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Paper microfluidic implementation of loop mediated isothermal amplification for early diagnosis of hepatitis C virus.

ABSTRACT: The early diagnosis of active hepatitis C virus (HCV) infection remains a significant barrier to the treatment of the disease and to preventing the associated significant morbidity and mortality seen, worldwide. Current testing is delayed due to the high cost, long turnaround times and high expertise needed in centralised diagnostic laboratories. Here we demonstrate a user-friendly, low-cost pan-genotypic assay, based upon reverse transcriptase loop mediated isothermal amplification (RT-LAMP). We developed a prototype device for point-of-care use, comprising a LAMP amplification chamber and lateral flow nucleic acid detection strips, giving a visually-read, user-friendly result in <40 min. The developed assay fulfils the current guidelines recommended by World Health Organisation and is manufactured at minimal cost using simple, portable equipment. Further development of the diagnostic test will facilitate linkage between disease diagnosis and treatment, greatly improving patient care pathways and reducing loss to follow-up, so assisting in the global elimination strategy.

SUBMITTER: Witkowska McConnell W

PROVIDER: S-EPMC8632961 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Paper microfluidic implementation of loop mediated isothermal amplification for early diagnosis of hepatitis C virus.

Witkowska McConnell Weronika W Davis Chris C Sabir Suleman R SR Garrett Alice A Bradley-Stewart Amanda A Jajesniak Pawel P Reboud Julien J Xu Gaolian G Yang Zhugen Z Gunson Rory R Thomson Emma C EC Cooper Jonathan M JM

Nature communications 20211130 1

The early diagnosis of active hepatitis C virus (HCV) infection remains a significant barrier to the treatment of the disease and to preventing the associated significant morbidity and mortality seen, worldwide. Current testing is delayed due to the high cost, long turnaround times and high expertise needed in centralised diagnostic laboratories. Here we demonstrate a user-friendly, low-cost pan-genotypic assay, based upon reverse transcriptase loop mediated isothermal amplification (RT-LAMP). W ...[more]

PMID: 34848705

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Project description:BackgroundContagious Bovine Pleuropneumonia (CBPP) is the most important chronic pulmonary disease of cattle on the African continent causing severe economic losses. The disease, caused by infection with Mycoplasma mycoides subsp. mycoides is transmitted by animal contact and develops slowly into a chronic form preventing an early clinical diagnosis. Because available vaccines confer a low protection rate and short-lived immunity, the rapid diagnosis of infected animals combined with traditional curbing measures is seen as the best way to control the disease. While traditional labour-intensive bacteriological methods for the detection of M. mycoides subsp. mycoides have been replaced by molecular genetic techniques in the last two decades, these latter approaches require well-equipped laboratories and specialized personnel for the diagnosis. This is a handicap in areas where CBPP is endemic and early diagnosis is essential.ResultsWe present a rapid, sensitive and specific diagnostic tool for M. mycoides subsp. mycoides detection based on isothermal loop-mediated amplification (LAMP) that is applicable to field conditions. The primer set developed is highly specific and sensitive enough to diagnose clinical cases without prior cultivation of the organism. The LAMP assay detects M. mycoides subsp. mycoides DNA directly from crude samples of pulmonary/pleural fluids and serum/plasma within an hour using a simple dilution protocol. A photometric detection of LAMP products allows the real-time visualisation of the amplification curve and the application of a melting curve/re-association analysis presents a means of quality assurance based on the predetermined strand-inherent temperature profile supporting the diagnosis.ConclusionThe CBPP LAMP developed in a robust kit format can be run on a battery-driven mobile device to rapidly detect M. mycoides subsp. mycoides infections from clinical or post mortem samples. The stringent innate quality control allows a conclusive on-site diagnosis of CBPP such as during farm or slaughter house inspections.

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Paper microfluidic implementation of loop mediated isothermal amplification for early diagnosis of hepatitis C virus.

Publications

Paper microfluidic implementation of loop mediated isothermal amplification for early diagnosis of hepatitis C virus.

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