scholarly journals Development of an RT-LAMP Assay for the Rapid Detection of SFTS Virus

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 693
Author(s):  
Shiori Sano ◽  
Shuetsu Fukushi ◽  
Souichi Yamada ◽  
Shizuko Harada ◽  
Hitomi Kinoshita ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Early Phase ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Viral Rna ◽  
Nucleic Acid Amplification ◽  
Target Sequence ◽  
Rt Pcr ◽  
Diagnostic Efficacy ◽  
Probe Set

Detection of severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) during the early phase of the disease is important for appropriate treatment, infection control, and prevention of further transmission. The reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a nucleic acid amplification method that amplifies the target sequence under isothermal conditions. Here, we developed an RT-LAMP with a novel primer/probe set targeting a conserved region of the SFTSV L segment after extraction of viral RNA (standard RT-LAMP). Both the Chinese and Japanese SFTSV strains, including various genotypes, were detected by the standard RT-LAMP. We also performed RT-LAMP using the same primer/probe set but without the viral RNA extraction step (called simplified RT-LAMP) and evaluated the diagnostic efficacy. The sensitivity and specificity of the simplified RT-LAMP were 84.9% (45/53) and 89.5% (2/19), respectively. The simplified RT-LAMP can detect SFTSV in human sera containing >103.5 copies/mL viral RNA. The two RT-LAMP positive but quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) negative samples were positive in the conventional RT-PCR, suggesting that there was no false positive reaction in the RT-LAMP. Both the standard and simplified RT-LAMP are useful for detecting the SFTSV genome in patients during the early phase of the disease.

scholarly journals Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

2020 ◽  
Author(s):  
Weihua Yang ◽  
Xiaofei Dang ◽  
Qingxi Wang ◽  
Mingjie Xu ◽  
Qianqian Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Virus Disease ◽  
Lamp Assay ◽  
Nucleic Acid Amplification ◽  
N Gene ◽  
Nucleic Acid Detection ◽  
Lamp Method ◽  
Rt Pcr ◽  
Rapid Amplification

AbstractCorona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 °C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

scholarly journals Improved RNA Extraction from Woody Plants for the Detection of Viral Pathogens by Reverse Transcription-Polymerase Chain Reaction

Plant Disease ◽  
1997 ◽  
Vol 81 (2) ◽  
pp. 222-226 ◽  
Author(s):  
Donald J. MacKenzie ◽  
Morven A. McLean ◽  
Srima Mukerji ◽  
Margaret Green
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Woody Plants ◽  
Rna Extraction ◽  
Viral Rna ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Apple Stem ◽  
Spin Column ◽  
Polymerase Chain

An efficient procedure for the extraction of high-quality RNA from woody plants without the use of phenol, organic solvents, or alcohol precipitation is described. The method employs commercially available spin-column matrices and mitigates the inhibitory effects of plant polysaccharides and polyphenolic compounds commonly observed on subsequent polymerase chain reaction amplification when conventional extraction methods are applied to woody plant species. The method described has been successfully used in the development of highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) techniques for the detection of a number of viruses in their woody hosts. The viruses detected included apple stem grooving capillovirus (ASGV), apple stem pitting virus, Prunus necrotic ringspot ilarvirus (PNRSV), grapevine fanleaf and Arabis mosaic nepoviruses, and grapevine leafroll-associated closterovirus type 3. The method described was equally effective for the extraction of viral RNA from either budwood, leaves, or flower blossoms as determined by the equivalent RT-PCR detection of ASGV and PNRSV from these tissues. Detection of viral RNA in samples of total plant RNA prepared using this method was found to be as sensitive as was previously described for the immunocapture RT-PCR technique.

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.

Microelectrothermofluidic packaging for single chip integrated viral RNA extraction and RT-PCR microdevices

2010 ◽  
Author(s):  
Tae Goo Kang ◽  
Hong Miao Ji ◽  
Siow Pin Melvin Tan ◽  
Guang Kai Ignatius Tay ◽  
Ming Yi Daniel Ang ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Viral Rna ◽  
Single Chip ◽  
Rt Pcr

scholarly journals Detection of Schmallenberg Virus RNA in Bull Semen in Poland

2016 ◽  
Vol 19 (3) ◽  
pp. 655-657 ◽  
Author(s):  
J. Kęsik-Maliszewska ◽  
M. Larska
Keyword(s):  
Extraction Method ◽  
Rna Extraction ◽  
Viral Rna ◽  
Schmallenberg Virus ◽  
Rt Pcr ◽  
Bovine Semen ◽  
Bull Semen ◽  
Virus Rna ◽  
Virus Excretion ◽  
Venereal Transmission

Abstract The detection of Schmallenberg virus (SBV) in the breeding bull semen raised the question of the possibility of venereal transmission of SBV which could result in cost-intensive restrictions in the trade of bovine semen. In order to evaluate the presence of SBV RNA in bovine semen, 131 bull semen samples from four locations in Poland collected between 2013 and 2015 were analysed by RT-PCR for viral RNA. SBV RNA was detected in 5.3% of the samples. The study has revealed that application of an appropriate RNA extraction method is crucial to detect virus excretion via semen.

scholarly journals Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6617
Author(s):  
Eva Rajh ◽  
Tina Šket ◽  
Arne Praznik ◽  
Petra Sušjan ◽  
Alenka Šmid ◽  
...  
Keyword(s):  
Oral Cavity ◽  
Reverse Transcription ◽  
Screening Program ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Diagnostic Sensitivity ◽  
Nucleic Acid Amplification ◽  
Nasopharyngeal Swab ◽  
Viral Spread ◽  
One Step

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

scholarly journals SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification

2020 ◽  
Vol 117 (39) ◽  
pp. 24450-24458 ◽  
Author(s):  
Brian A. Rabe ◽  
Constance Cepko
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Reverse Transcription ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Viral Rna ◽  
Rna Detection ◽  
Sample Stability ◽  
Rapid Inactivation ◽  
Diagnostic Capabilities ◽  
Purification Protocol

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.

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 Viral RNA Shedding and Transmission Potential of Asymptomatic and Pauci-symptomatic COVID-19 Patients

2020 ◽  
Author(s):  
Arghadip Samaddar ◽  
Ravisekhar Gadepalli ◽  
Vijaya Lakshmi Nag ◽  
Sanjeev Misra ◽  
Pankaj Bhardwaj ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Viral Rna ◽  
Exhaled Breath ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Transmission Potential ◽  
Polymerase Chain ◽  
Pcr Test

Abstract We studied the pattern and duration of viral RNA shedding in 32 asymptomatic and 11 pauci-symptomatic coronavirus disease 2019 (COVID-19) cases. Viral RNA shedding in exhaled breath progressively diminished and became negative after six days of a positive reverse transcription polymerase chain reaction (RT-PCR) test. Therefore, the duration of isolation can be minimised to six days.

scholarly journals Rapid isothermal amplification and portable detection system for SARS-CoV-2

2020 ◽  
Vol 117 (37) ◽  
pp. 22727-22735 ◽  
Author(s):  
Anurup Ganguli ◽  
Ariana Mostafa ◽  
Jacob Berger ◽  
Mehmet Y. Aydin ◽  
Fu Sun ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Alternative Pathway ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Complete Agreement ◽  
Rt Pcr ◽  
Viral Transport Medium

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

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