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Amplicon Competition Enables End-Point Quantitation of Nucleic Acids Following Isothermal Amplification.

ABSTRACT: It is inherently difficult to quantitate nucleic acid analytes with most isothermal amplification assays. We developed loop-mediated isothermal amplification (LAMP) reactions in which competition between defined numbers of "false" and "true" amplicons leads to order of magnitude quantitation by a single endpoint determination. These thresholded LAMP reactions were successfully used to directly and quantitatively estimate the numbers of nucleic acids in complex biospecimens, including directly from cells and in sewage, with the values obtained closely correlating with qPCR quantitations. Thresholded LAMP reactions are amenable to endpoint readout by cell phone, unlike other methods that require continuous monitoring, and should therefore prove extremely useful in developing one-pot reactions for point-of-care diagnostics without needing sophisticated material or informatics infrastructure.

SUBMITTER: Jiang YS

PROVIDER: S-EPMC5890436 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Amplicon Competition Enables End-Point Quantitation of Nucleic Acids Following Isothermal Amplification.

Jiang Yu Sherry YS Stacy Apollo A Whiteley Marvin M Ellington Andrew D AD Bhadra Sanchita S

Chembiochem : a European journal of chemical biology 20170717 17

It is inherently difficult to quantitate nucleic acid analytes with most isothermal amplification assays. We developed loop-mediated isothermal amplification (LAMP) reactions in which competition between defined numbers of "false" and "true" amplicons leads to order of magnitude quantitation by a single endpoint determination. These thresholded LAMP reactions were successfully used to directly and quantitatively estimate the numbers of nucleic acids in complex biospecimens, including directly fr ...[more]

PMID: 28628741

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Project description:In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However, it still requires temperature cycling, which is undesirable under resource-limited conditions. This makes isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), more attractive. A microfluidic digital RPA SlipChip is described here for simultaneous initiation of over one thousand nL-scale RPA reactions by adding a chemical initiator to each reaction compartment with a simple slipping step after instrument-free pipet loading. Two designs of the SlipChip, two-step slipping and one-step slipping, were validated using digital RPA. By using the digital RPA SlipChip, false-positive results from preinitiation of the RPA amplification reaction before incubation were eliminated. End point fluorescence readout was used for "yes or no" digital quantification. The performance of digital RPA in a SlipChip was validated by amplifying and counting single molecules of the target nucleic acid, methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA. The digital RPA on SlipChip was also tolerant to fluctuations of the incubation temperature (37-42 °C), and its performance was comparable to digital PCR on the same SlipChip design. The digital RPA SlipChip provides a simple method to quantify nucleic acids without requiring thermal cycling or kinetic measurements, with potential applications in diagnostics and environmental monitoring under resource-limited settings. The ability to initiate thousands of chemical reactions in parallel on the nanoliter scale using solvent-resistant glass devices is likely to be useful for a broader range of applications.

Project description:Malaria, a parasitic disease caused by Plasmodium spp. and transmitted by Anopheles mosquitoes, remains a major global health issue, with an estimated 249 million cases and 608,000 deaths in 2022. Rapid and accurate diagnosis and treatment are crucial for malaria control and elimination. However, limited access to sensitive molecular tests means that microscopic examination and rapid diagnostic tests (RDT) are the most used methods in endemic areas, despite their lower diagnostic accuracy. Therefore, there is a need for developing sensitive, simple, accurate, and rapid diagnostic tools suitable for field conditions. Herein, we aimed to explore the potential of the enzymatic recombinase amplification assay (ERA® Technology) as a remote laboratory test by evaluating and validating the GENEYE® ERA Plasmodium detection kit in Brazilian endemic areas. A cross-sectional cohort study was conducted between June and August of 2023 in the Brazilian Amazon. The study enrolled 323 participants residing in three malaria-affected regions: Cruzeiro do Sul and Mâncio Lima (Acre State) and Guajará (Amazonas State). The participants were tested for malaria by microscopy, rapid diagnostic tests (RDT), nested PCR (nPCR), quantitative real-time PCR (qPCR), and ERA. The sensitivity, specificity, and predictive values were assessed using nPCR as a gold standard. Plasmodium prevalence was 21.7%, 18.8%, 19.2%, 21.7%, and 21.7% by nPCR, microscopy, RDT, qPCR, and ERA respectively. Using nPCR as the standard, qPCR, and ERA showed a sensitivity of 100%. In comparison, microscopy and RDT showed a sensitivity of 87.1% and 88.6%, a negative predictive value (NPV) of 96.56 and 96.93, and kappa values of 0.91 and 0.92, respectively. For Plasmodium falciparum, the sensitivity of qPCR and ERA was 100% while the sensitivity of microscopy and RDT was 96.9% and 93.7%, and the NPV was 99.66 and 99.32, respectively. For Plasmodium vivax, only ERA showed the same sensitivity of nPCR. The sensitivity, NPV, and kappa values were 78.85%, 97.27, and 0.87 for qPCR and microscopy, and 84.21%, 97.94, and 0.9 for RDT. The data presented here show that the GENEYE® ERA Plasmodium detection kit offers a promising alternative to traditional malaria diagnostic methods. Its high sensitivity, specificity, fast processing time, and operational simplicity position it as a valuable point-of-care diagnostic tool, particularly in resource-limited and remote malaria-endemic areas. KEY POINTS: • GENEYE® ERA kit detects Plasmodium in under 25 min, no DNA purification needed. • The kit matches or exceeds the compared methods in sensitivity and specificity. • The kit is suitable for accurate testing in low-infrastructure, point-of-care settings.

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Amplicon Competition Enables End-Point Quantitation of Nucleic Acids Following Isothermal Amplification.

Publications

Amplicon Competition Enables End-Point Quantitation of Nucleic Acids Following Isothermal Amplification.

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