Dataset Information

Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens.

ABSTRACT: BACKGROUND: Eimeria parasites can cause the disease coccidiosis in poultry and even subclinical infection can incur economic loss. Diagnosis of infection predominantly relies on traditional techniques including lesion scoring and faecal microscopy despite the availability of sensitive molecular assays, largely due to cost and the requirement for specialist equipment. Despite longstanding proven efficacy these traditional techniques demand time and expertise, can be highly subjective and may under-diagnose subclinical disease. Recognition of the tight economic margins prevailing in modern poultry production and the impact of avian coccidiosis on poverty in many parts of the world has highlighted a requirement for a panel of straightforward and sensitive, but cost-effective, Eimeria species-specific diagnostic assays. RESULTS: Loop-mediated isothermal amplification (LAMP) is an uncomplicated, quick and relatively inexpensive diagnostic tool. In this study we have developed a panel of species-specific LAMP assays targeting the seven Eimeria species that infect the chicken. Each assay has been shown to be genuinely species-specific with the capacity to detect between one and ten eimerian genomes, equivalent to less than a single mature schizont. Development of a simple protocol for template DNA preparation from tissue collected post mortem with no requirement for specialist laboratory equipment supports the use of these assays in routine diagnosis of eimerian infection. Preliminary field testing supports this hypothesis. CONCLUSIONS: Development of a panel of sensitive species-specific LAMP assays introduces a valuable new cost-effective tool for use in poultry husbandry.

SUBMITTER: Barkway CP

PROVIDER: S-EPMC3217895 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens.

Barkway Christopher P CP Pocock Rebecca L RL Vrba Vladimir V Blake Damer P DP

BMC veterinary research 20111103

<h4>Background</h4>Eimeria parasites can cause the disease coccidiosis in poultry and even subclinical infection can incur economic loss. Diagnosis of infection predominantly relies on traditional techniques including lesion scoring and faecal microscopy despite the availability of sensitive molecular assays, largely due to cost and the requirement for specialist equipment. Despite longstanding proven efficacy these traditional techniques demand time and expertise, can be highly subjective and m ...[more]

PMID: 22053893

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Project description:Recently, a novel coronavirus (SARS-CoV-2; coronavirus disease 2019, COVID-19) has emerged, rapidly spreading and severely straining the capacity of the global health community. Many nations are employing combinations of containment and mitigation strategies, where early diagnosis of COVID-19 is vital in controlling illness progression and limiting viral spread within the population. Thus, rapid and accurate methods of early detection are vital to contain COVID-19 and prevent further spread and predicted subsequent infectious waves of viral recurrence in future. Immediately after its initial characterization, Chinese and American Centers for Disease Control and Prevention (CDCs) rapidly employed molecular assays for detection of COVID-19, mostly employing real-time polymerase chain reaction (RT-PCR) methods. However, such methods require specific expensive items of equipment and highly trained analysts, requiring upwards of 4-8 h to process. These requirements coupled with associated financial pressures may prevent effective deployment of such diagnostic tests. Loop mediated isothermal amplification(LAMP) is method of nucleic acid amplification which exhibits increased sensitivity and specificity are significantly rapid, and do not require expensive reagents or instruments, which aids in cost reduction for coronavirus detection. Studies have shown the successful application of LAMP assays in various forms to detect coronavirus RNA in patient samples, demonstrating that 1-10 copies of viral RNA template per reaction are sufficient for successful detection, ~100-fold more sensitive than conventional RT-PCR methods. Importantly, studies have also now demonstrated the effectiveness of LAMP methodology in the detection of SARS-CoV-2 RNA at significantly low levels, particularly following numerous improvements to LAMP assay protocols. We hypothesise that recent advancements in enhanced LAMP protocols assay perhaps represent the best chance for a rapid and robust assay for field diagnosis of COVID-19, without the requirement of specialized equipment and highly trained professionals to interpret results. Herein, we present our arguments with a view to disseminate such findings, to assist the combat of this virus that is proving so devastating. We hope that this strategy could be applied rapidly, and confirmed for viability with clinical samples, before being rolled out for mass-diagnostic testing in these current times.

Project description:The COVID-19 pandemic caused by SARS-CoV-2 has infected millions worldwide, therefore there is an urgent need to increase our diagnostic capacity to identify infected cases. Although RT-qPCR remains the gold standard for SARS-CoV-2 detection, this method requires specialised equipment in a diagnostic laboratory and has a long turn-around time to process the samples. To address this, several groups have recently reported the development of loop-mediated isothermal amplification (LAMP) as a simple, low cost and rapid method for SARS-CoV-2 detection. Herein we present a comparative analysis of three LAMP-based assays that target different regions of the SARS-CoV-2: ORF1ab RdRP, ORF1ab nsp3 and Gene N. We perform a detailed assessment of their sensitivity, kinetics and false positive rates for SARS-CoV-2 diagnostics in LAMP or RT-LAMP reactions, using colorimetric or fluorescent detection. Our results independently validate that all three assays can detect SARS-CoV-2 in 30 min, with robust accuracy at detecting as little as 1000 RNA copies and the results can be visualised simply by color changes. Incorporation of RT-LAMP with fluorescent detection further increases the detection sensitivity to as little as 100 RNA copies. We also note the shortcomings of some LAMP-based assays, including variable results with shorter reaction time or lower load of SARS-CoV-2, and false positive results in some experimental conditions and clinical saliva samples. Overall for RT-LAMP detection, the ORF1ab RdRP and ORF1ab nsp3 assays have faster kinetics for detection but varying degrees of false positives detection, whereas the Gene N assay exhibits no false positives in 30 min reaction time, which highlights the importance of optimal primer design to minimise false-positives in RT-LAMP. This study provides validation of the performance of LAMP-based assays as a rapid, highly sensitive detection method for SARS-CoV-2, which have important implications in development of point-of-care diagnostics for SARS-CoV-2.

Dataset Information

Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens.

Publications

Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens.

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