Strand Displacement Amplification for Multiplex Detection of Nucleic Acids

The identification of various targets such as bacteria, viruses, and other cells remains a prerequisite for point-of-care diagnostics and biotechnological applications. Nucleic acids, as encoding information for all forms of life, are excellent biomarkers for detecting pathogens, hereditary diseases, and cancers. To date, many techniques have been developed to detect nucleic acids. However, most of them are based on polymerase chain reaction (PCR) technology. These methods are sensitive and robust, but they require expensive instruments and trained personnel. DNA strand displacement amplification is carried out under isothermal conditions and therefore does not need expensive instruments. It is simple, fast, sensitive, specific, and inexpensive. In this chapter, we introduce the principles, methods, and updated applications of DNA strand displacement technology in the detection of infectious diseases. We also discuss how robust, sensitive, and specific nucleic acid detection could be obtained when combined with the novel CRISPR/Cas system

Diagnostic performance of isothermal strand displacement amplification of Mycobacterium tuberculosis IS 6110 in tissue samples

AbstractBackgroundVisualized histopathological findings in tissue samples are not specific for tuberculosis while mycobacterial cultures from such specimens have low yields and long turn around times. A rapid, sensitive method is therefore needed for detection of Mycobacterium tuberculosis in paucibacillary tissue samples.MethodologyIn this paper, a total of 158 tissue specimens, including 42 culture-positives, were tested for the presence of Mycobacterium tuberculosis by strand displacement amplification of DNA targeting the region of the insertion element IS 6110 and detected by a chemiluminescence based commercial platform (BDProbeTec™ ET System). The amplification results were correlated to histopathology, microscopy and microbiological culture.ResultsThe strand displacement amplification based assay showed low overall sensitivity (31.5%) but high specificity (97.5%) which varied across various tissue types. Only 35.7% of culture-positive biopsies were positive by the molecular assay. Some discrepancy were attributed to suboptimal performance of the traditional methods.ConclusionsThe assay is useful to rule in the disease in common tissue specimens (lung, pleura and lymph node); but less so in other tissue types. The poor sensitivity in tissue specimens necessitates careful interpretation of data generated by the assay in conjunction with a clinical suspicion of tuberculosis for making decision regarding empirical treatment. The complexity of the disease pathology along with the low bacillary load and clumping tendency require selection of more sensitive methods or gene targets

Cross Priming Amplification: Mechanism and Optimization for Isothermal DNA Amplification

CPA is a class of isothermal amplification reactions that is carried out by a strand displacement DNA polymerase and does not require an initial denaturation step or the addition of a nicking enzyme. At the assay temperature of 63°C, the formation of a primer-template hybrid at transient, spontaneous denaturation bubbles in the DNA template is favored over re-annealing of the template strands by the high concentration of primer relative to template DNA. Strand displacement is encouraged by the annealing of cross primers with 5′ ends that are not complementary to the template strand and the binding of a displacement primer upstream of the crossing primer. The resulting exponential amplification of target DNA is highly specific and highly sensitive, producing amplicons from as few as four bacterial cells. Here we report on the basic CPA mechanism – single crossing CPA – and provide details on alternative mechanisms

Direct detection of Mycobacterium tuberculosis in respiratory samples from patients in Scandinavia by polymerase chain reaction

ObjectiveTo investigate the use of DNA amplification by the polymerase chain reaction (PCR) for the detection of Mycobacterium tuberculosis directly in human respiratory specimens.MethodsThe PCR assay employed was the Amplicor M. tuberculosis Test (Roche Diagnostics, Switzerland), which uses the 16S rDNA as the target template. Nine hundred and sixty samples from 741 patients in two clinical microbiology laboratories in Norway and Sweden were processed by routine culture analysis and PCR.ResultsOf the 56 specimens containing cultivatable M. tuberculosis, 49 (87.5%) were detected by PCR. Among the 904 culture-negative specimens, 897 samples were negative also by PCR and seven (0.8%) were positive by PCR. In comparison with culture, the sensitivity, specificity, and positive and negative predictive values of PCR were 91.7%, 99.6%, 94.2% and 99.4% for laboratory 1 and 80.0%, 98.7%, 76.2% and 99.0% for laboratory 2, respectively. For both laboratories combined the values were 87.5%, 99.2%, 87.5% and 99.2%.ConclusionsThese results indicate that multiple (two or three) respiratory samples from each patient should be tested, to allow sufficient accuracy in detecting M. tuberculosis in the specimens. Still, the labor-intensive format of this test necessitates strong clinical indications and patient prioritization to provide a service feasible within the current limits of routine laboratories

Novel and newer nucleic acid amplification tests for the diagnosis of TB

Background: Current tools for TB diagnosis have suboptimal accuracy, perform poorly in diagnosing extra-pulmonary TB, and are not point of care; hence results have a slow turn-around time. Objective: This project evaluated the diagnostic accuracy of the promising novel loop mediated isothermal amplification (LAMP) assay on sputum, and that of the semi-automated Xpert MTB/RIF (Xpert) test on non-sputum specimens (bronchoalveolar lavage fluid [BALF], tracheal aspirates, and cerebrospinal fluid [CSF]) from South African patients with suspected TB (the accuracy of Xpert using these fluids was unknown at the time this work was performed). Methodology: Biological samples (sputum, tracheal aspirates, BALF, or CSF) were collected from patients with suspected TB. Liquid culture served as the reference standard for the diagnosis of definite TB. Accuracy was evaluated according to HIV and smear microscopy status, where appropriate. The relationship between test performance and bacterial load (culture time-to-positivity [TTP]) was also compared. For the evaluation of LAMP, 2 spot sputa of approximately 4 ml were collected from 301 patients (60 μl of sputum was used for the assay). For the evaluation of Xpert on BALF, 152 patients who were sputum scarce or smear-negative were recruited (1 ml of the BALF aliquot or a re-suspended pellet from 10 ml BALF was used). For the evaluation of Xpert on tracheal aspirates, 120 tracheal aspirates from patients enrolled in the intensive care unit (ICU) were tested. For the evaluation of Xpert on CSF, 235 patients with suspected TBM had a lumbar puncture with 1 ml of CSF or where available a re-suspended pellet from 3 ml of CSF evaluated using Xpert

Evaluation of rapid low-cost colorimetric methods for diagnosis of multidrug-resistant tuberculosis in limited-resource settings

One third of the world\u27s population is currently infected with tuberculosis (TB), a consuming airborne disease whose main causative agent is Mycobacterium tuberculosis. The majority of these patients are found in the world\u27s poorest areas. Treatment of TB is a lengthy and demanding process utilizing a cocktail of powerful drugs; however, multidrug resistant TB (MDR-TB) strains, defined by resistance to both isoniazid (INH) and rifampicin (RIF), are now emerging worldwide and threatening disease control efforts. The major problem facing efforts to combat MDR-TB spread is its early detection. Conventional fairly affordable methods for drug resistance detection are based on solid culture and are highly time consuming (3-6 weeks in addition to initial pathogen culturing). On the other hand, the more rapid liquid culture-based automated systems are costly to set up and maintain while the very rapid molecular assays (hours to few days) are simply too complex and unaffordable and non-sustainable in limited resource settings. The objective of this work was to evaluate the performance of two liquid culture-based colorimetric assays for detection of drug resistance; nitrate reducate assay (NRA) and colorimetric redox indicator (CRI) methods for detection of MDR-TB. The assays were tested on mycobacterial isolates from Egyptian patients and their performance was compared with microscopic observation drug susceptibility assay (MODS) and the commercial automated culture system MGIT 960. Concordance was 96.7% for CRI and 93.3%, at almost one-tenth of the MGIT cost, and close to that of MODS without the need for an inverted microscope. The NRA format used in this study is more convenient and higher in throughput than the initially developed format. Additionally, DNA was extracted from the mycobacterial isolates and16S rDNA was amplified and sequenced to gain insight on the molecular diversity of Egyptian strains. Moreover, the molecular basis of strain resistance was investigated by DNA sequencing of the genes most commonly containing resistance conferring mutations. Analysis of the 16S rDNA sequencing results confirmed the identity of the samples as mycobacterium tuberculosis and suggested possible presence of two different strains. On the other hand, the analysis of the resistance related genes found common resistance conferring mutations in the MDR samples

Novel Diagnostic approach for tuberculosis diagnosis

There is a clear need for a rapid, inexpensive point-of-care diagnostic test for tuberculosis (TB). The aim of this study was to analyze the performance of a novel rapid diagnostic test for TB, GeneXpert MTB/RIF, in symptomatic adults and to compare it to other commercially available nucleic acid amplification assays and to standard microbiological smear and culture. The GeneXpert system performs real time, nested PCR from sputum and provides a result within two hours of sampling. The result includes a semi-quantitative assessment of bacillary load in the sample and simultaneously detects rifampicin resistance. This study was part of a cross-sectional, multi-centre clinical trial. The Cape Town component of this study was conducted at three sites, one hospital based and other two community clinics, all with high TB/HIV coinfection rate. Among 43.2% of patients diagnosed with TB during the evaluation study, GeneXpert detected TB in 95.5% of all culture positive cases. In smear positive patients, sensitivity was 99.0% and in smear-negative, culture positive patients, 86.1%. Specificity in patients who were culture negative and clinically diagnosed as non-TB after follow up was 98.4%. Sensitivity and specificity of GeneXpert in detecting rifampicin resistance was 100% comparing to phenotypically detected drug resistance. GeneXpert is a highly promising novel tool for the rapid diagnosis of adult TB. Future studies are needed to establish the performance and impact of GeneXpert when performed at the level of the microscopy centre

Isothermal amplification methods most used in the diagnosis of infections

Nükleik asit amplifikasyonu, enfeksiyonların, tümörlerin, genetik anormalliklerin tanısında ve adli tıpta en fazla kullanılan yöntemlerdendir. İzotermal amplifikasyon yöntemleri, Polimeraz Zincir Reaksiyonu (PZR) metoduna göre sabit sıcaklıkta yapılmasıyla karmaşıklığı azaltır. Bu yöntemler, kullanılan enzim çeşitliliği, primerler, duyarlılıkları ile özgüllükleri bakımından farklılıklar göstermektedir. Bu derlemede izotermal amplifikasyon yöntemlerinden en sık kullanılanları, prensipleri, kullanım alanları ve getirdikleri yenilikler ele alınmıştır.Isothermal amplification of nucleic acid is one of the most commonly used methods to identify the infections, tumors, genetic abnormalities, and in forensic medicine. As isothermal amplification methods can be performed at one reaction temperature under simple conditions they are less complex in comparison to Polymerase Chain Reaction (PCR). These methods differ from each other with the diversity of enzymes, primers used, sensitivity and specificity. In this review, the most commonly used isothermal amplification methods, their principles, areas of use and innovations brought about are discussed

Development of diagnostic platform for detection of biological agents and toxic microalgae using isothermal amplification

L'objectiu principal d’aquesta tesis és desenvolupar sensors d’ADN portàtils i fàcils d’utilitzar per analitzar material genètic al lloc on sigui necessari, superant les limitacions de les tecnologies actuals. Per dur-ho a terme, els sensors d’ADN s’han dissenyat integrant en un únic dispositiu dues tecnologies: l’amplificació isotèrmica d’ADN, tant en fase líquida com en fase sòlida, i la tecnologia de microarrays. A la tesis es detallen els resultats obtinguts per assolir els objectius específics, que inclouen: el desenvolupament d’una plataforma d’anàlisi d’ADN en fase líquida per a la detecció simultània de diversos organismes. L’ús d’amplificació isotèrmica d’ADN en superfície (fase sòlida), combinant tant amb detecció òptica com detecció electroquímica per a la detecció de mostres reals. L’estudi i optimització de la química de superfície dels sensors d’ADN en fase sòlida, i finalment, l’amplificació isotèrmica d’ADN d’algues tòxiques emprant dNTPs modificats amb molècules redox, fet que permet mesurar directament l’ADN amplificat reduint el temps total d’anàlisi.El objetivo principal de la tesis es desarrollar sensores de ADN portátiles y fáciles de utilizar para analizar material genético allí donde sea requerido, superando las limitaciones de las tecnologías actuales. Para llevarlo a cabo, los sensores de ADN se han diseñado integrando en un único dispositivo dos tecnologías: la amplificación isotérmica de ADN tanto en fase líquida como en fase sólida, y la tecnología de microarrays. En la tesis se detallan los resultados obtenidos para lograr los objetivos específicos, que incluyen: el desarrollo de una plataforma de análisis de ADN en fase líquida para la detección simultánea de distintos organismos. El uso de amplificación isotérmica de ADN en superficie (fase sólida) combinado tanto con detección óptica como con detección electroquímica para la detección de muestras reales. El estudio y optimización de la química de superficie de los sensores de ADN en fase sólida, y finalmente, la amplificación isotérmica de ADN de algas tóxicas utilizando dNTPs modificados con moléculas redox, cosa que permite medir el ADN amplificado directamente, reduciendo así el tiempo tota de análisis.The main goal of this doctoral thesis is to present alternative approaches in the field of DNA biosensors, design and develop new isothermal amplification protocols compatible with a portable, easy-to use device that can be deployed for analysis of genetic material at the point-of-care/need, while overcoming some of the currently existing limitations. In order to achieve this objective, a variety of different strategies of effective solid-phase immobilisation and liquid-phase and solid-phase isothermal enzymatic amplification have been studied to achieve lower detection limits with rapid and easy to carry out assays. This work presents a convenient, flexible solution for detecting DNA with biosensors, exploiting a general concept of liquid-phase and solid-phase isothermal amplification and detection, thus integrating two nucleic acid tests, PCR and microarrays, in one single device. The thesis report the work performed to achieve the specific objectives of this doctoral thesis: the development of multiplexing platform for simultaneous detection of several targets, the use of solid-phase recombinase polymerase amplification strategy for DNA amplification with optical and electrochemical detection, the detection of real samples, the study of the surface chemistry and the combination of isothermal amplification with redox labelled dNTPs for the amplification and detection of toxic microalgae as an innovative method that permits incorporation of labels throughout the amplification process facilitating direct electrochemical detection of the DNA products and an inherent shortening of assay time