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

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

The Analyst ◽

10.1039/c5an01632k ◽

2015 ◽

Vol 140 (22) ◽

pp. 7540-7549 ◽

Cited By ~ 43

Author(s):

Bhushan J. Toley ◽

Isabela Covelli ◽

Yevgeniy Belousov ◽

Sujatha Ramachandran ◽

Enos Kline ◽

...

Keyword(s):

Nucleic Acid ◽

Kinetic Model ◽

Point Of Care ◽

Reaction Network ◽

Dna Amplification ◽

Nucleic Acid Amplification ◽

Strand Displacement ◽

Strand Displacement Amplification ◽

Simple Kinetic Model ◽

Sensitive Method

A new rapid and sensitive method of isothermal DNA amplification and a simple kinetic model of this reaction network.

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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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Detection of Mycobacterium tuberculosis DNA with thermophilic strand displacement amplification and fluorescence polarization

Clinical Chemistry ◽

10.1093/clinchem/42.10.1604 ◽

1996 ◽

Vol 42 (10) ◽

pp. 1604-1608 ◽

Cited By ~ 16

Author(s):

G T Walker ◽

C P Linn

Keyword(s):

Mycobacterium Tuberculosis ◽

Restriction Enzyme ◽

Fluorescence Polarization ◽

Target Sequence ◽

Strand Displacement ◽

Insertion Element ◽

Strand Displacement Amplification ◽

Simple Protocol ◽

Polymerase Binding

Abstract Strand displacement amplification (SDA) is an isothermal, in vitro method for diagnostics that amplifies a target DNA sequence by using a restriction enzyme and DNA polymerase. We have combined a new thermophilic form of SDA that involves restriction enzyme BsoBI and polymerase exo-Bca with fluorescence polarization for detection of Mycobacterium tuberculosis DNA by using the IS6110 insertion element as the target sequence. A 5'-fluorescein-labeled oligodeoxynucleotide detector probe hybridizes to the amplified product as it rises in concentration during SDA, and the single- to double-stranded conversion is monitored through an increase in fluorescence polarization. The associated change in polarization upon amplification of the target sequence is enhanced by specific polymerase binding to the double-stranded detector probe. Fewer than 10 M. tuberculosis genomes can be amplified and detected with an extremely simple protocol that takes only 20 min and uses relatively simple instrumentation and reagents, all of which can be purchased off-the-shelf.

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Quantitative nucleic acid amplification by digital PCR for clinical viral diagnostics

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2015-1101 ◽

2016 ◽

Vol 0 (0) ◽

Cited By ~ 4

Author(s):

Kuo Zhang ◽

Guigao Lin ◽

Jinming Li

Keyword(s):

Nucleic Acid ◽

Digital Pcr ◽

Nucleic Acid Amplification ◽

Target Sequence ◽

The Past ◽

Amplification Technique ◽

Diagnostic Applications ◽

Further Development ◽

Nucleic Acid Amplification Technique ◽

Viral Diagnostics

AbstractIn the past few years, interest in the development of digital PCR (dPCR) as a direct nucleic acid amplification technique for clinical viral diagnostics has grown. The main advantages of dPCR over qPCR include: quantification of nucleic acid concentrations without a calibration curve, comparable sensitivity, superior quantitative precision, greater resistance to perturbations by inhibitors, and increased robustness to the variability of the target sequence. In this review, we address the application of dPCR to viral nucleic acid quantification in clinical applications and for nucleic acid quantification standardization. Further development is required to overcome the current limitations of dPCR in order to realize its widespread use for viral load measurements in clinical diagnostic applications.

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Current Methods for Fluorescence-Based Universal Sequence-Dependent Detection of Nucleic Acids in Homogenous Assays and Clinical Applications

Clinical Chemistry ◽

10.1373/clinchem.2013.205211 ◽

2013 ◽

Vol 59 (11) ◽

pp. 1567-1582 ◽

Cited By ~ 20

Author(s):

Bernd Faltin ◽

Roland Zengerle ◽

Felix von Stetten

Keyword(s):

Critical Evaluation ◽

Cost Effective ◽

Clinical Applications ◽

Clinical Diagnostics ◽

Detection Methods ◽

Target Sequence ◽

Strand Displacement ◽

Strand Displacement Amplification ◽

Sequence Dependent ◽

Universal Detection

BACKGROUND Specific and sensitive nucleic acid (NA) testing in research and clinical diagnostics is usually performed by use of labeled oligonucleotide probes. However, the use of target-specific fluorogenic probes increases the cost of analysis. Therefore, universal sequence-dependent (USD) NA detection methods have been developed to facilitate cost-effective target detection using standardized reagents. CONTENT We provide a comprehensive review of the current methods for fluorescence-based USD NA detection. Initially, we focus on the emergence of these methods as a means to overcome the shortcomings of common NA detection methods, such as hydrolysis probes and molecular beacons. Thereafter, we provide a critical evaluation of the individual detection methods. These methods include (a) target amplification with bipartite primers introducing a universal detection tag to the amplicon (UniPrimer PCR, universal fluorescence energy transfer probe PCR, attached universal duplex probe PCR, and universal strand displacement amplification) or combined with bipartite probes comprising a universal detection region (mediator probe PCR, universal strand displacement amplification, universal quenching probe PCR) and (b) amplification-independent assays employing either a universal variant of the invader assay or universal NA hybridization sensors. We discuss differences between the methods and review clinical applications. SUMMARY The current methods for USD NA testing are cost-effective and flexible and have concordant analytical performance in comparison with common probe-based techniques. They can detect any target sequence by the simple use of a label-free, low-cost primer or probe combined with a universal fluorogenic reporter. The methods differ in the number of target specificities, capability of multiplexing, and incubation requirements (isothermal/thermocycling). Extensive clinical applications comprise detection of single-nucleotide polymorphisms, study of gene expression, in situ PCR, and quantification of pathogen load.

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Loop-Mediated Isothermal Amplification (LAMP) for Detection of Various Organisms: A Review

Bangladesh Journal of Medical Microbiology ◽

10.3329/bjmm.v11i2.51679 ◽

2017 ◽

Vol 11 (2) ◽

pp. 20-27

Author(s):

Arifa Akram

Keyword(s):

Nucleic Acid ◽

High Specificity ◽

Disease Diagnosis ◽

Isothermal Amplification ◽

Nucleic Acid Amplification ◽

Lamp Method ◽

Isolation And Identification ◽

Strand Displacement Amplification ◽

Loop Mediated Isothermal Amplification ◽

Prophylactic Measures

Disease diagnosis is important for implementation of proper therapeutic and prophylactic measures. Traditionally, disease diagnosis was depends upon isolation and identification of the causative organisms. This was followed by serology and after that molecular method. Molecular tests are valuable when early diagnosis is important. For this purpose, nucleic acid amplification (PCR, nucleic acid sequence-based amplification, self-sustained sequence replication, strand displacement amplification) is one of the most valuable tools not only for the diagnosis of infectious diseases but also used in advanced level research. The Loop-Mediated Isothermal Amplification (LAMP) is a unique nucleic acid amplification technique for diagnosis of various pathogens introduced at 2000 by Notomi and his colleagues which is simple, easy, rapid and cost effective when compared to PCR due to its high specificity, sensitivity, and rapidity. It uses a set of six primers and a DNA polymerase with stranddisplacement activity. Major advantage of LAMP method is its cost-effectiveness as it can be done simply by using waterbath or heating block. Bangladesh J Med Microbiol 2017; 11 (2): 20-27

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Genomic Variations in SARS-CoV-2 Strains at the Target Sequences of Nucleic Acid Amplification Tests

Archives of Medical Science ◽

10.5114/aoms/133120 ◽

2021 ◽

Author(s):

Canhui Cao ◽

Ruidi Yu ◽

Shaoqing Zeng ◽

Dan Liu ◽

Wenjian Gong ◽

...

Keyword(s):

Nucleic Acid ◽

False Negative ◽

False Negative Rate ◽

Nucleic Acid Amplification ◽

Whole Genome Sequencing Data ◽

Target Sequence ◽

Nucleic Acid Amplification Tests ◽

Probe Target ◽

Genomic Variations ◽

Target Sequences

IntroductionNucleic acid amplification is the main method used to detect infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the false-negative rate of nucleic acid tests cannot be ignored.Material and methodsHerein, we demonstrated genomic variations at the target sequences for the tests and the geographical distribution of the variations across countries by analyzing the whole-genome sequencing data of SARS-CoV-2 strains from the 2019 Novel Coronavirus Resource (2019nCoVR) database.ResultsAmong the 21 pairs of primer sequences in regions ORF1ab, S, E, and N, the total length of primer and probe target sequences was 938bp, with 131(13.97%) variant loci in 2415 (38.96%) isolates. Primer targets in the N region contained the most variations that were distributed among the most isolates, and the E region contained the least. Single nucleotide polymorphisms were the most frequent variation, with C to T transitions being detected in the most variant loci. G to A transitions and G to C transversions were the most common and had the highest isolate density. Genomic variations at the three mutation sites N: 28881, N: 28882, and N: 28883 were the most commonly detected, including in 608 SARS-CoV-2 strains from 33 countries, especially in the United Kingdom, Portugal, and Belgium.ConclusionsOur work comprehensively analyzed genomic variations on the target sequences of the nucleic acid amplification tests, offering evidence to optimize primer and probe target sequence selection, thereby improving the performance of the SARS-CoV-2 diagnostic test.

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