scholarly journals RT qLAMP--Direct Detection of SARS-CoV-2 in Raw Sewage

2020 ◽  
Author(s):  
Jerry E. Ongerth ◽  
Richard E. Danielson
Keyword(s):  
Process Development ◽  
Direct Detection ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Health Department ◽  
N Gene ◽  
Virus Concentration ◽  
Primer Selection ◽  
Specific Amplification ◽  
Primer Sets

The project purpose was to examine ability of a loop-mediated isothermal amplification (LAMP) assay to quantify SARS-CoV-2 in raw sewage, directly, using no preliminary sample processing for virus concentration and RNA extraction. An objective was to take advantage of extensive recently published work to facilitate process development, principally primer selection, and to use readily available off the shelf materials with conventional lab procedure and equipment. Well-developed and referenced primers for ORF1a, E, and N-gene targets were selected and applied, using commercially available synthetic RNA standards, and raw sewage from a local wastewater agency serving 650,000. County health department monitoring provided current COVID-19 data. Testing defined performance characteristics for each primer set, with significant differences between them. Specific amplification of SARS-CoV-2 RNA was observed using each of the primer sets, with E-gene and N-gene primers most effective. Positive analysis results from all raw sewage samples corresponded to calculated concentrations of virus in 5-10 μL raw sewage aliquots for 25 μL reactions. Results show that even at low reported case rates e.g. 1-10/100,000, SARS-CoV-2 is present in raw sewage at > 1-5/ μL, permitting direct LAMP-based detection. Use of RT qLAMP will facilitate wastewater-based epidemiology as an important component for COVID-19 control.

scholarly journals One-step RNA extraction for RT-qPCR detection of 2019-nCoV

2020 ◽  
Author(s):  
Monica Sentmanat ◽  
Evguenia Kouranova ◽  
Xiaoxia Cui
Keyword(s):  
Real Time ◽  
Rna Extraction ◽  
Virus Transmission ◽  
Extraction Methods ◽  
Viral Rna ◽  
Taqman Probe ◽  
N Gene ◽  
Structural Protein ◽  
Diagnostic Panel ◽  
Primer Sets

ABSTRACTThe global outbreak of coronavirus disease 2019 (COVID-19) has placed an unprecedented burden on healthcare systems as the virus spread from the initial 27 reported cases in the city of Wuhan, China to a global pandemic in under three month[1]. Resources essential to monitoring virus transmission have been challenged with a demand for expanded surveillance. The CDC 2019-nCoV Real-Time Diagnostic Panel uses a real-time reverse transcription polymerase chain reaction (RT-PCR) consisting of two TaqMan probe and primer sets specific for the 2019-nCoV N gene, which codes for the nucleocapsid structural protein that encapsulates viral RNA, for the qualitative detection of 2019-nCoV viral RNA in respiratory samples. To isolate RNA from respiratory samples, the CDC lists RNA extraction kits from four manufacturers. In anticipation of a limited supply chain of RNA extraction kits and the need for test scalability, we sought to identify alternative RNA extraction methods. Here we show that direct lysis of respiratory samples can be used in place of RNA extraction kits to run the CDC 2019-nCoV Real-Time Diagnostic assay with the additional benefits of higher throughput, lower cost, faster turnaround and possibly higher sensitivity and improved safety.

scholarly journals Integration of RT-LAMP and Microfluidic Technology for Detection of SARS-CoV-2 in Wastewater as an Advanced Point-of-care Platform

2021 ◽  
Author(s):  
Ahmed Donia ◽  
Muhammad Furqan Shahid ◽  
Aftab Ahmad ◽  
Aneela Javed ◽  
Muhammad Nawaz ◽  
...  
Keyword(s):  
Point Of Care ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Surrogate Marker ◽  
Lamp Assay ◽  
N Gene ◽  
Microfluidic Chips ◽  
Microfluidic Technology ◽  
Wastewater Samples ◽  
The One

Development of lab-on-a-chip (LOC) system based on integration of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and microfluidic technology is expected to speed up SARS-CoV-2 diagnostics allowing early intervention. In the current work, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-LAMP assays were performed on extracted RNA of 7 wastewater samples. RT-LAMP assay was also performed on wastewater samples without RNA extraction. Current detection of SARS-CoV-2 is mainly by RT-qPCR of ORF (ORF1ab) and N genes so we targeted both to find the best surrogate marker for SARS-CoV-2 detection. We also performed RT-LAMP with/without RNA extraction inside microfluidic device to target both genes. Positivity rates of RT-qPCR and RT-LAMP performed on extracted RNA were 100.0% (7/7) and 85.7% (6/7), respectively. RT-qPCR results revealed that all 7 wastewater samples were positive for N gene (Ct range 37-39), and negative for ORF1ab, suggesting that N gene could be used as a surrogate marker for detection of SARS-CoV-2. RT-LAMP of N and ORF (ORF1a) genes performed on wastewater samples without RNA extraction indicated that all 7 samples remains pink (negative). The color remains pink in all microchannels except the one which subjected to RT-LAMP for targeting N region after RNA extraction (yellowish/orange color). This study shows for the first time that SARS-CoV-2 was successfully detected from wastewater samples using RT-LAMP in microfluidic chips.

scholarly journals Rapid and accurate detection of three genes in SARS-CoV-2 using RT-LAMP method

2020 ◽  
Author(s):  
Weihua Yang ◽  
Xiaofei Dang ◽  
Qingxi Wang ◽  
Mingjie Xu ◽  
Qianqian Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Lamp Assay ◽  
High Specificity ◽  
N Gene ◽  
Clinical Samples ◽  
Lamp Method ◽  
E Gene ◽  
Primer Sets

Abstract Background Corona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. It has the characteristics of rapid transmission, difficult treatment, rapid deterioration, and high mortality rate. Reliable and rapid diagnosis of SARS-CoV-2 infection is critical to treat patients and control transmission in time. Methods To detect SARS-CoV-2 nucleic acid faster and more convenient, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. ORF1ab gene, E gene and N gene were selected as the target genes, because ORF1ab gene is the most specific and N gene is the most sensitive, while E gene has both sensitivity and specificity. Over thirty primer sets for them were designed, then the primer sets with rapid response and high specificity were screened out and their loop primers were designed and selected in order to improve the reaction speed further. Using these primers, RNA reverse transcription and nucleic acid amplification were performed in one step at 63 ℃ isothermal conditions, and the results can be judged by naked eyes through color change within 30 minutes. Finally, the optimal primer set for each gene was verified with over 208 clinical samples. Results ORF1ab gene, E gene and N gene were detected simultaneously by this method. ORF1ab gene has high specificity and the same sensitivity as RT-PCR currently used in clinic. Although N gene is less specific than ORF1ab, it is 80 times more sensitive than ORF1ab. The sensitivity and specificity of E gene are between them. Simultaneous detection of these three genes can neither cause false positives nor miss samples with low virus concentration, ensuring the sensitivity and specificity of SARS-CoV-2 detection. BLAST comparing results showed the primers for the three genes were highly specific for SARS-CoV-2. And the sequencing results of RT-LAMP products showed that the amplified fragments were unique to SARS-CoV-2. The accuracy of RT-LAMP assay was 99% as detecting 208 clinical specimens. Conclusions 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 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 Direct detection of SARS-CoV-2 using non-commercial RT-LAMP reagents on heat-inactivated samples

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alisa Alekseenko ◽  
Donal Barrett ◽  
Yerma Pareja-Sanchez ◽  
Rebecca J. Howard ◽  
Emilia Strandback ◽  
...  
Keyword(s):  
Large Scale ◽  
Direct Detection ◽  
Dna Polymerases ◽  
Scale Up ◽  
Lamp Assay ◽  
Cost Effective ◽  
Reaction Conditions ◽  
Clinical Patient ◽  
Reverse Transcriptases ◽  
Large Scale Testing

AbstractRT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.

scholarly journals SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

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.

LAMP assay for distinguishing Fusarium oxysporum and Fusarium commune in lotus (Nelumbo nucifera) rhizomes

Plant Disease ◽  
2021 ◽  
Author(s):  
Sheng Deng ◽  
Xin Ma ◽  
Yifan Chen ◽  
Hui Feng ◽  
Dongmei Zhou ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Fusarium Species ◽  
Lamp Assay ◽  
Rapid Identification ◽  
Nelumbo Nucifera ◽  
Disease Spread ◽  
Molecular Diagnostic ◽  
Primer Sets ◽  
Rot Disease ◽  
Rhizome Rot

The yields of edible rhizome from the cultivation of the perennial hydrophyte lotus (Nelumbo nucifera) can be severely reduced by rhizome rot disease caused by Fusarium species. There is a lack of rapid field-applicable methods for detection of these pathogens on lotus plants displaying symptoms of rhizome-rot. Fusarium commune (91%) and Fusarium oxysporum (9%) were identified at different frequencies from lotus samples showing symptoms of rhizome-rot. As these two species can cause different severity of disease and their morphology is very similar, molecular-diagnostic based methods to detect these two species were developed. Based on the comparison of the mitochondrial genome of the two species, three specific DNA loci targets were found. The designed primer sets for conventional PCR, qPCR and loop-mediated isothermal amplification (LAMP) precisely distinguished the above two species when isolated from lotus and other plants. The LAMP detection limits were 10 pg/μl and 1 pg/μl of total DNA for F. commune and F. oxysporum, respectively. We also carried out field-mimicked experiments on lotus seedlings and rhizomes (including inoculated samples and field diseased samples), and the results indicated that the LAMP primer sets and the supporting portable methods are suitable for the rapid diagnosis of the lotus disease in the field. The LAMP-based detection method will aid in the rapid identification of whether F. oxysporum or F. commune are infecting lotus plants with symptoms of rhizome-rot, and can facilitate efficient pesticide use and prevent the disease spread through vegetative propagation of Fusarium-infected lotus rhizomes.

scholarly journals Detection and partial characterization of an isolate of Groundnut ringspot virus in Solanum sessiliflorum

2002 ◽  
Vol 27 (3) ◽  
pp. 249-253 ◽  
Author(s):  
ALESSANDRA J. BOARI ◽  
EUNIZE MACIEL-ZAMBOLIM ◽  
DOUGLAS D. LAU ◽  
GAUS S. A. LIMA ◽  
ELLIOT W. KITAJIMA ◽  
...  
Keyword(s):  
Amazon Basin ◽  
Rna Extraction ◽  
Northern Region ◽  
Ringspot Virus ◽  
N Gene ◽  
Electron Microscopic ◽  
Systemic Necrosis ◽  
Thin Sections ◽  
Local Lesions ◽  
Stem Necrosis

The cubiu (Solanum sessiliflorum) fruit, originating in the Amazon basin, is commonly used in that region for food, medicine, and cosmetics. In an experimental culture of cubiu, in order to evaluate its adaptation to conditions in the Northern region of the state of Rio de Janeiro, it was observed plants with mosaic symptoms. A cubiu plant was collected and analyzed to identify the etiological agent. After mechanical passage through a local lesion host, a host range test was performed. The virus induced chlorotic local lesions in Chenopodium quinoa, necrotic local lesions in Gomphrena globosa, mosaic in S. sessiliflorum, leaf and stem necrosis in tomato (Lycopersicon esculentum) 'Rutgers', mosaic and leaf distortion in Datura stramonium and Physalis floridana, and necrotic local lesions followed by systemic necrosis and plant death in four Nicotiana species. Electron microscopic observations of ultra thin sections from infected cubiu leaves showed the presence of spheroidal, membrane-bound particles typical of tospovirus species. Analysis of the nucleocapsid protein from concentrated virus particles indicated the presence of a 28 kDa protein. RT-PCR was performed after total RNA extraction from infected IPA-6 tomato leaves. A fragment of approximately 0,8 kbp corresponding to the N gene was amplified, cloned and sequenced. The N protein from the cubiu isolate was 95% homologous to the Groundnut ringspot virus (GRSV) protein, and no more than 85% homologous to those from Zucchini lethal chlorosis virus (ZLCV) and Chrysanthemun stem necrosis virus (CSNV), Tomato spotted wilt virus (TSWV), and Tomato chlorotic spot virus (TCSV). This is the first report of the occurrence of GRSV (or any other plant virus) in cubiu.

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.

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