Isothermal strand displacement amplification (iSDA): a rapid and sensitive method of nucleic acid amplification for point-of-care diagnosis

Nucleic acid amplification techniques (NAATs) have been demonstrated to make significant improvements in the diagnosis of tuberculosis (TB), particularly in the time to diagnosis and the diagnosis of smear-negative TB. The BD ProbeTec strand displacement amplification (SDA) system for the diagnosis of pulmonary and non-pulmonary tuberculosis was evaluated. A total of 689 samples were analysed from patients with clinically suspected TB. Compared with culture, the sensitivity and specificity for pulmonary samples were 98 and 89 %, and against final clinical diagnosis 93 and 92 %, respectively. This assay has undergone limited evaluation for non-respiratory samples and so 331 non-respiratory samples were tested, identifying those specimens that were likely to yield a useful result. These were CSF (n = 104), fine needle aspirates (n = 64) and pus (n = 41). Pleural fluid (n = 47) was identified as a poor specimen. A concern in using the SDA assay was that low-positive samples were difficult to interpret; 7.8 % of specimens fell into this category. Indeed, 64 % of the discrepant results, when compared to final clinical diagnosis, could be assigned as low-positive samples. Specimen type did not predict likelihood of a sample being in the low-positive zone. Although the manufacturers do not describe the concept of a low-positive zone, we have found that it aids clinical diagnosis.

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Development of an Automated, Non-Enzymatic Nucleic Acid Amplification Test

Micromachines ◽

10.3390/mi12101204 ◽

2021 ◽

Vol 12 (10) ◽

pp. 1204

Author(s):

Zackary A. Zimmers ◽

Alexander D. Boyd ◽

Hannah E. Stepp ◽

Nicholas M. Adams ◽

Frederick R. Haselton

Keyword(s):

Nucleic Acid ◽

Internal Control ◽

Magnetic Beads ◽

Point Of Care ◽

Dna Amplification ◽

Magnetic Bead ◽

Nucleic Acid Amplification ◽

Diagnostic Strategies ◽

Low Resource ◽

Target Dna

Among nucleic acid diagnostic strategies, non-enzymatic tests are the most promising for application at the point of care in low-resource settings. They remain relatively under-utilized, however, due to inadequate sensitivity. Inspired by a recent demonstration of a highly-sensitive dumbbell DNA amplification strategy, we developed an automated, self-contained assay for detection of target DNA. In this new diagnostic platform, called the automated Pi-powered looping oligonucleotide transporter, magnetic beads capture the target DNA and are then loaded into a microfluidic reaction cassette along with the other reaction solutions. A stepper motor controls the motion of the cassette relative to an external magnetic field, which moves the magnetic beads through the reaction solutions automatically. Real-time fluorescence is used to measure the accumulation of dumbbells on the magnetic bead surface. Left-handed DNA dumbbells produce a distinct signal which reflects the level of non-specific amplification, acting as an internal control. The autoPiLOT assay detected as little as 5 fM target DNA, and was also successfully applied to the detection of S. mansoni DNA. The autoPiLOT design is a novel step forward in the development of a sensitive, user-friendly, low-resource, non-enzymatic diagnostic test.

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Patent Evaluation: Nucleic acid amplification of a target sequence by strand displacement amplification

Current Opinion on Therapeutic Patents ◽

10.1517/13543776.3.10.1540 ◽

1993 ◽

Vol 3 (10) ◽

pp. 1540-1541

Keyword(s):

Nucleic Acid ◽

Nucleic Acid Amplification ◽

Target Sequence ◽

Strand Displacement ◽

Strand Displacement Amplification ◽

Patent Evaluation

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Can nucleic acid amplification tests be used to test for chlamydia and gonorrhoea in microbicide trials?

International Journal of STD & AIDS ◽

10.1258/095646207781439766 ◽

2007 ◽

Vol 18 (8) ◽

pp. 543-545

Author(s):

Patricia Rizzo-Price ◽

Paul D Stamper ◽

Billie Jo Wood ◽

Steven J Reynolds ◽

Thomas C Quinn ◽

...

Keyword(s):

Clinical Trials ◽

Nucleic Acid ◽

Nucleic Acid Amplification ◽

Urine Samples ◽

Strand Displacement ◽

Inhibitory Effects ◽

Strand Displacement Amplification ◽

Nucleic Acid Amplification Tests ◽

No Inhibition ◽

Topical Microbicides

Microbicides may interfere with detection of Chlamydia trachomatis ( Ct) and Neisseria gonorrhoeae ( Ng) in urine samples from women who use microbicides. The inhibitory effects of BufferGel, PRO2000 and PRO2000 placebo, in urine samples, were determined by nucleic acid amplification tests (NAATs). Uninfected urine was inoculated with different concentrations (105–101 organisms/mL); microbicides were added to achieve final concentrations from 5% to 0.1%. Specimens were tested using strand displacement amplification (SDA) for Ct and Ng. Samples with BufferGel demonstrated no inhibition. Samples with PRO2000 showed inhibition at the 5% concentration when tested for Ct, whereas for Ng, PRO2000 showed inhibition at 5%, 2% and some 1% concentrations. The placebo showed no inhibition when detecting Ct, and variable inhibition at the 5% and 2% concentrations for Ng. The potential inhibitory effects of microbicides on the NAATs selected for detection of Ct and Ng should be considered in clinical trials involving topical microbicides.

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Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring

Sensors ◽

10.3390/s21020602 ◽

2021 ◽

Vol 21 (2) ◽

pp. 602

Author(s):

Sandra Leonardo ◽

Anna Toldrà ◽

Mònica Campàs

Keyword(s):

Food Safety ◽

Environmental Monitoring ◽

Rolling Circle Amplification ◽

Dna Amplification ◽

Isothermal Amplification ◽

In Situ Monitoring ◽

Strand Displacement ◽

Strand Displacement Amplification ◽

Bacterial Detection ◽

Efficient Detection

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.

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Nitrocellulose-bound achromopeptidase for point-of-care nucleic acid tests

Scientific Reports ◽

10.1038/s41598-021-85481-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Georgios Chondrogiannis ◽

Shirin Khaliliazar ◽

Anna Toldrà ◽

Pedro Réu ◽

Mahiar M. Hamedi

Keyword(s):

Nucleic Acid ◽

Sample Preparation ◽

Point Of Care ◽

Dna Amplification ◽

Recombinase Polymerase Amplification ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Enzymatic Function ◽

Modern Biotechnology ◽

Nucleic Acid Tests

AbstractEnzymes are the cornerstone of modern biotechnology. Achromopeptidase (ACP) is a well-known enzyme that hydrolyzes a number of proteins, notably proteins on the surface of Gram-positive bacteria. It is therefore used for sample preparation in nucleic acid tests. However, ACP inhibits DNA amplification which makes its integration difficult. Heat is commonly used to inactivate ACP, but it can be challenging to integrate heating into point-of-care devices. Here, we use recombinase polymerase amplification (RPA) together with ACP, and show that when ACP is immobilized on nitrocellulose paper, it retains its enzymatic function and can easily and rapidly be activated using agitation. The nitrocellulose-bound ACP does, however, not leak into the solution, preventing the need for deactivation through heat or by other means. Nitrocellulose-bound ACP thus opens new possibilities for paper-based Point-of-Care (POC) devices.

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Achieving room temperature DNA amplification by dialling in destabilization

Chemical Communications ◽

10.1039/c5cc01548k ◽

2015 ◽

Vol 51 (44) ◽

pp. 9101-9104 ◽

Cited By ~ 8

Author(s):

B. Safeenaz Alladin-Mustan ◽

Catherine J. Mitran ◽

Julianne M. Gibbs-Davis

Keyword(s):

Nucleic Acid ◽

Room Temperature ◽

Point Of Care ◽

Dna Amplification ◽

Heating Element ◽

Nucleic Acid Sequences

The ability to amplify nucleic acid sequences at room temperature without the need for any heating element has been achieved, which has promise in bio-diagnostics employed at the point of care.

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