional ◽  
Cost Effective ◽  
Reaction Scheme ◽  
Ease Of Use ◽  
Isothermal Amplification ◽  
N Protein ◽  
Loop Mediated Isothermal Amplification ◽  
3D Printed

AbstractBy the third week of June 2020, more than 8,500,000 positive cases of COVID-19 and more than 450,000 deaths had been officially reported worldwide. The COVID-19 pandemic arrived in Latin America, India, and Africa—territories in which the mounted infrastructure for diagnosis is greatly underdeveloped. Here, we demonstrate the combined use of a three-dimensional (3D)-printed incubation chamber for commercial Eppendorf PCR tubes, and a colorimetric embodiment of a loop-mediated isothermal amplification (LAMP) reaction scheme for the detection of SARS-CoV-2 nucleic acids. We used this strategy to detect and amplify SARS-CoV-2 genetic sequences using a set of in-house designed initiators that target regions encoding the N protein. We were able to detect and amplify SARS-CoV-2 nucleic acids in the range of 62 to 2 × 105 DNA copies by this straightforward method. Using synthetic SARS-CoV-2 samples and a limited number of RNA extracts from patients, we also demonstrate that colorimetric LAMP is a quantitative method comparable in diagnostic performance to RT-qPCR. We envision that LAMP may greatly enhance the capabilities for COVID-19 testing in situations where RT-qPCR is not feasible or is unavailable. Moreover, the portability, ease of use, and reproducibility of this strategy make it a reliable alternative for deployment of point-of-care SARS-CoV-2 detection efforts during the pandemics.

scholarly journals Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Bruna de Oliveira Coelho ◽  
Heloisa Bruna Soligo Sanchuki ◽  
Dalila Luciola Zanette ◽  
Jeanine Marie Nardin ◽  
Hugo Manuel Paz Morales ◽  
...  
Keyword(s):  
Viral Load ◽  
Internal Control ◽  
Reverse Transcription ◽  
Color Change ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
False Negative ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

Abstract Background SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. Methods To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. Results First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. Conclusion This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

scholarly journals Loop Mediated Isothermal Amplification: Principles and Applications in Plant Virology

Plants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 461 ◽  
Author(s):  
Stefano Panno ◽  
Slavica Matić ◽  
Antonio Tiberini ◽  
Andrea Giovanni Caruso ◽  
Patrizia Bella ◽  
...  
Keyword(s):  
Plant Protection ◽  
Point Of Care ◽  
Low Cost ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Plant Virology ◽  
Food Industries ◽  
Loop Mediated Isothermal Amplification ◽  
Application Fields ◽  
Agriculture And Food

In the last decades, the evolution of molecular diagnosis methods has generated different advanced tools, like loop-mediated isothermal amplification (LAMP). Currently, it is a well-established technique, applied in different fields, such as the medicine, agriculture, and food industries, owing to its simplicity, specificity, rapidity, and low-cost efforts. LAMP is a nucleic acid amplification under isothermal conditions, which is highly compatible with point-of-care (POC) analysis and has the potential to improve the diagnosis in plant protection. The great advantages of LAMP have led to several upgrades in order to implement the technique. In this review, the authors provide an overview reporting in detail the different LAMP steps, focusing on designing and main characteristics of the primer set, different methods of result visualization, evolution and different application fields, reporting in detail LAMP application in plant virology, and the main advantages of the use of this technique.

scholarly journals Loop-Mediated Isothermal Amplification as Point-of-Care Diagnosis for Neglected Parasitic Infections

2020 ◽  
Vol 21 (21) ◽  
pp. 7981
Author(s):  
Catalina Avendaño ◽  
Manuel Alfonso Patarroyo
Keyword(s):  
Neglected Tropical Diseases ◽  
Point Of Care ◽  
Low Cost ◽  
High Specificity ◽  
World Health Organisation ◽  
Isothermal Amplification ◽  
World Health ◽  
Parasitic Infections ◽  
River Blindness ◽  
Loop Mediated Isothermal Amplification

The World Health Organisation (WHO) has placed twenty diseases into a group known as neglected tropical diseases (NTDs), twelve of them being parasitic diseases: Chagas’ disease, cysticercosis/taeniasis, echinococcosis, food-borne trematodiasis, human African trypanosomiasis (sleeping sickness), leishmaniasis, lymphatic filariasis, onchocerciasis (river blindness), schistosomiasis, soil-transmitted helminthiasis (ascariasis, hookworm, trichuriasis), guinea-worm and scabies. Such diseases affect millions of people in developing countries where one of the main problems concerning the control of these diseases is diagnosis-based due to the most affected areas usually being far from laboratories having suitable infrastructure and/or being equipped with sophisticated equipment. Advances have been made during the last two decades regarding standardising and introducing techniques enabling diagnoses to be made in remote places, i.e., the loop-mediated isothermal amplification (LAMP) technique. This technique’s advantages include being able to perform it using simple equipment, diagnosis made directly in the field, low cost of each test and the technique’s high specificity. Using this technique could thus contribute toward neglected parasite infection (NPI) control and eradication programmes. This review describes the advances made to date regarding LAMP tests, as it has been found that even though several studies have been conducted concerning most NPI, information is scarce for others.

scholarly journals Specific and sensitive, ready-to-use universal fungi detection by visual color using ITS1 loop-mediated isothermal amplification combined hydroxynaphthol blue

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11082
Author(s):  
Ilada Choopara ◽  
Yothin Teethaisong ◽  
Narong Arunrut ◽  
Sudaluck Thunyaharn ◽  
Wansika Kiatpathomchai ◽  
...  
Keyword(s):  
Point Of Care ◽  
Colorimetric Detection ◽  
High Sensitivity ◽  
Fungal Species ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Fungal Isolates ◽  
Simple Detection ◽  
Hydroxynaphthol Blue ◽  
Life Threatening

Being ubiquitous, fungi are common opportunistic pathogens to humans that can lead to invasive and life-threatening infections in immunocompromised individuals. Eukaryote-resembling cell membrane and filamentous branches make the fungal diagnosis difficult. This study therefore developed a ready-to-use ITS1 loop-mediated isothermal amplification combined with hydroxynaphthol blue (LAMP-HNB) for rapid, sensitive and specific colorimetric detection of universal fungi in all phyla. The ITS1 LAMP-HNB could identify every evolutionary phylum of fungi according to sequence analyses. We tested a total of 30 clinically relevant fungal isolates (representing three major human pathogenic phyla of fungi, namely Zygomycota, Ascomycota and Basidiomycota) and 21 non-fungal isolates, and the ITS1 LAMP-HNB properly identified all isolates, with a detection limit of as low as 4.6 ag (9.6 copies), which was identical to ITS1 and 18S rDNA PCR. The assays were also validated on the feasibility of point-of-care diagnostic with real food (dry peanuts, chili and garlics) and blood samples. Furthermore, the shelf life of our ready-to-use ITS1 LAMP activity (≥50%) was more than 40 days at 30 °C with 3–5% polyvinyl alcohol or glycerol additive. The results supported the ready-to-use ITS1 LAMP-HNB for simple detection of fungi contamination with high sensitivity in local and resource-constrained areas to prevent opportunistic fungal species infections.

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