Detection of Non-Amplified Mycobacterium tuberculosis Genomic DNA Using Piezoelectric DNA-Based Biosensors

Piezoelectric DNA-based biosensor technology was developed as a new method for detection of M. tuberculosis. This method consists of immobilizing a thiol-modified oligonucleotide probe on the gold electrode surface of a quartz crystal, using a self-assembled monolayer method. The advantage of this study is that a non-amplified genomic bacterial DNA target was used. Instead, the genomic DNA was digested by restriction enzyme to obtain DNA fragments containing the target sequence. The fabricated biosensor was evaluated through an examination of 200 samples. No cross hybridization were observed against M. avium complex and other microorganisms. This target DNA preparation, without PCR amplification, will reduce time, costs, and the tedious step of amplification

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

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.info:eu-repo/semantics/publishedVersio

Noble Metal Nanoparticles for Biosensing Applications

In the last decade the use of nanomaterials has been having a great impact in biosensing. In particular, the unique properties of noble metal nanoparticles have allowed for the development of new biosensing platforms with enhanced capabilities in the specific detection of bioanalytes. Noble metal nanoparticles show unique physicochemical properties (such as ease of functionalization via simple chemistry and high surface-to-volume ratios) that allied with their unique spectral and optical properties have prompted the development of a plethora of biosensing platforms. Additionally, they also provide an additional or enhanced layer of application for commonly used techniques, such as fluorescence, infrared and Raman spectroscopy. Herein we review the use of noble metal nanoparticles for biosensing strategies—from synthesis and functionalization to integration in molecular diagnostics platforms, with special focus on those that have made their way into the diagnostics laboratory

Direct detection of mycobacterium tuberculosis complex using gold nanoparticles

Tuberculosis (TB) is still one of the most significant causes of morbidity and mortality worldwide. According to WHO, TB causes 2 million deaths and more than 9 million new cases annually; the overwhelming majority of TB cases occur in developing countries where accurate diagnosis of TB remains a challenge. This work aims to develop a rapid nano-gold assay for specific detection of mycobacterium tuberculosis complex (MTBC). In the first version of the assay, DNA was extracted from clinical isolates grown on LJ media. 16s rDNA regions were amplified by PCR then the genus and species of MTB were confirmed by semi-nested PCR. Spherical gold nanoparticles (AuNPs; 13 nm) were synthesized by citrate reduction method of HAuCl4 and characterized by spectrophotometry and SEM. In the first assay, the 16srDNA amplicons were denatured (95 oC, 30 s) then allowed to anneal (48 oC, 30 s) with genus- and species-specific oligotargeters in a hybridization buffer contaning NaCl (40 nM). This was followed by the addition of unmodified AuNPs (14 nM). In case of a positive specimen, the AuNPs aggregated and the solution color changed from red to blue. The solution retained red color in case of negative specimen. This assay was further optimized to specificially differentiate MTBC from other mycobacterial strains. In the second version of the assay, MTBC was directly detected in the extracted genomic DNA. Species-specific oligotargeter was added to genomic DNA and denatured for 3 min at 95 oC followed by annealing at 48 oC for 1 min. AuNPs were added and solution color changed from red to blue in case of MTBC-positive specimens. The assay detection limit was 1 ng for PCR product and 40 ng for genomic DNA. The assay showed 100% sensitivity and specificity (n = 27) as compared with automated liquid culture system (MGIT) and semi-nested PCR. Following DNA extraction according to standard procedures, the assay turnaround time is about 1 hour. In conclusion, we have developed a nano-gold assay prototype for direct detection of MTBC as a low cost alternative to current amplification-based detection platforms. The developed assay is simple, sensitive, rapid, and shows a great potential in the clinical diagnosis of TB especially in developing countries with low resource settings

Microfluidic technologies for genomic interrogation of mycobacterium tuberculosis clinical isolates using the polymerase chain reaction (PCR) and high resolution melting analysis (HRMA).

Master of Medical Science in Medical Microbiology. University of KwaZulu-Natal, Medical School 2015.Background: A number of Mycobacterium tuberculosis (Mtb) genes have been shown to be under positive selection pressure in the presence of anti-TB therapy. This results in the selection of drug resistant phenotypes associated with genetic changes—which can be point mutations, deletions and/or insertions. Some mutations from multiple genes have been documented to be associated with reduced susceptibility to anti-TB drugs such as rifampicin, ethambutol, carpreomycin and fluoroquinolones. The list is continuously updated as new mutations are discovered and validated. In principle therefore, there is an urgent need to design robust molecular diagnostics and more efficacious therapeutic strategies that are able to indicate diverse genetic mechanisms behind drug resistance in individual isolates Materials and Methods: We used the LightForge system we developed at K-RITH. This LightForge system is a fluorescence detection based, highly scalable microfluidic platform. It interrogates Mycobacterium tuberculosis strains using Real-Time PCR and High Resolution Melt Analysis (HRMA) on a chip. Results and Discussion: We have used this LightForge system to identify clinical Mtb strains resistant to rifampicin—a frontline drug used to treat tuberculosis, relative to a susceptible strain H37RV, based on mutations in the rpoB gene. This system has the potential to contribute towards a low-cost solution to diagnosis of multidrug resistant tuberculosis—a current critical global healthcare challenge. The interrogation of clinical Mtb isolates—including R35, KZN 605 and Tkk 01-062—using the LightForge system has detected mutations linked to rifampicin resistance including single nucleotide polymorphisms (SNPs) in a congruous manner with commercial systems. Conclusions: In preparation for diagnosis of clinical samples, this LightForge approach is now being expanded to incorporate detection of genetic markers linked with resistance to other TB drugs that include fluoroquinolones and isoniazid based on mutations in gyrA, katG and Mab-inhA regions of the Mtb genome. The scalability of LightForge can also be harnessed to conduct digital PCR (dPCR), a critical tool for detecting genetic heterogeneity in Mtb

tmRNA kasutamine markermolekulina bakterite tuvastamisel mikrokiibi ja biosensor tehnoloogia kaudu

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Bakterite uurimiseks on traditsiooniliselt kasutatud erinevaid kultuuris kasvatamise meetodeid. Kuigi mainitud meetodid on töökindlad ja täpsed, on nad samas ka küllaltki töö- ja ajamahukad ning ei võimalda kõikide bakterite uurimist. Seetõttu kasutatakse tänapäeval tihti nende uurimiseks erinevaid molekulaarbioloogilisi meetodeid, mis põhinevad spetsiifiliste nukleiinhappe järjestuste tuvastamisel ja kirjeldamisel. Käesolevas töös tutvustatakse erinevaid tehnoloogiaid, mida kasutatakse nukleiinahappe põhises bakteriaalses diagnostikas. Põhirõhk on erinevatel nukleiinhapete paljundamise meetoditel ning hübridisatsiooni-põhistel detektsiooni tehnoloogiatel. Käsitletud on erinevate mikrokiibi ja biosensor tehnoloogiate põhimõtteid ning nende võimalikke kasutusviise bakterite tuvastamisel. Lisaks antakse ülevaade DNA ja RNA järjestustest, mida saab kasutada markerjärjestusena erinevate bakterite tuvastamisel ja üksteisest eristamisel. Pikemalt tutvustatakse tmRNA molekule, mida kasutatakse markerjärjestusena käesoleva doktoritöö raames välja töötatud diagnostiliste meetodite puhul. tmRNA on kõikides bakterites leiduv, keskmiselt 300-400 nukleotiidi pikkune spetsiifiline RNA molekul, mis abistab rakus valgusünteesi mehhanismi, ning mille molekuli järjestuse põhjal on võimalik tuvastada ning eristada erinevaid bakteriliike ja ka kõrgemaid taksonoomilisi üksusi. Töö praktilises osas kirjeldatakse kahte erinevat meetodit, kus tmRNA detektsiooni kaudu tuvastatakse erinevaid baktereid. Nendest esimene põhineb tmRNA molekulide spetsiifilisel paljundamisel NASBA tehnoloogia abil, millele järgneb märgistatud tmRNA molekulide tuvastatamine ja täpne identifitseerimine mikrokiibi tehnoloogia abil. Teise puhul toimub tmRNA-de detektsioon märkevaba reaal-ajas toimiva biosensor süsteemi abil, mis põhineb optilisel mikroring resonaator tehnoloogial. Kuigi mõlema meetodi puhul kasutati testsüsteemina erinevaid hingamisteede haigusi põhjustavaid baktereid ning nende vastavaid liigispetsiifilisi tmRNA molekule, on kirjeldatud tehnoloogiad lihtsasti rakendatavad ka teiste RNA järjestuste ning erinevate bakteri-liikide korral.There is a growing need for faster and more reliable approaches for microorganism detection and identification that could complement or replace conventional rather time- and labor-consuming culture-based technologies. A common tactics nowadays is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to specific nucleic acid profiles that are detected and identified. Theoretically every bacterial species and strain contain unique characteristic target regions that can be used for their specific identification. In the first part of current thesis a literature overview is given about the different technologies that are used for nucleic acid-based bacterial detection. Main focus is on nucleic acid amplification and hybridization-based detection methods with emphasis on microarray and biosensor technologies, and their practical application in bacterial diagnostics. In second part of the literature overview, a description of different DNA and RNA molecules that have been targeted for bacterial detection and identification is reviewed. Longer explanation is given about the trans-translation mediating RNA molecule called tmRNA that is used as a target marker molecule in the current thesis. The research section describes two different methods that apply tmRNA for bacterial detection and identification. Firstly, a microarray-based technology is described where target tmRNA molecules are amplified using Nucleic Acid Sequence Based Amplification (NASBA) and labeled fluorescently prior the hybridization experiment. The developed method was applied for tmRNA detection from bacterial total RNA samples. In second part of the research tmRNA molecules are specifically targeted using real-time label-free biosensing platform that is based on the optical microring resonator technology. Potential quantitative nature and sensitivity of the biosensor is demonstrated using in vitro synthesized tmRNA molecules

Nanomaterials for Healthcare Biosensing Applications

In recent years, an increasing number of nanomaterials have been explored for their applications in biomedical diagnostics, making their applications in healthcare biosensing a rapidly evolving field. Nanomaterials introduce versatility to the sensing platforms and may even allow mobility between different detection mechanisms. The prospect of a combination of different nanomaterials allows an exploitation of their synergistic additive and novel properties for sensor development. This paper covers more than 290 research works since 2015, elaborating the diverse roles played by various nanomaterials in the biosensing field. Hence, we provide a comprehensive review of the healthcare sensing applications of nanomaterials, covering carbon allotrope-based, inorganic, and organic nanomaterials. These sensing systems are able to detect a wide variety of clinically relevant molecules, like nucleic acids, viruses, bacteria, cancer antigens, pharmaceuticals and narcotic drugs, toxins, contaminants, as well as entire cells in various sensing media, ranging from buffers to more complex environments such as urine, blood or sputum. Thus, the latest advancements reviewed in this paper hold tremendous potential for the application of nanomaterials in the early screening of diseases and point-of-care testing

Molecular and Biotechnological Approaches in the Diagnosis of Leprosy

Leprosy is a worldwide health problem, which needs the development of new and innovative strategies to be controlled. Early diagnosis of leprosy is an important contribution to reducing the incidence of the disease; thus, the development of biotechnology platforms, which include the mapping of antigens with potential to be used in immunodiagnostic and molecular methods for the detection of Mycobacterium leprae, is an important tool to confirm the clinical diagnostic. Molecular biology and biotechnological methods have been used to assist in the diagnosis of this disease, each one with its advantages and drawbacks. Enzyme-linked immunosorbent assay (ELISA) is the used method for leprosy diagnosis, and it allows the detection of infection-related antigens. Alternatively, due to their versatility to perform the same functions as the protein and non-protein natural antigens, mimetic peptides are considered an important tool. On the other hand, lateral flow assay (LFA) and optical and electrochemical biosensors are rapid and portable methods, capable of performing diagnosis in the field without sample preparation. This chapter presents such techniques, their uses in the diagnosis and detection of M. leprae, as well as the potential for the development of new techniques and strategies that can help to control and understand mycobacteriosis

Emerging (Bio)Sensing Technology for Assessing and Monitoring Freshwater Contamination - Methods and Applications

Ecological Water Quality - Water Treatment and ReuseWater is life and its preservation is not only a moral obligation but also a legal requirement. By 2030, global demands will exceed more than 40 % the existing resources and more than a third of the world's population will have to deal with water shortages (European Environmental Agency [EEA], 2010). Climate change effects on water resources will not help. Efforts are being made throughout Europe towards a reduced and efficient water use and prevention of any further deterioration of the quality of water (Eurostat, European Comission [EC], 2010). The Water Framework Directive (EC, 2000) lays down provisions for monitoring, assessing and classifying water quality. Supporting this, the Drinking Water sets standards for 48 microbiological and chemical parameters that must be monitored and tested regularly (EC, 1998). The Bathing Water Directive also sets concentration limits for microbiological pollutants in inland and coastal bathing waters (EC, 2006), addressing risks from algae and cyanobacteria contamination and faecal contamination, requiring immediate action, including the provision of information to the public, to prevent exposure. With these directives, among others, the European Union [EU] expects to offer its citizens, by 2015, fresh and coastal waters of good quality

Cellulose Based Genoassays for the Detection of Pathogen DNA

Simple, reliable and cost-effective methods for detecting pathogens are a vital part of diagnostics inside and outside the clinic, in particular in the developing world. Paper based colorimetric techniques are a promising approach for biosensors and bioassays as they can be used at the point of sampling and require little equipment. This study reports on the development of a colorimetric cellulose bioassay that can detect pathogen DNA with covalently attached single-stranded DNA probes. Chemical activation of cellulose via tosylation and oxidation was investigated. The successful activation of cellulose was characterised by Fourier transform infrared spectroscopy, scanning electron microscopy and elemental analysis. Sulfhydryl and amine functionalised oligonucleotide probes complementary to a segment of IS6110 element in Mycobacterium tuberculosis genome were covalently immobilised on the cellulose strips for recognition of target nucleic acid. The detection of biotinylated target oligonucleotides was achieved with horseradish peroxidase (HRP) linked to streptavidin that binds biotin with high affinity. HRP catalysed the oxdidation of tetramethylbenzidine by hydrogen peroxide. The successful assay was confirmed by the appearance of blue coloured spots on cellulose strips incubated with biotinylated target oligonucleotides complementary to the surface attached probe. The study also showed that tosylated cellulose is more reliable for the detection of targets. Initial experiments have shown sensitivity upto 0.1 µM and considerable specificity. High probe immobilization efficiencies (>90%) have been observed. The assay was also effectively demonstrated with mycobacterial DNA. Additionally, the development of a label free assay based on a dual-probe approach was investigated, but did not yield conclusive results. The developed assay has the potential for use as a simple test for the detection of pathogen DNA in clinical samples since it requires minimal equipment and is cost effective. In addition, it also shows the potential use of tosylated cellulose as a prospective surface for attaching other types of biomolecules in an active conformation.