scholarly journals Rapid SARS-CoV-2 testing in primary material based on a novel multiplex LAMP assay

2020 ◽  
Author(s):  
Bernhard Schermer ◽  
Francesca Fabretti ◽  
Maximilian Damagnez ◽  
Veronica Di Cristanziano ◽  
Eva Heger ◽  
...  
Keyword(s):  
Point Of Care ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Airport Security ◽  
Primary Material ◽  
Nucleic Acid Isolation ◽  
Emergency Units ◽  
One Step ◽  
Polymerase Chain ◽  
Standard Lamp

AbstractBackgroundRapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose.MethodsTo avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including loop-mediated isothermal amplification (LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK).ResultsWhilst specificity of standard LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel LAMP assays with SHERLOCK.ConclusionIn summary, this approach reveals one-step multiplexed LAMP assays as a prime-option for the development of easy and cheap POC test kits.

scholarly journals LAMP-based foldable microdevice platform for the rapid detection of Magnaporthe oryzae and Sarocladium oryzae in rice seed

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
M. K. Prasannakumar ◽  
P. Buela Parivallal ◽  
Devanna Pramesh ◽  
H. B. Mahesh ◽  
Edwin Raj
Keyword(s):  
Magnaporthe Oryzae ◽  
Plant Pathogens ◽  
Point Of Care ◽  
Lamp Assay ◽  
Rice Seed ◽  
Highly Sensitive ◽  
Polymerase Chain ◽  
Rice Seeds ◽  
Template Dna ◽  
Assay Conditions

AbstractRice blast (caused by Magnaporthe oryzae) and sheath rot diseases (caused by Sarocladium oryzae) are the most predominant seed-borne pathogens of rice. The detection of both pathogens in rice seed is essential to avoid production losses. In the present study, a microdevice platform was designed, which works on the principles of loop-mediated isothermal amplification (LAMP) to detect M. oryzae and S. oryzae in rice seeds. Initially, a LAMP, polymerase chain reaction (PCR), quantitative PCR (qPCR), and helicase dependent amplification (HDA) assays were developed with primers, specifically targeting M. oryzae and S. oryzae genome. The LAMP assay was highly efficient and could detect the presence of M. oryzae and S. oryzae genome at a concentration down to 100 fg within 20 min at 60 °C. Further, the sensitivity of the LAMP, HDA, PCR, and qPCR assays were compared wherein; the LAMP assay was highly sensitive up to 100 fg of template DNA. Using the optimized LAMP assay conditions, a portable foldable microdevice platform was developed to detect M. oryzae and S. oryzae in rice seeds. The foldable microdevice assay was similar to that of conventional LAMP assay with respect to its sensitivity (up to 100 fg), rapidity (30 min), and specificity. This platform could serve as a prototype for developing on-field diagnostic kits to be used at the point of care centers for the rapid diagnosis of M. oryzae and S. oryzae in rice seeds. This is the first study to report a LAMP-based foldable microdevice platform to detect any plant pathogens.

scholarly journals Ultrarapid, Ultrasensitive One-Step Kinetic Immunoassay for C-Reactive Protein (CRP) in Whole Blood Samples: Measurement of the Entire CRP Concentration Range with a Single Sample Dilution

2002 ◽  
Vol 48 (2) ◽  
pp. 269-277 ◽  
Author(s):  
Piia Tarkkinen ◽  
Tom Palenius ◽  
Timo Lövgren
Keyword(s):  
Whole Blood ◽  
Solid Phase ◽  
Point Of Care ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Cardiac Complications ◽  
Fluorescence Signal ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
One Step

Abstract Background: Recently, measurement of very low concentrations of C-reactive protein (CRP) has gained popularity as a potential new means for predicting the risk of future cardiac complications. In this study, we demonstrate the feasibility of a kinetic, one-step microparticle assay for quantitative determination of extremely low and high CRP concentrations in the limited timeframe typical for point-of-care testing. Methods: A noncompetitive, kinetic CRP immunoassay was developed that uses individual, porous microparticles as the solid phase. The microparticles were covalently coated with a monoclonal capture antibody, and the monoclonal detection antibody was labeled with europium. The one-step binding reaction was stopped by washing after 2 min of incubation, and the fluorescence signal of individual particles was measured. Results: The analytical detection limit (mean of zero calibrator + 3 SD) was 0.00016 mg/L CRP. Clinical samples were diluted 400-fold before assay to cover the CRP concentration range of 0.064–1200 mg/L. The assay correlated well with the Dade Behring N High Sensitivity CRP assay (for 0–10 mg/L, r = 0.969, Sy|x = 0.68, n = 54; for 0–350 mg/L, r = 0.969, Sy|x = 11.7, n = 100). The within- and between-run CVs based on calculated concentrations were, respectively, 9–16% and 14% at 0.11 mg/L, 4.5–12% and 8.2% at 4.2 mg/L, and 3.5–6.3% and 4.4% at 105 mg/L, with a CV <15% at 0.2 mg/L and above. Conclusions: Use of the kinetic microparticle approach combined with time-resolved fluorometry allows ultrasensitive quantification of CRP in whole blood in 2 min with a linear assay range spanning more than four orders of magnitude.

scholarly journals A Simple Colorimetric Molecular Detection of Novel Coronavirus (COVID-19), an Essential Diagnostic Tool for Pandemic Screening

2020 ◽  
Author(s):  
Pazhanimuthu Annamalai ◽  
Madhu Kanta ◽  
Pazhanivel Ramu ◽  
Baskar Ravi ◽  
Kokilavani Veerapandian ◽  
...  
Keyword(s):  
Molecular Detection ◽  
Point Of Care ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Cost Effective ◽  
Cost Effective Method ◽  
Recent Outbreak ◽  
Pcr Method ◽  
Novel Coronavirus ◽  
Virus Isolates

AbstractThe recent outbreak of the newly emerged novel coronavirus (SARS-CoV-2) presents a big challenge for public health laboratories as virus isolates are not available while there is an increasing evidence that the epidemic is more widespread than initially thought, as well as spreading internationally across borders through travellers does already happen warranting a methodology for the rapid detection of the infection to control SARS-CoV-2. Aim: We intended to develop and deploy a robust and rapid diagnostic methodology using LAMP assay for use in point of care settings to detect SARS-COV-2 infection. Methodology: In the present study, we have developed a validated rapid diagnostic procedure to detect SARS-CoV-2 using LAMP assay, its design relying on isothermal amplification of the nucleic acids of the SARS-CoV-2. Results: The LAMP assay developed detects SARS-CoV-2 infection rapidly with high sensitivity and reliability. The data generated by LAMP assay were comparable and at par with the data generated by real-time PCR method. Conclusion: The present study demonstrates that the LAMP assay developed was a rapid, reliable, sensitive and cost effective method to detect SARS-CoV-2 infection in a point of care as well as in laboratory settings.

scholarly journals Real-Time Fluorometric Isothermal LAMP Assay for Detection of Chlamydia pecorum in Rapidly Processed Ovine Abortion Samples: A Veterinary Practitioner’s Perspective

Pathogens ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1157
Author(s):  
Tom Clune ◽  
Susan Anstey ◽  
Vasilli Kasimov ◽  
Caroline Jacobson ◽  
Martina Jelocnik
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Lamp Assay ◽  
Test Results ◽  
Tissue Samples ◽  
Chlamydia Pecorum ◽  
Foetal Liver ◽  
Polymerase Chain ◽  
Foetal Lung ◽  
Positive Agreement

Traditional methods of detecting Chlamydia pecorum in tissue samples such as polymerase chain reaction or cell culture are laborious and costly. We evaluated the use of a previously developed C. pecorum LAMP assay using minimally processed ovine samples. Cotyledon (n = 16), foetal liver (n = 22), foetal lung (n = 2), and vaginal (n = 6) swabs, in addition to cotyledon (n = 6) and foetal liver (n = 8) tissue samples, were rapidly processed and used for LAMP testing without DNA extraction. Overall, LAMP test results were highly congruent with the in-house reference qPCR, with 80.43% (37/46; 72.73% positive agreement (PA); 84.75% negative agreement (NA)) overall agreeance for swab samples, and 85.71% (12/14; 80% PA; 88.89% NA) overall agreeance for tissue samples. Out of the 11 total discrepant results, discrepancy was mainly observed in samples (n = 10) with less than 100 copies/µL C. pecorum DNA. While sensitivity could be improved, the simplicity, low cost, and accuracy of detection makes this test amenable for use at point-of-care for detecting C. pecorum in sheep.

scholarly journals Field Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Platform for the Detection of Schistosoma japonicum Infection in Oncomelania hupemsis Snails

2018 ◽  
Author(s):  
Zhi-Qiang Qin ◽  
Jing Xu ◽  
Ting Feng ◽  
Shan Lv ◽  
Yin-Jun Qian ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Point Of Care ◽  
Infected Snail ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Field Evaluation ◽  
Isothermal Amplification ◽  
Pcr Analysis ◽  
Schistosomiasis Control ◽  
Loop Mediated Isothermal Amplification

Schistosoma infection in snails can be monitored by microscopy or indirectly by sentinel mice. As both these approaches sometimes miss infections, more sensitive tests are needed, particularly in low-level transmission settings. In this study, the loop-mediated isothermal amplification (LAMP) technique, designed to detect a specific 28S ribosomal S. japonicum gene with high sensitivity, was compared to microscopy using snail samples from 51 areas endemic for schistosomiasis in five Chinese provinces. The results were compared with those by polymerase chain reaction (PCR) adding DNA sequencing as a reference when needed. The testing of pooled snail samples showed that a dilution factor of 1/50, i.e., one infected snail plus 49 non-infected ones, would still result in a positive reaction after the recommended number of amplification cycles. Testing a total of 232 pooled samples, emanating from 4,006 snail specimens, with the LAMP assay showed a 6.5% rate of infection, while traditional microscopy found only 0.04% positive samples in the same materials. Parallel PCR analysis confirmed the diagnostic accuracy of the LAMP assay, with DNA sequencing even giving LAMP a slight lead. Microscopy and the LAMP test were carried out at local schistosomiasis-control stations demonstrating that the potential of the latter assay to serve as a point-of-care (POC) test with results available within 60–90 minutes, while the more complicated PCR test had to be carried out at the National Institute of Parasitic Diseases (NIPD) in Shanghai, China. In conclusion, LAMP was found to be clearly superior to microscopy and as good as, or better, than PCR. Application of LAMP testing would be useful for surveillance and risk prediction as it requires less time than other techniques and can be used under field conditions, which improves and accelerates schistosomiasis control.

scholarly journals Detection of SARS-CoV-2 in Saliva by RT-LAMP During a Screening of Workers in Brazil, Including Pre-Symptomatic Carriers

2021 ◽  
Author(s):  
Carlos dos Santos ◽  
Kézia de Oliveira ◽  
Geovana Mendes ◽  
Lívia Silva ◽  
Marcio de Souza Jr. ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Asymptomatic Carrier ◽  
Point Of Care ◽  
Quantitative Polymerase Chain Reaction ◽  
Lamp Assay ◽  
Diagnostic Methods ◽  
Public And Private ◽  
Polymerase Chain ◽  
Increasing Demand

The coronavirus pandemic has been causing damage to many nations, as public and private health systems deteriorate by the increasing demand. Some infected patients have culturable severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) even though not presenting any symptoms, and therefore, are probably able to transmit it. Correctly diagnosing and isolating infected patients is an important step towards preventing new infections. Current diagnostic methods rely mainly on reverse transcription quantitative polymerase chain reaction (RT-qPCR). Methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) have risen as viable alternatives, as they are cheaper and require less infrastructure, they have the potential to be applied in low-resource scenarios and even at point-of-care. Here we report a colorimetric RT‑LAMP assay capable of detecting SARS-CoV-2 in ribonucleic acid (RNA) from saliva. In some cases, the test was able to detect viral RNA before symptom onset and even in a self-reported asymptomatic carrier. It had a limit of detection of 300 copies per reaction and showed a sensitivity of 80%, a specificity of 100%, a general accuracy of 99.59%, and a Cohen’s kappa of 0.887. The possibility of detecting positive cases even before the clinical manifestation shows great potential and can contribute to controlling the pandemic.

scholarly journals A Syringe-Based and Centrifugation-Free DNA Extraction Procedure for the Rapid Detection of Bacteria

Chemosensors ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 167
Author(s):  
Taehwi Yoon ◽  
Seokjoon Kim ◽  
Jung Ho Kim ◽  
Ki Soo Park
Keyword(s):  
Nucleic Acid ◽  
Dna Extraction ◽  
Extraction Procedure ◽  
Food Poisoning ◽  
Pcr Amplification ◽  
High Sensitivity ◽  
Diagnostic Systems ◽  
Extraction Device ◽  
One Step ◽  
Polymerase Chain

Several bacteria are known to cause food poisoning; therefore, diagnostic systems that detect bacteria are essential. Nucleic acid-based testing methods that involve polymerase chain reaction (PCR) amplification are of great interest due to their high sensitivity and specificity. Herein, we developed a syringe-based one-step DNA extraction device that streamlines the extraction of genomic DNA (gDNA) from bacteria within 2 min, enabling versatile application of nucleic acid-based testing in the field. Notably, the bolt-nut structured case coupled with the syringe enables control of the volume of solution dispensed for enabling DNA extraction without the need for bulky centrifuge equipment. Using the proposed system, the gDNA of a model bacterium, Escherichia coli, was extracted at a good quantity and quality and amplified via PCR. The DNA extracted was comparable to that extracted via a centrifugation-based procedure. In addition, bacteria that were artificially spiked in common samples, including a work cloth, a work bench, and meat, were successfully detected with high accuracy.

A loop-mediated isothermal DNA amplification (LAMP) assay for detection of the clubroot pathogen Plasmodiophora brassicae

Plant Disease ◽  
2021 ◽  
Author(s):  
Xinyu Yang ◽  
Lin Sun ◽  
Huiying Sun ◽  
Yingzhe Hong ◽  
Zihao Xia ◽  
...  
Keyword(s):  
Plasmodiophora Brassicae ◽  
Lamp Assay ◽  
Primary Source ◽  
Dna Amplification ◽  
High Sensitivity ◽  
Minimal Amount ◽  
Source Of Infection ◽  
Resting Spores ◽  
Polymerase Chain ◽  
Plant Bioassays

Clubroot caused by Plasmodiophora brassicae is a serious threat to cruciferous crops around the world. The resting spores of P. brassicae are primary source of infection and can survive in soil for many years. Detection of resting spores in soil is essential for forecasting clubroot prevalence. Detection of P. brassicae has been relying on plant bioassays or polymerase chain reaction (PCR)-based methods. The loop-mediated isothermal DNA amplification (LAMP) is a promising approach for microorganism detection with the advantage of high sensitivity, being accurate and convenient to visualize. In this study, we developed a LAMP assay for detection of P. brassicae in soil, roots and seeds. This method can detect P. brassicae at a minimal amount of 1 fg plasmid DNA or 10 resting spores in the soil. Compared to conventional PCR, the LAMP was more sensitive in detection P. brassicae at the lower levels in soil samples. In conclusion, we elaborated a sensitive, accurate and easy-to-use LAMP assay to detect P. brassicae, which will facilitate to plan sustainable clubroot management.

scholarly journals Performance and Application of Commercially Available Loop-Mediated Isothermal Amplification (LAMP) Kits in Malaria Endemic and Non-Endemic Settings

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 336
Author(s):  
Ulrika Morris ◽  
Berit Aydin-Schmidt
Keyword(s):  
Point Of Care ◽  
High Sensitivity ◽  
Rapid Diagnostic Tests ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Point Of Care Test ◽  
Search Terms ◽  
Polymerase Chain ◽  
Meta Analyses

Loop-mediated isothermal amplification (LAMP) is a sensitive molecular tool suitable for use as a near point-of-care test for the diagnosis of malaria. Recent meta-analyses have detailed high sensitivity and specificity of malaria LAMP when compared to microscopy, rapid diagnostic tests, and polymerase chain reaction in both endemic and non-endemic settings. Despite this, the use of malaria LAMP has primarily been limited to research settings to date. In this review, we aim to assess to what extent commercially available malaria LAMP kits have been applied in different settings, and to identify possible obstacles that may have hindered their use from being adopted further. In order to address this, we conducted a literature search in PubMed.gov using the search terms (((LAMP) OR (Loop-mediated isothermal amplification)) AND ((Malaria) OR (Plasmodium))). Focusing primarily on studies employing one of the commercially available kits, we then selected three key areas of LAMP application for further review: the performance and application of LAMP in malaria endemic settings including low transmission areas; LAMP for malaria screening during pregnancy; and malaria LAMP in returning travelers in non-endemic settings.

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