Dataset Information

Critical review of methods for isothermal amplification of nucleic acids for environmental analysis.

ABSTRACT: The past 30 years have seen the emergence and proliferation of isothermal amplification methods (IAMs) for rapid, sensitive detection and quantification of nucleic acids in a variety of sample types. These methods share dependence on primers and probes with quantitative PCR, but they differ in the specific enzymes and instruments employed, and are frequently conducted in a binary, rather than quantitative format. IAMs typically rely on simpler instruments than PCR analyses due to the maintenance of a single temperature throughout the amplification reaction, which could facilitate deployment of IAMs in a variety of environmental and field settings. This review summarizes the mechanisms of the most common IAM methods and their use in studies of pathogens, harmful algae and fecal indicators in environmental waters, feces, wastewater, reclaimed water, and tissues of aquatic animals. Performance metrics of sensitivity, specificity and limit of detection are highlighted, and the potential for use in monitoring and regulatory contexts is discussed.

SUBMITTER: Nieuwkerk DM

PROVIDER: S-EPMC7726081 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Critical review of methods for isothermal amplification of nucleic acids for environmental analysis.

Nieuwkerk Dana M DM Korajkic Asja A Valdespino Erika L EL Herrmann Michael P MP Harwood Valerie J VJ

Journal of microbiological methods 20201105

The past 30 years have seen the emergence and proliferation of isothermal amplification methods (IAMs) for rapid, sensitive detection and quantification of nucleic acids in a variety of sample types. These methods share dependence on primers and probes with quantitative PCR, but they differ in the specific enzymes and instruments employed, and are frequently conducted in a binary, rather than quantitative format. IAMs typically rely on simpler instruments than PCR analyses due to the maintenance ...[more]

PMID: 33159993

Similar Datasets

Project description:Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a 4- to 8-h growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70-120 min) genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP) to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx). The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads and direct LAMP amplification without DNA purification from clinical blood, urine, wound, sputum and stool samples. The In-Dx panel was tested using two methods of detection: (1) real-time thermocycler fluorescent detection of LAMP amplification and (2) visual discrimination of color change in the presence of Eriochrome Black T (EBT) dye following amplification. In total, 239 duplicate samples were collected (31 blood, 122 urine, 73 mucocutaneous wound/swab, 11 sputum and two stool) from 229 prospectively enrolled hospital patients with suspected clinical infection and analyzed both at the hospital and by In-Dx. Sensitivity (Se) of the In-Dx panel targets pathogens from urine samples by In-Dx was 91.1% and specificity (Sp) was 97.3%, with a positive predictive value (PPV) of 53.7% and a negative predictive value (NPV) of 99.7% as compared to clinical microbial detection methods. Sensitivity of detection of the In-Dx panel from mucocutaneous swab samples was 65.5% with a Sp of 99.3%, and a PPV of 84% and NPV of 98% as compared to clinical microbial detection methods. Results indicate the LAMP-based In-Dx panel allows rapid and precise diagnosis of clinical infections by targeted pathogens across multiple culture types for point-of-care utilization.

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.

Dataset Information

Critical review of methods for isothermal amplification of nucleic acids for environmental analysis.

Publications

Critical review of methods for isothermal amplification of nucleic acids for environmental analysis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets