scholarly journals The detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP) in developing country

10.51511/pr.9 ◽  
2021 ◽  
Vol 1 (1) ◽  
pp. 9
Author(s):  
Muhammad Rizky Wibowo ◽  
Erna Harfiani ◽  
Sarmoko Sarmoko ◽  
Yudhi Nugraha
Keyword(s):  
Reverse Transcription ◽  
Rural Areas ◽  
Genetic Material ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
World Health ◽  
Rt Pcr ◽  
Loop Mediated Isothermal Amplification ◽  
Alternative Detection ◽  
Health Organization

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) has infected the human system resulting in Covid-19, and has spread rapidly worldwide. Therefore, a fast, simple, cost-effective, and accurate detecting tool is required. The standard diagnostic tool of the World Health Organization is the reverse transcription-polymerase chain reaction (RT-PCR). This method detects the presence of viral genetic material in the human body with accurate results. However, it has several limitations in terms of equipment, personnel, duration, and cost. Therefore, a fast, simple, and sensitive alternative detection, is required, one of which is the reverse transcription loop-mediated isothermal amplification (RT-LAMP) that functions under isothermal conditions. This method is battery-driven, hence, easy to move closer to the patient. Conclusively, the RT-LAMP test for SARS CoV-2 diagnosis produces comparable sensitivity to a standard RT-PCR and is more suitable for resource-poor settings, such as rural areas of developing countries.

scholarly journals Evaluation of RT-qPCR and Loop-Mediated Isothermal Amplification (LAMP) Assays for the Detection of SARS-CoV-2 in Argentina

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 659
Author(s):  
María Dolores Fellner ◽  
Romina Bonaventura ◽  
Jorge Basiletti ◽  
Martín Avaro ◽  
Estefanía Benedetti ◽  
...  
Keyword(s):  
World Health Organization ◽  
Reverse Transcription ◽  
Diagnostic Performance ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Isothermal Amplification ◽  
World Health ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Health Organization

Our aim was to evaluate the analytical and clinical performance of the SARS-CoV-2 molecular detection kits used in Argentina. Nine real-time reverse-transcription polymerase chain reaction (RT-qPCR) and three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were evaluated using the World Health Organization (WHO) recommended test as reference method. A secondary standard calibrated for the E, N and RdRp genes against the Pan American Health Organization—World Health Organization—International Standard was used to calculate the limit of detection (LoD). A panel of artificial clinical samples, 32 positive and 30 negative for SARS-CoV-2, were analyzed to estimate the kappa concordance (κ) and the diagnostic performance. Differences among the LoD values for the target genes amplified by each kit were >1 log copies/reaction. The κ for the RT-qPCR kits was greater than 0.9, whereas that for the RT-LAMP assays ranged from 0.75 to 0.93. The clinical performance of RT-qPCR kits showed 100% specificity and high sensitivity, although with variations according to the gene analyzed. The E and N genes provided greater clinical sensitivity, whereas the RdRp gene increased the clinical specificity. The RT-LAMP assays revealed a variable diagnostic performance. The information provided can be useful to choose the most appropriate diagnostic test and may contribute to the establishment of a consensus in the diagnosis of SARS-CoV-2 in Argentina and the region.

scholarly journals Preliminary study of coronavirus disease 2019 on pets in pandemic in Denpasar, Bali, Indonesia

2021 ◽  
pp. 2979-2983
Author(s):  
Hamong Suharsono ◽  
Ali Ghufron Mukti ◽  
Ketut Suryana ◽  
I. Wayan Masa Tenaya ◽  
Dilasdita Kartika Pradana ◽  
...  
Keyword(s):  
Close Contact ◽  
Genetic Material ◽  
Rna Isolation ◽  
World Health ◽  
Intermediate Hosts ◽  
Rt Pcr ◽  
Pcr Products ◽  
Preliminary Study ◽  
Health Organization ◽  
Respiratory Droplets

Background and Aim: Coronavirus disease 2019 (COVID-19) is an acute infectious respiratory disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has spread rapidly globally, resulting in a pandemic. In humans, the main routes of transmission are respiratory droplets and close contact with infected individuals or through contact with an object infected with the virus, followed by touching mouth, nose, or eyes. It is assumed that SARS-CoV-2 was originated in wild animals and was then transmitted to humans. Although some wildlife and domestic animals can be naturally or experimentally infected with the virus, the intermediate hosts that transmitted it to humans are still unknown. Understanding the dynamics of SARS-CoV-2 associated with possible zoonotic transmission of intermediate hosts is considered critical. Reportedly, cats or dogs living with COVID-19-positive humans tested positive for the disease, suggesting that the virus was transmitted to the animals from humans. Information regarding the epidemiological investigation and comprehensive studies is limited. Therefore, it is still unclear how high is the correlation of infection in humans and pet animals, especially those living together. The aim of this study was to investigate the possibility of SARS-CoV-2 infection in the pets of patients with COVID-19 who were hospitalized at the Wangaya hospital, Denpasar, Bali, Indonesia. Materials and Methods: A total of seven clinically asymptomatic pets (six dogs of different races and sexes and a cat [age, 360-2920 days]) were included in this study. These animals belonged to patients with confirmed SARS-CoV-2 infection from August to November 2020. Nasal swab and nasopharyngeal samples were collected from the pets individually under anesthetic condition and were collected 6-12 days after confirmed SARS-CoV-2 infection in owners and hospitalization at the Wangaya Hospital. The swab samples were then processed for RNA isolation and tested using reverse transcription-polymerase chain reaction (RT-PCR) for SARS-CoV-2, in accordance with the World Health Organization manual 2020. Results: RT-PCR results for all seven RNA samples, prepared from the swab samples, were negative. For the samples, all PCR products were below the threshold limit, suggesting no genetic material belonging to the samples tested. Conclusion: This was the first preliminary study of COVID-19 on pets in pandemic using RT-PCR. The study tested a very limited quantity of samples, and all of them were negative. However, the way in which the samples were prepared was considered appropriate. Therefore, in further studies, testing of more samples of pets of more individuals with confirmed SARS-CoV-2 infection is required.

scholarly journals Reverse Transcription Loop-Mediated Isothermal Amplification for the Detection of Porcine Reproductive and Respiratory Syndrome Virus

2009 ◽  
Vol 21 (3) ◽  
pp. 350-354 ◽  
Author(s):  
Albert Rovira ◽  
Juan Abrahante ◽  
Michael Murtaugh ◽  
Muñoz-Zanzi Claudia
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Restriction Analysis ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Serum Samples ◽  
Loop Mediated Isothermal Amplification

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine. The objective of the current study is to investigate the feasibility of using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of PRRSV. The RT-LAMP is a recently described DNA amplification technique reported to be simple, inexpensive, fast, and accurate. The RT-LAMP reaction was set up using 2 sets of primers that were designed to detect North American and European strains of PRRSV and performed successfully in a simple heat block. The specificity of the amplified product was demonstrated by restriction analysis. The RT-LAMP was able to detect 5 different PRRSV isolates. However, the limit of detection ranged between 10 2 and 10 4 50% tissue culture infective dose/ml. The RT-LAMP was further evaluated using serum samples from animals of known infection status. The ability of RT-LAMP to detect PRRSV in serum from acutely infected animals was evaluated with 114 serum samples from 18 experimentally inoculated boars. Forty-nine of these samples tested positive by RT-LAMP, while 94 were positive by reverse transcription polymerase chain reaction (RT-PCR). The diagnostic specificity, evaluated with 100 known negative serum samples, was estimated as 99%. The feasibility of RT-LAMP to detect PRRSV was demonstrated in the current study. The RT-LAMP reaction could be performed in just 1 hr with a simple and inexpensive heat block. However, the sensitivity of this technique was significantly lower than that of RT-PCR.

scholarly journals Identificação molecular de SARS-CoV-2 em superfícies ambientais em estabelecimentos de saúde de uma universidade pública no Brasil

2021 ◽  
Vol 2 (4) ◽  
pp. 431-441
Author(s):  
Caio Ricardo Eich ◽  
Barbara Scariot Colombelli ◽  
Kattlyn Larissa Candido ◽  
Luciana Oliveira De Fariña
Keyword(s):  
Internal Control ◽  
Local Population ◽  
Genetic Material ◽  
Virus Transmission ◽  
Health Surveillance ◽  
World Health ◽  
Rt Pcr ◽  
Surveillance Studies ◽  
Risk Zones ◽  
Health Organization

Em 11 de março de 2020, a Organização Mundial de Saúde (OMS) decretou a pandemia do COVID-19, causado pelo vírus SARS-CoV-2, responsável por mais de 4,5 milhões de mortes até o momento. Esta nova realidade exigiu respostas por parte das autoridades e da população, a fim de mitigar a propagação do vírus e evitar o colapso do sistema de saúde, assim como estudos de vigilância em saúde, que possibilitaram um melhor entendimento dos mecanismos de transmissão do vírus e possibilitaram identificar zonas de risco dentro de cidades ou ambientes públicos. Este estudo tem o objetivo de identificar a presença do SARS-CoV-2 dentro da Universidade Estadual do Oeste do Paraná, a qual fornece serviços de saúde para a população local, assim como realizar um controle interno no Laboratório de Bioquímica Molecular (LaBioqMol) da universidade, onde são realizados testes de RT-PCR semanalmente. Foram coletadas 21 amostras de áreas frequentemente tocadas por pessoas, cuja presença do RNA viral e de material genético humano foi identificada por RT-PCR. Em nenhuma das amostras foi detectado a presença do vírus. Entretanto, em 8 (38,1%) das amostras foi verificada a amplificação do gene RNaseP, indicando a presença de células humana. Este estudo auxilia no controle e garantia de qualidade do LaBioqMol e fortalece a visão de que a contaminação do ambiente pelo SARS-CoV-2 é provavelmente menos frequente do que foi anteriormente sugerido no início da pandemia.   On March 11, 2020, the World Health Organization (WHO) decreed the pandemic of COVID-19, caused by the SARS-CoV-2 virus, responsible for more than 4.5 million deaths to date. This new reality demanded responses from the authorities and the population in order to mitigate the spread of the virus and avoid the collapse of the health system, as well as health surveillance studies, which enabled a better understanding of the mechanisms of virus transmission and made it possible to identify risk zones within cities or public environments. This study aims to identify the presence of SARS-CoV-2 within the Universidade Estadual do Oeste do Paraná, which provides health services to the local population, as well as to perform an internal control at the university's Molecular Biochemistry Laboratory (LaBioqMol), where RT-PCR tests are performed weekly. Twenty-one samples were collected from areas frequently touched by people, and the presence of viral RNA and human genetic material was identified by RT-PCR. In none of the samples was the presence of the virus detected. However, in 8 (38.1%) of the samples the RNaseP gene amplification was verified, indicating the presence of human cells. This study assists in quality control and assurance at LaBioqMol and strengthens the view that environmental contamination by SARS-CoV-2 is probably less frequent than was previously suggested at the beginning of the pandemic.

scholarly journals Rapid and Sensitive Detection of Tomato Brown Rugose Fruit Virus in Tomato and Pepper Seeds by Reverse Transcription Loop-Mediated Isothermal Amplification Assays (Real Time and Visual) and Comparison With RT-PCR End-Point and RT-qPCR Methods

2021 ◽  
Vol 12 ◽  
Author(s):  
Domenico Rizzo ◽  
Daniele Da Lio ◽  
Alessandra Panattoni ◽  
Chiara Salemi ◽  
Giovanni Cappellini ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
The European Union ◽  
Rt Pcr ◽  
Loop Mediated Isothermal Amplification ◽  
End Point ◽  
Specificity And Sensitivity ◽  
The Status

Tomato brown rugose fruit virus (ToBRFV) represents an emerging viral threat to the productivity of tomato and pepper protected cultivation worldwide. This virus has got the status of quarantine organism in the European Union (EU) countries. In particular, tomato and pepper seeds will need to be free of ToBRFV before entering the EU and before coming on the market. Thus, lab tests are needed. Here, we develop and validate a one-step reverse transcription LAMP platform for the detection of ToBRFV in tomato and pepper leaves, by real-time assay [reverse transcription loop-mediated isothermal amplification (RT-LAMP)] and visual screening (visual RT-LAMP). Moreover, these methods can also be applied successfully for ToBRFV detection in tomato and pepper seeds. The diagnostic specificity and sensitivity of both RT-LAMP and visual RT-LAMP are both 100%, with a detection limit of nearly 2.25 fg/μl, showing the same sensitivity as RT-qPCR Sybr Green, but 100 times more sensitive than end-point RT-PCR diagnostic methods. In artificially contaminated seeds, the proposed LAMP assays detected ToBRFV in 100% of contaminated seed lots, for up to 0.025–0.033% contamination rates in tomato and pepper, respectively. Our results demonstrate that the proposed LAMP assays are simple, inexpensive, and sensitive enough for the detection of ToBRFV, especially in seed health testing. Hence, these methods have great potential application in the routine detection of ToBRFV, both in seeds and plants, reducing the risk of epidemics.

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 Rapid Detection of Peste des Petits Ruminants Virus (PPRV) Nucleic Acid Using a Novel Low-Cost Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for Future Use in Nascent PPR Eradication Programme

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 699 ◽  
Author(s):  
Mahapatra ◽  
Howson ◽  
Fowler ◽  
Batten ◽  
Flannery ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Lamp Assay ◽  
Early Warning Systems ◽  
Isothermal Amplification ◽  
Effective Control ◽  
Peste Des Petits Ruminants ◽  
Rt Pcr ◽  
Eradication Programme ◽  
Loop Mediated Isothermal Amplification

Peste des petits ruminants (PPR) is a disease of small ruminants caused by peste des petits ruminants virus (PPRV), and is endemic in Asia, the Middle East and Africa. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) have improved the sensitivity and rapidity of diagnosing PPR. However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for PPR would improve the fast implementation of control policies, particularly when PPR has been targeted to be eradicated by 2030. Loop-mediated isothermal amplification (LAMP) assays are simple to use, rapid, and have sensitivity and specificity within the range of RT-qPCR; and can be performed in the field using disposable consumables and portable equipment. This study describes the development of a novel RT-LAMP assay for the detection of PPRV nucleic acid by targeting the N-protein gene. The RT-LAMP assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I-IV) of PPRV. The test displayed 100% concordance with RT-qPCR when considering an RT-qPCR cut-off value of CT >40. Further, the RT-LAMP assay was evaluated using experimental and outbreak samples without prior RNA extraction making it more time and cost-effective. This assay provides a solution for a pen-side, rapid and inexpensive PPR diagnostic for use in the field in nascent PPR eradication programme.

scholarly journals Reverse Transcription Loop-Mediated Isothermal Amplification of DNA for Detection of Potato virus Y

Plant Disease ◽  
2005 ◽  
Vol 89 (6) ◽  
pp. 605-610 ◽  
Author(s):  
Xianzhou Nie
Keyword(s):  
Potato Virus ◽  
Reverse Transcription ◽  
Potato Virus Y ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Time Saving ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

A reverse transcription loop-mediated isothermal amplification of DNA (RT-LAMP) for detection of Potato virus Y (PVY) was developed. In this procedure, a set of four primers matching a total of six sequences of the coat protein (CP) gene of PVY were designed in such a way that a loop could be formed and elongated during DNA amplification. Using PVY CP complementary DNA clones as templates, the LAMP reaction was optimized by adjusting the concentrations of MgSO4, dNTPs, and Bst DNA polymerase. The effects of fragment length of target DNA on LAMP also were investigated. Two-step and one-step RT-LAMPs were performed using RNA extracts of various PVY cultures, and the results were correlated with two-step reverse transcription polymerase chain reaction (RT-PCR) for detection of PVY. Further, the turbidity caused by precipitation of magnesium pyrophosphate formed in positive RT-LAMP reactions was used to measure the amplification by utilizing a time-saving spectrophotometric method. The one-step RT-LAMP-turbidity method gave results comparable with the two-step RT-PCR method for detection of PVY from potato leaf and tuber samples. Of the total 240 samples, 234 were diagnosed similarly by both methods.

scholarly journals Detecting SARS-CoV-2 at point of care: preliminary data comparing loop-mediated isothermal amplification (LAMP) to polymerase chain reaction (PCR)

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Marc F. Österdahl ◽  
Karla A. Lee ◽  
Mary Ni Lochlainn ◽  
Stuart Wilson ◽  
Sam Douthwaite ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Predictive Value ◽  
Point Of Care ◽  
Isothermal Amplification ◽  
Service Improvement ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Polymerase Chain

Abstract Background A cost effective and efficient diagnostic tool for COVID-19 as near to the point of care (PoC) as possible would be a game changer in the current pandemic. We tested reverse transcription loop mediated isothermal amplification (RT-LAMP), a method which can produce results in under 30 min, alongside standard methods in a real-life clinical setting. Methods This prospective service improvement project piloted an RT-LAMP method on nasal and pharyngeal swabs on 21 residents of a high dependency care home, with two index COVID-19 cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (RT-PCR). We recorded vital signs of patients to correlate clinical and laboratory information and calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of a single swab using RT-LAMP compared with the current standard, RT-PCR, as per Standards for Reporting Diagnostic Accuracy Studies (STARD) guidelines. Results The novel method accurately detected 8/10 RT-PCR positive cases and identified a further 3 positive cases. Eight further cases were negative using both methods. Using repeated RT-PCR as a “gold standard”, the sensitivity and specificity of a single novel test were 80 and 73% respectively. PPV was 73% and NPV was 83%. Incorporating retesting of low signal RT-LAMP positives improved the specificity to 100%. We also speculate that hypothermia may be a significant early clinical sign of COVID-19. Conclusions RT-LAMP testing for SARS-CoV-2 was found to be promising, fast and to work equivalently to RT-PCR methods. RT-LAMP has the potential to transform COVID-19 detection, bringing rapid and accurate testing to the PoC. RT-LAMP could be deployed in mobile community testing units, care homes and hospitals to detect disease early and prevent spread.

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