scholarly journals Development and Validation of a Rapid, Single-Step Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) System Potentially to Be Used for Reliable and High-Throughput Screening of COVID-19

2020 ◽  
Vol 10 ◽  
Author(s):  
Minghua Jiang ◽  
Weihua Pan ◽  
Amir Aratehfar ◽  
Wenjie Fang ◽  
Liyan Ling ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Step ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Lamp System ◽  
Development And Validation

scholarly journals Development and validation of a rapid, single-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) system potentially to be used for reliable and high-throughput screening of COVID-19

2020 ◽  
Author(s):  
Minghua Jiang ◽  
Weihua Pan ◽  
Amir Aratehfar ◽  
Wenjie Fang ◽  
Liyan Ling ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
High Throughput ◽  
Predictive Value ◽  
High Throughput Screening ◽  
Lamp Assay ◽  
Single Step ◽  
Clinical Samples ◽  
Qrt Pcr ◽  
Pcr Assays ◽  
Development And Validation

AbstractObjectivesDevelopment and validation of a single-step and accurate reverse transcriptase loop-mediated isothermal amplification technique (RT-LAMP) for rapid identification of SARS COV-2 relative to commercial quantitative reverse transcriptase real-time PCR (qRT-PCR) assays to allow prompt initiation of proper medical care and containment of virus spread.MethodsPrimers showing optimal in-silico features were subjected to analytical sensitivity and specificity to assess the limit of detection (LOD) and cross-reaction with closely- and distantly-related viral species, and clinically prominent bacterial and fungal species. In order to evaluate the clinical utility, our RT-LAMP was subjected to a large number of clinical samples, including 213 negative and 47 positive patients, relative to two commercial quantitative RT-PCR assays.ResultsThe analytical specificity and sensitivity of our assay was 100% and 500 copies/ml when serial dilution performed in both water and sputum. Subjecting our RT-LAMP assay to clinical samples showed a high degree of specificity (99.5%), sensitivity (91.4%), positive predictive value (97.7%), and negative predictive value (98.1%) when used relative to qRT-PCR. Our RT-LAMP assay was two times faster than qRT-PCR and is storable at room temperature. A suspected case that later became positive tested positive using both our RT-LAMP and the two qRT-PCR assays, which shows the capability of our assay for screening purposes.ConclusionsWe present a rapid RT-LAMP assay that could extend the capacity of laboratories to process two times more clinical samples relative to qRT-PCR and potentially could be used for high-throughput screening purposes when demand is increasing at critical situations.

scholarly journals A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jonas Schmidt ◽  
Sandro Berghaus ◽  
Frithjof Blessing ◽  
Folker Wenzel ◽  
Holger Herbeck ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Mass Screening ◽  
High Throughput ◽  
Clinical Laboratory ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Rt Pcr ◽  
Loop Mediated Isothermal Amplification ◽  
Clinical Diagnostic

AbstractShortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen’s kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar’s test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.

Integration of reverse transcriptase loop-mediated isothermal amplification with an immunochromatographic strip on a centrifugal microdevice for influenza A virus identification

Lab on a Chip ◽  
2015 ◽  
Vol 15 (3) ◽  
pp. 718-725 ◽  
Author(s):  
J. H. Jung ◽  
B. H. Park ◽  
S. J. Oh ◽  
G. Choi ◽  
T. S. Seo
Keyword(s):  
Reverse Transcriptase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Virus Detection ◽  
Isothermal Amplification ◽  
Immunochromatographic Strip ◽  
Virus Identification ◽  
Loop Mediated Isothermal Amplification

In this paper, we demonstrated an integrated centrifugal microdevice which could perform reverse transcriptase loop-mediated isothermal amplification and immunochromatographic strip based amplicon analysis for rapid, sensitive, and multiplex influenza A virus detection.

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