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 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 A Rapid, Simple, Inexpensive, and Mobile Colorimetric Assay COVID-19-LAMP for Mass On-Site Screening of COVID-19

2020 ◽  
Vol 21 (15) ◽  
pp. 5380 ◽  
Author(s):  
Franklin Wang-Ngai Chow ◽  
Tony Tat-Yin Chan ◽  
Anthony Raymond Tam ◽  
Suhui Zhao ◽  
Weiming Yao ◽  
...  
Keyword(s):  
Mass Screening ◽  
Color Change ◽  
Colorimetric Assay ◽  
Point Of Care ◽  
Low Cost ◽  
Lamp Assay ◽  
Virus Infections ◽  
Economic Activities ◽  
Amplification Assay ◽  
Rapid Deployment

To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.

scholarly journals Is Point-of-Care testing feasible and safe in care homes in England? An exploratory usability and accuracy evaluation of Point-of-Care Polymerase Chain Reaction test for SARS-COV-2

2020 ◽  
Author(s):  
Massimo Micocci ◽  
Adam L Gordon ◽  
Mikyung Kelly Seo ◽  
A. Joy Allen ◽  
Kerrie Davies ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Point Of Care ◽  
Care Home ◽  
Care Homes ◽  
Polymerase Chain Reaction Test ◽  
List Type ◽  
Point Of Care Testing ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Positive Agreement

AbstractIntroductionReliable rapid testing on COVID-19 is needed in care homes to reduce the risk of outbreaks and enable timely care. Point-of-care testing (POCT) in care homes could provide rapid actionable results. This study aimed to examine the usability and test performance of point of care polymerase chain reaction (PCR) for COVID-19 in care homes.MethodsPoint-of-care PCR for detection of SARS-COV2 was evaluated in a purposeful sample of four UK care homes. Test agreement with laboratory real-time PCR and usability and use errors were assessed.ResultsPoint of care and laboratory polymerase chain reaction (PCR) tests were performed on 278 participants. The point of care and laboratory tests returned uncertain results or errors for 17 and 5 specimens respectively. Agreement analysis was conducted on 256 specimens. 175 were from staff: 162 asymptomatic; 13 symptomatic. 69 were from residents: 59 asymptomatic; 10 symptomatic. Asymptomatic specimens showed 83.3% (95% CI: 35.9%-99.6%) positive agreement and 98.7% negative agreement (95% CI: 96.2%-99.7%), with overall prevalence and bias-adjusted kappa (PABAK) of 0.965 (95% CI: 0.932 – 0.999). Symptomatic specimens showed 100% (95% CI: 2.5%-100%) positive agreement and 100% negative agreement (95% CI: 85.8%-100%), with overall PABAK of 1. No usability-related hazards emerged from this exploratory study.ConclusionApplications of point-of-care PCR testing in care homes can be considered with appropriate preparatory steps and safeguards. Agreement between POCT and laboratory PCR was good. Further diagnostic accuracy evaluations and in-service evaluation studies should be conducted, if the test is to be implemented more widely, to build greater certainty on this initial exploratory analysis.Key pointsPoint of care tests (POCT) in care homes are feasible and could increase testing capacity for the control of COVID-19 infection.The test of agreement between POCT and laboratory PCR for care home residents and the staff was good.Adoption of POCT in care homes can be considered with appropriate preparatory steps and safeguards in place.Repetitive errors and test malfunctioning can be mitigated with bespoke training for care home staff.Integrated care pathways should be investigated to test the high variability of the context of use.

scholarly journals Handyfuge-LAMP: low-cost and electricity-free centrifugation for isothermal SARS-CoV-2 detection in saliva

2020 ◽  
Author(s):  
Ethan Li ◽  
Adam Larson ◽  
Anesta Kothari ◽  
Manu Prakash
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Lamp Assay ◽  
Sample Collection ◽  
Rna Detection ◽  
Open Hardware ◽  
Point Of Care Diagnostics ◽  
Simple Protocol ◽  
Specialized Equipment ◽  
The Cost

AbstractPoint of care diagnostics for COVID-19 detection are vital to assess infection quickly and at the source so appropriate measures can be taken. The loop-mediated isothermal amplification (LAMP) assay has proven to be a reliable and simple protocol that can detect small amounts of viral RNA in patient samples (<10 genomes per μL) (Nagamine, Hase, and Notomi 2002). Recently, Rabe and Cepko at Harvard published a sensitive and simple protocol for COVID-19 RNA detection in saliva using an optimized LAMP assay (Rabe and Cepko, 2020).This LAMP protocol has the benefits of being simple, requiring no specialized equipment; rapid, requiring less than an hour from sample collection to readout; and cheap, costing around $1 per reaction using commercial reagents. The pH based colorimetric readout also leaves little ambiguity and is intuitive. However, a shortfall in many nucleic acid-based methods for detection in saliva samples has been the variability in output due to the presence of inhibitory substances in saliva. Centrifugation to separate the reaction inhibitors from inactivated sample was shown to be an effective way to ensure reliable LAMP amplification. However, a centrifuge capable of safely achieving the necessary speeds of 2000 RPM for several minutes often costs hundreds of dollars and requires a power supply.We present here an open hardware solution- Handyfuge - that can be assembled with readily available components for the cost of <5 dollars a unit and could be used together with the LAMP assay for point of care detection of COVID-19 RNA from saliva. The device is then validated using the LAMP protocol from Rabe and Cepko. With the use of insulated coolers for reagent supply chain and delivery, the assay presented can be completed without the need for electricity or any laboratory scale infrastructure.

scholarly journals Antigen-based testing but not real-time PCR correlates with SARS-CoV-2 virus culture

2020 ◽  
Author(s):  
Andrew Pekosz ◽  
Charles K. Cooper ◽  
Valentin Parvu ◽  
Maggie Li ◽  
Jeffrey C. Andrews ◽  
...  
Keyword(s):  
Real Time ◽  
Infectious Virus ◽  
Virus Isolation ◽  
Low Cost ◽  
Test Results ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Effective Implementation ◽  
Virus Culture ◽  
Polymerase Chain

SUMMARYIndividuals can test positive for SARS-CoV-2 by real-time polymerase chain reaction (RT-PCR) after no longer being infectious.1-8 Positive SARS-CoV-2 antigen-based testing exhibits a temporal pattern that corresponds with active, replicating virus and could therefore be a more accurate predictor of an individual’s potential to transmit SARS-CoV-2.2,3,9 Using the BD Veritor System for Rapid Detection of SARS-CoV-2 later flow antigen detection test, we demonstrate a higher concordance of antigen-positive test results with the presence of cultured, infectious virus when compared to RT-PCR. When compared to infectious virus isolation, the sensitivity of antigen-based testing is similar to RT-PCR. The correlation between SARS-CoV-2 antigen and SARS-CoV-2 culture represents a significant advancement in determining the risk for potential transmissibility beyond that which can be achieved by detection of SARS-CoV-2 genomic RNA. Coupled with a rapid time-to-result, low cost, and scalability, antigen-based testing should facilitate effective implementation of testing and public health interventions that will better contain COVID-19.

scholarly journals Nucleic Acids Analytical Methods for Viral Infection Diagnosis: State-of-the-Art and Future Perspectives

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1585
Author(s):  
Emanuele Luigi Sciuto ◽  
Antonio Alessio Leonardi ◽  
Giovanna Calabrese ◽  
Giovanna De Luca ◽  
Maria Anna Coniglio ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Analytical Methods ◽  
Point Of Care ◽  
Low Cost ◽  
Genetic Material ◽  
Treatment And Prevention ◽  
Polymerase Chain ◽  
Silicon Based ◽  
User Friendly ◽  
Diagnosis Of Infection

The analysis of viral nucleic acids (NA), DNA or RNA, is a crucial issue in the diagnosis of infections and the treatment and prevention of related human diseases. Conventional nucleic acid tests (NATs) require multistep approaches starting from the purification of the pathogen genetic material in biological samples to the end of its detection, basically performed by the consolidated polymerase chain reaction (PCR), by the use of specialized instruments and dedicated laboratories. However, since the current NATs are too constraining and time and cost consuming, the research is evolving towards more integrated, decentralized, user-friendly, and low-cost methods. These will allow the implementation of massive diagnoses addressing the growing demand of fast and accurate viral analysis facing such global alerts as the pandemic of coronavirus disease of the recent period. Silicon-based technology and microfluidics, in this sense, brought an important step up, leading to the introduction of the genetic point-of-care (PoC) systems. This review goes through the evolution of the analytical methods for the viral NA diagnosis of infection diseases, highlighting both advantages and drawbacks of the innovative emerging technologies versus the conventional approaches.

scholarly journals Nucleic acid amplification by a transparent graphene Visual-PCR chip and a disposable thermocycler

2019 ◽  
Author(s):  
Guozhi Zhu ◽  
Miao Qiao
Keyword(s):  
Nucleic Acids ◽  
Color Change ◽  
Point Of Care ◽  
Low Cost ◽  
Nucleic Acid Amplification ◽  
Driving Voltage ◽  
Conductive Films ◽  
Cooling Fan ◽  
Resource Limited ◽  
Polymerase Chain

ABSTRACTPolymerase chain reaction (PCR) is a method widely used to amplify trace amount of nucleic acids. It needs a process of thermocycling (repeated alternation of temperature). Traditional thermocycler relies on bulk size of metal block to achieve thermocycling, which results in high cost and the lack of portability. Here, a PCR chip made of graphene Transparent Conductive Films (TCFs) was employed. The thermocycling of the chip was fulfilled by a temperature programed microcontroller and a cooling fan under a low driving voltage (12V). A 35 cycles PCR was accomplished within 13 minutes using the chip and the thermocycler. The transparency of the graphene PCR chip enables the PCR reaction to be visually monitored by naked eye for a color change. The PCR chip and the thermocycler have a low cost at $2.5 and $6 respectively, and thus are feasible for Point-of-care testing (POCT) of nucleic acids in a disposable manner. The whole platform makes it possible to perform a low-cost testing of nucleic acids for varieties of purposes outside of laboratories or at resource limited locations.

scholarly journals Loop-Mediated Isothermal Amplification (LAMP) for the Diagnosis of Zika Virus: A Review

Viruses ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 19 ◽  
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Keith Pardee ◽  
Lindomar Pena
Keyword(s):  
Zika Virus ◽  
Molecular System ◽  
Point Of Care ◽  
Low Cost ◽  
Quantitative Polymerase Chain Reaction ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Freeze Dried ◽  
Polymerase Chain

The recent outbreak of Zika virus (ZIKV) in the Americas and its devastating developmental and neurological manifestations has prompted the development of field-based diagnostics that are rapid, reliable, handheld, specific, sensitive, and inexpensive. The gold standard molecular method for lab-based diagnosis of ZIKV, from either patient samples or insect vectors, is reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The method, however, is costly and requires lab-based equipment and expertise, which severely limits its use as a point-of-care (POC) tool in resource-poor settings. Moreover, given the lack of antivirals or approved vaccines for ZIKV infection, a POC diagnostic test is urgently needed for the early detection of new outbreaks and to adequately manage patients. Loop-mediated isothermal amplification (LAMP) is a compelling alternative to RT-qPCR for ZIKV and other arboviruses. This low-cost molecular system can be freeze-dried for distribution and exhibits high specificity, sensitivity, and efficiency. A growing body of evidence suggests that LAMP assays can provide greater accessibility to much-needed diagnostics for ZIKV infections, especially in developing countries where the ZIKV is now endemic. This review summarizes the different LAMP methods that have been developed for the virus and summarizes their features, advantages, and limitations.

scholarly journals A Complete Protocol for Rapid and Low-Cost Fabrication of Polymer Microfluidic Chips Containing Three-Dimensional Microstructures Used in Point-of-Care Devices

Micromachines ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 624 ◽  
Author(s):  
Trieu Nguyen ◽  
Aaydha Chidambara Vinayaka ◽  
Dang Duong Bang ◽  
Anders Wolff
Keyword(s):  
Salmonella Enteritidis ◽  
Solid Phase ◽  
Point Of Care ◽  
Low Cost ◽  
Three Dimensional ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Microfluidic Chips ◽  
Industrial Companies ◽  
Polymerase Chain

This protocol provides insights into the rapid, low-cost, and largescale fabrication of polymer microfluidic chips containing three-dimensional microstructures used in point-of-care devices for applications such as detection of pathogens via molecular diagnostic methods. The details of the fabrication methods are described in this paper. This study offers suggestions for researchers and experimentalists, both at university laboratories and in industrial companies, to prevent doom fabrication issues. For a demonstration of bio-application in point-of-care testing, the 3D microarrays fabricated are then employed in multiplexed detection of Salmonella (Salmonella Typhimurium and Salmonella Enteritidis), based on a molecular detection technique called solid-phase polymerase chain reaction (SP-PCR).

scholarly journals Cross-contamination in the Molecular Detection of Bartonella from Paraffin-embedded Tissues

2009 ◽  
Vol 46 (5) ◽  
pp. 940-944 ◽  
Author(s):  
M. Varanat ◽  
R. G. Maggi ◽  
K. E. Linder ◽  
S. Horton ◽  
E. B. Breitschwerdt
Keyword(s):  
Molecular Detection ◽  
Liquid Paraffin ◽  
Cross Contamination ◽  
Test Results ◽  
Sample Collection ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain ◽  
Formalin Fixed

The genus Bartonella comprises a group of gram-negative, fastidious bacteria. Because of diagnostic limitations of culture and serologic testing, polymerase chain reaction (PCR) has become a powerful tool for the detection of Bartonella spp. in blood and tissue samples. However, because many wild and domestic animals harbor Bartonella spp., transfer of Bartonella DNA during sample collection or histologic processing could result in false-positive PCR test results. In this study, we describe evidence of Bartonella DNA dissemination and transfer in the necropsy room and during the subsequent processing of formalin-fixed paraffin-embedded tissues. Bartonella DNA was amplified from different areas of the necropsy room, from the liquid paraffin in the tissue processor, and from different parts of the microtome. Unless stringent procedures are established and followed to avoid cross-contamination, the molecular detection of Bartonella spp. from tissue samples obtained at necropsy or processed in a multispecies histopathology laboratory will not be reliable.

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