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Allele-Specific Recombinase Polymerase Amplification to Detect Sickle Cell Disease in Low-Resource Settings.

ABSTRACT: Sickle cell disease (SCD) is a group of common, life-threatening disorders caused by a point mutation in the β globin gene. Early diagnosis through newborn and early childhood screening, parental education, and preventive treatments are known to reduce mortality. However, the cost and complexity of conventional diagnostic methods limit the feasibility of early diagnosis for SCD in resource-limited areas worldwide. Although several point-of-care tests are commercially available, most are antibody-based tests, which cannot be used in patients who have recently received a blood transfusion. Here, we describe the development of a rapid, low-cost nucleic acid test that uses real-time fluorescence to detect the point mutation encoding hemoglobin S (HbS) in one round of isothermal recombinase polymerase amplification (RPA). When tested with a set of clinical samples from SCD patients and healthy volunteers, our assay demonstrated 100% sensitivity for both the β^A globin and β^S globin alleles and 94.7 and 97.1% specificities for the β^A globin allele and β^S globin allele, respectively (n = 91). Finally, we demonstrate proof-of-concept sample-to-answer genotyping of genomic DNA from capillary blood using an alkaline lysis procedure and direct input of diluted lysate into RPA. The workflow is performed in <30 min at a cost of <$5 USD on a commercially available benchtop fluorimeter and an open-source miniature fluorimeter. This study demonstrates the potential utility of a rapid, sample-to-answer nucleic acid test for SCD that may be implemented near the point of care and could be adapted to other disease-causing point mutations in genomic DNA.

SUBMITTER: Natoli ME

PROVIDER: S-EPMC7992048 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundViral diseases are a major problem in shrimp aquaculture facilities as these diseases reduce growth rates, which inevitably lead to production and profit losses. Hepatopancreatic parvoviruses (HPV) are common diseases in shrimp that appear to be associated with high or low levels of replication in specific genetic lineages. Selective breeding may result in resistance to HPV and improved body traits such as body weight, meat yield and shrimp colour, facilitating shrimp farming. HPV virus titre is commonly determined by quantitative PCR (qPCR), which is a time-consuming method requiring laboratory equipment unsuitable for field implementation. The aim of this study was to develop a simple, robust, rapid and reliable method to detect HPV in low-resource environments.MethodsWe developed a rapid shrimp HPV test that uses (1) a simple three-step sample preparation protocol, followed by (2) isothermal recombinase polymerase amplification (RPA) and lateral flow strip detection (LFD). Analytical sensitivity testing was performed in a background banana shrimp sample matrix, and retrospective testing of Fenneropenaeus merguiensis hepatopancreas tissues (n = 33) with known qPCR viral titres was used to determine diagnostic sensitivity and specificity.ResultsThe rapid shrimp HPV test could detect as little as 35 genome-equivalent copies per reaction in homogenized F. merguiensis banana shrimp. Retrospective testing of stored tissues (n = 33) indicated 100% diagnostic sensitivity (95% confidence interval, CI: 86-100%) and 100% specificity (95% CI: 66-100%) for detection of HPV.ConclusionThe rapid shrimp HPV test could be completed in only 40 minutes, and required only homogenization pestles, some pipettors, and a small heating block for single temperature incubation at 39°C. Critically, our procedure eliminated the time-consuming purification of nucleic acids from samples and when combined with RPA-LFD offers a user-friendly HPV detection format that can potentially be performed on-site. Our approach represents a major step forward in the development of a simple and sensitive end-point method for quick determination of unfavourable HPV virus numbers in shrimp, and has great potential to advance on-site management of shrimps in aquaculture.

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Allele-Specific Recombinase Polymerase Amplification to Detect Sickle Cell Disease in Low-Resource Settings.

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