Dataset Information

Diagnostic utility and validation of a newly developed real time loop mediated isothermal amplification method for the detection of SARS CoV-2 infection.

ABSTRACT:

Background

Detecting SARS-CoV-2 using a simple real time molecular assay will be helpful for the mitigation efforts in low / middle income countries during the pandemic. We have developed and validated a rapid and simple real time loop mediated isothermal amplification assay (LAMP) for screening of SARS-CoV-2 infection in known infected and non-infected individuals.

Methods

Six sets of primers were designed targeting the N-gene of the SARS-CoV-2 (Accession ID MN994468). LAMP reactions were performed using Warm Start 2X Master Mix and real-time PCR machine at 65 °C for 60 cycles with 15 s for each cycle. Results were read by visualizing turbidity under ultraviolet light and real time fluorescence detection through FAM channel of the real time PCR machine. We tested a total of 320 including 240 SARS CoV-2 positive (Ct values <40) and 80 SARS CoV-2 negative samples as tested by a real time RT-PCR using the newly developed LAMP assay.

Results

A total of 206 out of 240 SARS CoV-2 positive samples were tested positive by the newly developed LAMP assay with a sensitivity of 86%. All 80 SARS CoV-2 negative samples were tested negative by the newly developed LAMP assay with a specificity of 100%.

Conclusion

The newly developed real time LAMP assay has a sensitivity of 86% and specificity of 100% compared to the real time RT-PCR for the detection of SARS CoV-2. The new assay will be useful to screen large number of samples if adopted to minimize the time and cost.

SUBMITTER: Iqbal BN

PROVIDER: S-EPMC9069985 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Publications

Diagnostic utility and validation of a newly developed real time loop mediated isothermal amplification method for the detection of SARS CoV-2 infection.

Iqbal Bushran N BN Arunasalam Shiyamalee S Divarathna Maduja V M MVM Jabeer Aaom A Sirisena Pdnn P Senaratne Thamarasi T Muthugala Rohitha R Noordeen Faseeha F

Journal of clinical virology plus 20220505 3

<h4>Background</h4>Detecting SARS-CoV-2 using a simple real time molecular assay will be helpful for the mitigation efforts in low / middle income countries during the pandemic. We have developed and validated a rapid and simple real time loop mediated isothermal amplification assay (LAMP) for screening of SARS-CoV-2 infection in known infected and non-infected individuals.<h4>Methods</h4>Six sets of primers were designed targeting the N-gene of the SARS-CoV-2 (Accession ID MN994468). LAMP react ...[more]

PMID: 35540180

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Project description:BackgroundKoi Herpesvirus (KHV) affects both juvenile and adult common carp and koi, and is especially lethal to fry. The high mortalities caused by the disease have had a negative impact on the international koi trade. Different diagnostic techniques have been used to detect KHV, including: isolation of the virus in cell culture, electron microscopy, several PCR tests, ELISA and in situ hybridisation. All of these methods are time consuming, laborious and require specialised equipment.ResultsA rapid field diagnosis of KHV in common and koi carp was developed using loop-mediated isothermal amplification (LAMP). The LAMP reaction rapidly amplified nucleic acid with high specificity and efficiency under isothermal conditions using a simple water bath. Two methods of extracting DNA from host tissue were compared: extraction by boiling and by using a commercial extraction kit. A set of six primers--two inner primers, two outer primers and two loop primers--was designed from a KHV amplicon. The reaction conditions were optimised for detection of KHV in 60 min at 65 degrees C using Bst (Bacillus stearothermophilus) DNA polymerase. When visualised by gel electrophoresis, the products of the KHV LAMP assay appeared as a ladder pattern, with many bands of different sizes from 50 base-pairs (bp) up to the loading well. The KHV LAMP product could also be simply detected visually by adding SYBR Green I to the reaction tube and observing a colour change from orange to green. All samples positive for KHV by visual detection were confirmed positive by gel electrophoresis. The KHV LAMP had the same sensitivity as a standard PCR assay for the detection of KHV.ConclusionThis paper describes an accelerated LAMP assay for diagnosis of KHV. The entire procedure took only 90 minutes to produce a result: 15 minutes for DNA extraction; 60 min for the LAMP reaction; 2 min for visual detection using SYBR Green I. The test can be used under field conditions because the only equipment it requires is a water bath.

Project description:Recently, a novel coronavirus (SARS-CoV-2; coronavirus disease 2019, COVID-19) has emerged, rapidly spreading and severely straining the capacity of the global health community. Many nations are employing combinations of containment and mitigation strategies, where early diagnosis of COVID-19 is vital in controlling illness progression and limiting viral spread within the population. Thus, rapid and accurate methods of early detection are vital to contain COVID-19 and prevent further spread and predicted subsequent infectious waves of viral recurrence in future. Immediately after its initial characterization, Chinese and American Centers for Disease Control and Prevention (CDCs) rapidly employed molecular assays for detection of COVID-19, mostly employing real-time polymerase chain reaction (RT-PCR) methods. However, such methods require specific expensive items of equipment and highly trained analysts, requiring upwards of 4-8 h to process. These requirements coupled with associated financial pressures may prevent effective deployment of such diagnostic tests. Loop mediated isothermal amplification(LAMP) is method of nucleic acid amplification which exhibits increased sensitivity and specificity are significantly rapid, and do not require expensive reagents or instruments, which aids in cost reduction for coronavirus detection. Studies have shown the successful application of LAMP assays in various forms to detect coronavirus RNA in patient samples, demonstrating that 1-10 copies of viral RNA template per reaction are sufficient for successful detection, ~100-fold more sensitive than conventional RT-PCR methods. Importantly, studies have also now demonstrated the effectiveness of LAMP methodology in the detection of SARS-CoV-2 RNA at significantly low levels, particularly following numerous improvements to LAMP assay protocols. We hypothesise that recent advancements in enhanced LAMP protocols assay perhaps represent the best chance for a rapid and robust assay for field diagnosis of COVID-19, without the requirement of specialized equipment and highly trained professionals to interpret results. Herein, we present our arguments with a view to disseminate such findings, to assist the combat of this virus that is proving so devastating. We hope that this strategy could be applied rapidly, and confirmed for viability with clinical samples, before being rolled out for mass-diagnostic testing in these current times.

Dataset Information

Diagnostic utility and validation of a newly developed real time loop mediated isothermal amplification method for the detection of SARS CoV-2 infection.

Background

Methods

Results

Conclusion

Publications

Diagnostic utility and validation of a newly developed real time loop mediated isothermal amplification method for the detection of SARS CoV-2 infection.

Similar Datasets

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