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 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 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.

Rapid and simple detection of Ebola virus by reverse transcription-loop-mediated isothermal amplification

2007 ◽  
Vol 141 (1) ◽  
pp. 78-83 ◽  
Author(s):  
Yohei Kurosaki ◽  
Ayato Takada ◽  
Hideki Ebihara ◽  
Allen Grolla ◽  
Naoki Kamo ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Ebola Virus ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Simple Detection

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 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.

scholarly journals Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Subtype H7N9 Avian Influenza Virus

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Hongmei Bao ◽  
Yuhui Zhao ◽  
Yunhe Wang ◽  
Xiaolong Xu ◽  
Jianzhong Shi ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Reverse Transcription ◽  
Influenza A ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
H7n9 Virus ◽  
Loop Mediated Isothermal Amplification ◽  
H7n9 Influenza Virus ◽  
Virus Rna ◽  
H7n9 Influenza

A novel influenza A (H7N9) virus has emerged in China. To rapidly detect this virus from clinical samples, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7N9 virus. The minimum detection limit of the RT-LAMP assay was 0.01 PFU H7N9 virus, making this method 100-fold more sensitive to the detection of the H7N9 virus than conventional RT-PCR. The H7N9 virus RT-LAMP assays can efficiently detect different sources of H7N9 influenza virus RNA (from chickens, pigeons, the environment, and humans). No cross-reactive amplification with the RNA of other subtype influenza viruses or of other avian respiratory viruses was observed. The assays can effectively detect H7N9 influenza virus RNA in drinking water, soil, cloacal swab, and tracheal swab samples that were collected from live poultry markets, as well as human H7N9 virus, in less than 30 min. These results suggest that the H7N9 virus RT-LAMP assays were efficient, practical, and rapid diagnostic methods for the epidemiological surveillance and diagnosis of influenza A (H7N9) virus from different resource samples.

A microfluidic chip capable of generating and trapping emulsion droplets for digital loop-mediated isothermal amplification analysis

Lab on a Chip ◽  
2018 ◽  
Vol 18 (2) ◽  
pp. 296-303 ◽  
Author(s):  
Yu-Dong Ma ◽  
Kang Luo ◽  
Wen-Hsin Chang ◽  
Gwo-Bin Lee
Keyword(s):  
Nucleic Acids ◽  
Microfluidic Chip ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Novel Device ◽  
Dna Molecule ◽  
Emulsion Droplets ◽  
Droplet Array ◽  
Single Dna Molecule

A microfluidic droplet array chip was designed to execute the digital LAMP. This novel device was capable of 1) creating emulsion droplets, 2) sorting them into a trapping array, and 3) executing LAMP for only 40 min. Nucleic acids could be accurately quantified and the limit of detection was only single DNA molecule.

scholarly journals Clinical assessment and validation of a rapid and sensitive SARS-CoV-2 test using reverse-transcription loop-mediated isothermal amplification without the need for RNA extraction

2020 ◽  
Author(s):  
Melis N Anahtar ◽  
Graham E G McGrath ◽  
Brian A Rabe ◽  
Nathan A Tanner ◽  
Benjamin A White ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Color Change ◽  
Limit Of Detection ◽  
Massachusetts General Hospital ◽  
Rna Extraction ◽  
Isothermal Amplification ◽  
Sample Collection ◽  
Assay Sensitivity ◽  
Loop Mediated Isothermal Amplification ◽  
Concentration Method

Abstract Background Amid the enduring pandemic, there is an urgent need for expanded access to rapid, sensitive, and inexpensive COVID-19 testing worldwide without specialized equipment. We developed a simple test that uses colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) to detect SARS-CoV-2 in forty minutes from sample collection to result. Methods We tested 135 nasopharyngeal specimens from patients evaluated for COVID-19 infection at Massachusetts General Hospital. Specimens were either added directly to RT-LAMP reactions, inactivated by a combined chemical and heat treatment step, or inactivated then purified with a silica-particle based concentration method. Amplification was performed with two SARS-CoV-2-specific primer sets and an internal specimen control; the resulting color change was visually interpreted. Results Direct RT-LAMP testing of unprocessed specimens could only reliably detect samples with abundant SARS-CoV-2 (>3,000,000 copies/mL), with sensitivities of 50% (95% CI, 28 to 72) and 59% (95% CI, 43 to 73) in samples collected in universal transport medium and saline, respectively, compared to qPCR. Adding an up-front RNase inactivation step markedly improved the limit of detection to at least 25,000 copies/mL, with 87.5% (95% CI, 72 to 95) sensitivity and 100% specificity (95% CI, 87 to 100). Using both inactivation and purification increased the assay sensitivity by ten-fold, achieving a limit of detection comparable to commercial real-time PCR-based diagnostics. Conclusion By incorporating a fast and inexpensive sample preparation step, RT-LAMP accurately detects SARS-CoV-2 with limited equipment for about US$6 per sample, making this a potentially ideal assay to increase testing capacity especially in resource-limited settings.

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