Loop Mediated Isothermal Amplification: Principles and Applications in Plant Virology

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.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21217981 ◽

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.

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Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes

10.1101/2020.12.21.20248288 ◽

2020 ◽

Author(s):

Daniel Urrutia-Cabrera ◽

Roxanne Hsiang-Chi Liou ◽

Jianxiong Chan ◽

Sandy Shen-Chi Hung ◽

Alex W Hewitt ◽

...

Keyword(s):

Comparative Analysis ◽

Reaction Time ◽

False Positive ◽

Point Of Care ◽

Low Cost ◽

Isothermal Amplification ◽

Fluorescent Detection ◽

Experimental Conditions ◽

Diagnostic Laboratory ◽

Loop Mediated Isothermal Amplification

AbstractThe COVID-19 pandemic caused by SARS-CoV-2 has infected millions worldwide and there is an urgent need to increase our diagnostic capacity to identify infected cases. Although RT-qPCR remains the gold standard for SARS-CoV-2 detection, this method requires specialised equipment in a diagnostic laboratory and has a long turn-around time to process the samples. To address this, several groups have recently reported development of loop-mediated isothermal amplification (LAMP) as a simple, low cost and rapid method for SARS-CoV-2 detection. Herein we present a comparative analysis of three LAMP-based assays that target different regions of the SARS-CoV-2: ORF1ab RdRP, ORF1ab nsp3 and Gene N. We perform a detailed assessment of their sensitivity, kinetics and false positive rates for SARS-CoV-2 diagnostics in LAMP or RT-LAMP reactions, using colorimetric or fluorescent detection. Our results independently validate that all three assays can detect SARS-CoV-2 in 30 minutes, with robust accuracy at detecting as little as 1000 RNA copies and the results can be visualised simply by color changes. We also note the shortcomings of these LAMP-based assays, including variable results with shorter reaction time or lower load of SARS-CoV-2, and false positive results in some experimental conditions. Overall for RT-LAMP detection, the ORF1ab RdRP and ORF1ab nsp3 assays have higher sensitivity and faster kinetics for detection, whereas the Gene N assay exhibits no false positives in 30 minutes reaction time. This study provides validation of the performance of LAMP-based assays for SARS-CoV-2 detection, which have important implications in development of point-of-care diagnostic for SARS-CoV-2.

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Highly performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification.

10.1101/2020.08.01.20166538 ◽

2020 ◽

Author(s):

Pierre Garneret ◽

Etienne Coz ◽

Elian Martin ◽

Jean-Claude Manuguerra ◽

Elodie Brient-Litzler ◽

...

Keyword(s):

Point Of Care ◽

Low Cost ◽

Rna Extraction ◽

Dna Amplification ◽

Developed Countries ◽

Temperature Cycling ◽

Isothermal Amplification ◽

Nucleic Acid Amplification ◽

Clinical Samples ◽

Molecular Testing

In order to respond to the urgent request of massive testing, developed countries perform nucleic acid amplification tests (NAAT) of SARS-CoV-2 in centralized laboratories. Real-time RT - PCR (Reverse transcription - Polymerase Chain Reaction) is used to amplify the viral RNA and enable its detection. Although PCR is 37 years old, it is still considered, without dispute, as the gold standard. PCR is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings. In the present work, by harnessing progress made in the past two decades in DNA amplification, microfluidics and membrane technologies, we succeeded to create a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT - LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or highly fluorescent probes. Depending on the viral load, the detection takes between twenty minutes and one hour. Using pools of naso-pharyngal clinical samples, we estimated a sensitivity comparable to RT-qPCR (up to a Cycle threshold of 39, equivalent to <0.1 TCID50 per mL) and a 100% specificity, for other human coronaviruses and eight respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called COVIDISC to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment paves the way towards a large dissemination of this device. The perspective of a reliable SARS-CoV-2 point of care detection, highly performing, that would deliver on-site results in less than one hour opens up a new efficient approach to manage the pandemics.

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Advances in loop-mediated isothermal amplification: integrated with several point-of-care diagnostic methods

Analytical Methods ◽

10.1039/c4ay00330f ◽

2014 ◽

Vol 6 (19) ◽

pp. 7585-7589 ◽

Cited By ~ 9

Author(s):

Pin Gong ◽

Taotao Zhang ◽

Fuxin Chen ◽

Lan Wang ◽

Sai Jin ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Point Of Care ◽

Low Cost ◽

Isothermal Amplification ◽

Diagnostic Methods ◽

Diagnostic Systems ◽

Loop Mediated Isothermal Amplification

Recent outbreaks linked to pathogenic bacteria heighten the need to develop rapid, sensitive, portable and low-cost pathogen diagnostic systems.

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Loop-Mediated Isothermal Amplification (LAMP) for the Diagnosis of Zika Virus: A Review

Viruses ◽

10.3390/v12010019 ◽

2019 ◽

Vol 12 (1) ◽

pp. 19 ◽

Cited By ~ 11

Author(s):

Severino Jefferson Ribeiro da Silva ◽

Keith Pardee ◽

Lindomar Pena

Keyword(s):

Zika Virus ◽

Molecular System ◽

Point Of Care ◽

Low Cost ◽

Quantitative Polymerase Chain Reaction ◽

High Specificity ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification ◽

Freeze Dried ◽

Polymerase Chain

The recent outbreak of Zika virus (ZIKV) in the Americas and its devastating developmental and neurological manifestations has prompted the development of field-based diagnostics that are rapid, reliable, handheld, specific, sensitive, and inexpensive. The gold standard molecular method for lab-based diagnosis of ZIKV, from either patient samples or insect vectors, is reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The method, however, is costly and requires lab-based equipment and expertise, which severely limits its use as a point-of-care (POC) tool in resource-poor settings. Moreover, given the lack of antivirals or approved vaccines for ZIKV infection, a POC diagnostic test is urgently needed for the early detection of new outbreaks and to adequately manage patients. Loop-mediated isothermal amplification (LAMP) is a compelling alternative to RT-qPCR for ZIKV and other arboviruses. This low-cost molecular system can be freeze-dried for distribution and exhibits high specificity, sensitivity, and efficiency. A growing body of evidence suggests that LAMP assays can provide greater accessibility to much-needed diagnostics for ZIKV infections, especially in developing countries where the ZIKV is now endemic. This review summarizes the different LAMP methods that have been developed for the virus and summarizes their features, advantages, and limitations.

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Rapid and reliable distinguish of Morinda officinalis How by loop-mediated isothermal amplification (LAMP) assay

10.21203/rs.3.rs-120446/v1 ◽

2020 ◽

Author(s):

Shuhan Wang ◽

Aihua Sui ◽

Yafei Han ◽

Quan Zhou ◽

Hang Xu ◽

...

Keyword(s):

Low Cost ◽

Lamp Assay ◽

Its2 Sequence ◽

Isothermal Amplification ◽

Nucleic Acid Amplification ◽

Nuclear Ribosomal Dna ◽

Traditional Medicines ◽

Loop Mediated Isothermal Amplification ◽

Amplification Method ◽

Human Disorders

Abstract The medicinal plant Morinda officinalis How (MO), especially the root, has been frequently used in traditional medicines around the world as an herbal drug for treating variable human disorders and diseases. Various adulterations of MO were found for economic or production limitations. However, authentication of MO from its adulterants by LAMP has not yet been established. The present study introduces a commercially available nucleic acid amplification method, loop-mediated isothermal amplification (LAMP) assay for the distinguish of MO from its adulterants. In this method, we combined DNA barcodes technology to design 2 pairs independent LAMP primers, which based on the internal transcribed spacer 2 (ITS2) sequence of MO’s nuclear ribosomal DNA. Our results showed that the LAMP could amplify the samples as expected and successfully identify target MO, and the limit for DNA template preciseness was verified as 1 × 10− 1 pg/µl. All the visual or real-time turbidity detection was performed within 60 min at approximately 63 °C. The result showed that the LAMP assay and primers we designed have high accuracy and efficiency for the differentiation of MO and its adulterants. Our results illustrated that the proposed low-cost, fast and reliable LAMP assay without the need for expensive equipment or specialized techniques could be a good way for MO rapid authentication.

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Visual and Rapid Diagnosis of Neisseria gonorrhoeae Using Loop-Mediated Isothermal Amplification Combined With a Polymer Nanoparticle–Based Biosensor in Clinical Application

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.702134 ◽

2021 ◽

Vol 8 ◽

Author(s):

Xu Chen ◽

Qingxue Zhou ◽

Xueli Wu ◽

Shuoshi Wang ◽

Rui Liu ◽

...

Keyword(s):

Limit Of Detection ◽

Detection System ◽

Point Of Care ◽

Low Cost ◽

Isothermal Amplification ◽

Clinical Samples ◽

Global Public Health ◽

Fixed Temperature ◽

Polymer Nanoparticle ◽

Loop Mediated Isothermal Amplification

Neisseriagonorrhoeae is a host-adapted human pathogen that causes sexually transmitted gonorrhea and remains to be a serious global public health challenge, especially in low- and middle-income regions. It is vital to devise a reliable, simple, cost-saving, and easy-to-use assay for detecting the N. gonorrhoeae agent. In the current study, we firstly report a novel approach, loop-mediated isothermal amplification linked with a polymer nanoparticle–based biosensor (LAMP-PNB), that was used for identifying N. gonorrhoeae in clinical samples. The results showed that the LAMP primers based on the orf1 gene were valid for development of the N. gonorrhoeae-LAMP-PNB assay. The detection system with optimal conditions could be performed at a fixed temperature of 64°C for 40 min. The whole process, including genomic DNA preparation (approximately 10 min), LAMP reaction (40 min), and PNB reporting (approximately 2 min), could be accomplished within 60 min. The limit of detection (LoD) of the N. gonorrhoeae-LAMP-PNB assay was 50 copies per test. The specificity of the current assay was 100%, and no cross-reactions to non–N. gonorrhoeae isolates were observed. These results confirmed that the N. gonorrhoeae-LAMP-PNB technique is a reliable, specific, sensitive, rapid, low-cost, and easy-to-use method for detecting gonococci isolates. More importantly, this assay has great potential to develop a point-of-care (POC) testing method in clinical practice, especially in resource-constrained regions.

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

Molecular Medicine ◽

10.1186/s10020-021-00289-0 ◽

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.

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