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 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 Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Keith Pardee ◽  
Udeni B. R. Balasuriya ◽  
Lindomar Pena
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Serum Samples ◽  
Low Resource ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

scholarly journals Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Matthew A Lalli ◽  
Joshua S Langmade ◽  
Xuhua Chen ◽  
Catrina C Fronick ◽  
Christopher S Sawyer ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Colorimetric Assay ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Viral Detection ◽  
Loop Mediated Isothermal Amplification

Abstract Background Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. Methods To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. Results The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44–104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Conclusions Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

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 Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

2021 ◽  
Vol 12 ◽  
Author(s):  
Pedro A. Alves ◽  
Ellen G. de Oliveira ◽  
Ana Paula M. Franco-Luiz ◽  
Letícia T. Almeida ◽  
Amanda B. Gonçalves ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Isothermal Amplification ◽  
Alternative Methods ◽  
Molecular Diagnostic ◽  
Clinical Validation ◽  
Rna Detection ◽  
Loop Mediated Isothermal Amplification ◽  
Viral Transport Medium

The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 ± 2.7 viral genomic copies/μL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65°C for 30 min. When compared to reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3–99.5%] sensitivity and 100% (95% CI = 94.5–100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non–SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction–free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

scholarly journals A Stoichiometric and Pseudo Kinetic Model of Loop Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Navjot Kaur ◽  
Nikhil Thota ◽  
Bhushan Toley
Keyword(s):  
Kinetic Model ◽  
Reaction Pathway ◽  
Reaction Network ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Reaction Products ◽  
Theoretical Understanding ◽  
Loop Mediated Isothermal Amplification ◽  
Commercial Applications

<p>Loop mediated isothermal amplification (LAMP) is one of the most popular isothermal DNA amplification techniques for research and commercial applications. The LAMP mechanism is powered by strategic primer design and a strand displacement polymerase, generating products that fold over, creating loops. LAMP leads to generation of products of increasing length over time. These products containing multiple loops are conventionally called cauliflower structures. Existing literature on LAMP provides extremely limited understanding of progression of cascades of reactions involved in the reaction and it is believed that cauliflower structures of increasing length constitute a majority of the product formed in LAMP. This study presents a first of its kind stoichiometric and pseudo kinetic model to comprehend LAMP reactions in deeper depth by (i) classifying LAMP reaction products into uniquely identifiable categories, (ii) generating a condensed reaction network to depict millions of interconnected reactions occurring during LAMP, and (iii) elucidating the pathways for amplicon generation. Despite the inherent limitations of conventional stoichiometric modelling for polymerization type reactions (the network rapidly becomes too large and intractable), our model provides new theoretical understanding of the LAMP reaction pathway. The model shows that while longer length products are formed, it is the smaller length recycle amplicons that contribute more towards the exponential increase in the amount of double stranded DNA. Prediction of concentration of different types of LAMP amplicons will also contribute substantially towards informing design of probe-based assays. </p>

scholarly journals A quite sensitive fluorescent loop-mediated isothermal amplification for rapid detection of respiratory syncytial virus

2019 ◽  
Vol 13 (12) ◽  
pp. 1135-1141 ◽  
Author(s):  
Yihong Hu ◽  
Zhenzhou Wan ◽  
Yonglin Mu ◽  
Yi Zhou ◽  
Jia Liu ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
High Morbidity ◽  
Rt Pcr ◽  
Loop Mediated Isothermal Amplification ◽  
Syncytial Virus ◽  
Syto 9

Introduction: Human respiratory syncytial virus (hRSV) is a common respiratory virus closely related to respiratory tract infection (RTI). Rapid and accurate detection of hRSV is urgently needed to reduce the high morbidity and mortality due to hRSV infection. Methodology: Here, we established a highly sensitive and specific reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of A and B group hRSV simultaneously. The specific primer sets for hRSV A and B groups were designed in the M and M2-2 gene, respectively. SYTO 9 was used as the fluorescent dye for real-time monitoring of the amplification of hRSV RNA without cross reaction between hRSV A and B. Results: The limit of detection (LOD) of our new method was 281.17 50% tissue culture infective doses (TCID50)/mL for hRSV A and 1.58 TCID50/mL for hRSV B. Using 90 clinical samples, a comparison to traditional RT-PCR was performed to validate this assay. The positivity rate of RT-LAMP and RT-PCR were 67.8% and 55.6%, respectively, and the positivity rate of RT-LAMP was significantly higher than RT-PCR (χ2 test, P < 0.01). Conclusions: Compared with traditional RT-PCR method, the newly developed fluorescent RT-LAMP combined with well-designed primers and SYTO 9 is quite sensitive, specific, rapid and well applicable to hRSV clinical diagnosis.

scholarly journals Fast and Parallel Detection of Four Ebola Virus Species on a Microfluidic-Chip-Based Portable Reverse Transcription Loop-Mediated Isothermal Amplification System

Micromachines ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 777 ◽  
Author(s):  
Xue Lin ◽  
Xiangyu Jin ◽  
Bin Xu ◽  
Ruliang Wang ◽  
Rongxin Fu ◽  
...  
Keyword(s):  
Microfluidic Chip ◽  
Reverse Transcription ◽  
Ebola Virus ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Virus Species ◽  
Fluorescence Detector ◽  
Parallel Detection ◽  
Loop Mediated Isothermal Amplification

Considering the lack of official vaccines and medicines for Ebola virus infection, reliable diagnostic methods are necessary for the control of the outbreak and the spread of the disease. We developed a microfluidic-chip-based portable system for fast and parallel detection of four Ebola virus species. The system is based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and consists of four specific LAMP primers, a disc microfluidic chip, and a portable real-time fluorescence detector. It could specifically and parallelly distinguish four species of the Ebola virus after only one sampling, including the Zaire Ebola virus, the Sudan Ebola virus, the Bundibugyo Ebola virus, and the Tai Forest Ebola virus, without cross-contamination. The limit of detection was as small as 10 copies per reaction, while the total consumption of sample and reagent was 0.94 μL per reaction. The final results could be obtained in 50 min after one addition of sample and reagent mixture. This approach provides simplicity, high sensitivity, and multi-target parallel detection at a low cost, which could enable convenient and effective on-site detections of the Ebola virus in the outdoors, remote areas, and modern hospitals.

scholarly journals A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

2020 ◽  
Vol 21 (8) ◽  
pp. 2826 ◽  
Author(s):  
Renfei Lu ◽  
Xiuming Wu ◽  
Zhenzhou Wan ◽  
Yingxue Li ◽  
Xia Jin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
N Gene ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity

COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 μL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 μL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

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