Dokaz virusa newcastleske bolesti molekularnim postupkom FTA-PCR

The feasibility of using Flinders Technology Associates (FTA) filter papers to store the Newcastle disease virus (NDV), infected allantoic fluid (AF) and tissue samples, for the molecular detection of NDV by reverse transcriptase - polymerase chain reaction (RT-PCR) - was investigated. An FTA card is a cotton based cellulose membrane, with lyophilized chemicals that lyse the viruses and bacteria. The viral RNA was detectable from FTA cards up to a concentration of 107.6 EID50/100 μL (a 100 times dilution of 109.6 EID50/100 μL of initial stock). The inactivated virus remained stable on the cards for up to 30 days, both at room temperature and 4 ºC. NDV was detected by RT-PCR from all the FTA imprints of the caecal tonsils, kidney, proventriculus, spleen, trachea, faecal swabs and intestinal lesions of NDV-suspected birds. NDV was inactivated upon contact with FTA, as shown by the inability of the virus to propagate in embryonated eggs and its inability to infect chicken embryo fibroblast culture. In conclusion, FTA cards are suitable for collecting and transporting NDV infected samples, without cold storage. The virus inactivated in FTA cards, however, is a suitable source of viral RNA for molecular detection and characterization.Istražena je mogućnost uporabe Flinders tehnologije s filtrirnim papirom za pohranjivanje alantoisne tekućine zaražene virusom newcastleske bolesti i uzoraka tkiva radi molekularnog dokazivanja toga virusa lančanom reakcijom polimerazom uz prethodnu reverznu transkripciju (RT-PCR). Flinders kartica celulozna je membrana pripravljena na osnovi pamuka s liofiliziranim kemikalijama koje liziraju viruse i bakterije. Virusna RNA mogla se na takvoj kartici dokazati u količini od 107,6 EID50/100 μL (100 puta manje razrjeđenje od 109,6 EID50/100 μL osnovne suspenzije). Inaktivirani virus bio je stabilan na karticama tijekom 30 dana pri sobnoj temperaturi i 4 ºC. Virus je bio dokazan RT-PCR-om u svima Flinders tehnologijom pripravljenim otiscima cekalnih tonzila, bubrega, voljke, slezene, dušnika, obriscima fecesa i crijevnih lezija ptica sumnjivih na newcastlesku bolest. Virus je bio inaktiviran nakon dodira s FT fifi ltrirnim papirom što je bilo potvrđeno činjenicom da se nije više mogao uzgojiti u kokošjim embrijima ni na kulturi fibroblasta podrijetlom od kokošjih embrija. Zaključuje se da su FT papirići prikladni za uzimanje i prijenos uzoraka koji sadrže virus newcastleske bolesti bez potrebe pohranjivanja u hladnom prostoru. Virus inaktiviran na FT papiriću (kartici) odgovarajući je izvor virusne RNA za njegov molekularni dokaz i identifikaciju

Dokaz virusa newcastleske bolesti molekularnim postupkom FTA-PCR

The feasibility of using Flinders Technology Associates (FTA) filter papers to store the Newcastle disease virus (NDV), infected allantoic fluid (AF) and tissue samples, for the molecular detection of NDV by reverse transcriptase - polymerase chain reaction (RT-PCR) - was investigated. An FTA card is a cotton based cellulose membrane, with lyophilized chemicals that lyse the viruses and bacteria. The viral RNA was detectable from FTA cards up to a concentration of 107.6 EID50/100 μL (a 100 times dilution of 109.6 EID50/100 μL of initial stock). The inactivated virus remained stable on the cards for up to 30 days, both at room temperature and 4 ºC. NDV was detected by RT-PCR from all the FTA imprints of the caecal tonsils, kidney, proventriculus, spleen, trachea, faecal swabs and intestinal lesions of NDV-suspected birds. NDV was inactivated upon contact with FTA, as shown by the inability of the virus to propagate in embryonated eggs and its inability to infect chicken embryo fibroblast culture. In conclusion, FTA cards are suitable for collecting and transporting NDV infected samples, without cold storage. The virus inactivated in FTA cards, however, is a suitable source of viral RNA for molecular detection and characterization.Istražena je mogućnost uporabe Flinders tehnologije s filtrirnim papirom za pohranjivanje alantoisne tekućine zaražene virusom newcastleske bolesti i uzoraka tkiva radi molekularnog dokazivanja toga virusa lančanom reakcijom polimerazom uz prethodnu reverznu transkripciju (RT-PCR). Flinders kartica celulozna je membrana pripravljena na osnovi pamuka s liofiliziranim kemikalijama koje liziraju viruse i bakterije. Virusna RNA mogla se na takvoj kartici dokazati u količini od 107,6 EID50/100 μL (100 puta manje razrjeđenje od 109,6 EID50/100 μL osnovne suspenzije). Inaktivirani virus bio je stabilan na karticama tijekom 30 dana pri sobnoj temperaturi i 4 ºC. Virus je bio dokazan RT-PCR-om u svima Flinders tehnologijom pripravljenim otiscima cekalnih tonzila, bubrega, voljke, slezene, dušnika, obriscima fecesa i crijevnih lezija ptica sumnjivih na newcastlesku bolest. Virus je bio inaktiviran nakon dodira s FT fifi ltrirnim papirom što je bilo potvrđeno činjenicom da se nije više mogao uzgojiti u kokošjim embrijima ni na kulturi fibroblasta podrijetlom od kokošjih embrija. Zaključuje se da su FT papirići prikladni za uzimanje i prijenos uzoraka koji sadrže virus newcastleske bolesti bez potrebe pohranjivanja u hladnom prostoru. Virus inaktiviran na FT papiriću (kartici) odgovarajući je izvor virusne RNA za njegov molekularni dokaz i identifikaciju

Brzi lateks aglutinacijski test za serološki dokaz serotipa 4 ptičjeg adenovirusa upotrebom rekombinantnog antigena

Hydropericardium hepatitis syndrome (HPS) is a newly emerging disease of poultry, which is caused by fowl adenovirus serotype 4. The virus was propagated in a primary chicken embryo liver culture. The cytopathic effect was observed from the third passage onwards. DNA was isolated from the infected culture and used as a template for amplification of a partial hexon gene (700 bp) using hexon gene specific primers. The amplified product was cloned into pProEX HT b vector and the ligated product was transformed into DH5α cells. The recombinant clones were analyzed by colony PCR and plasmid isolation, followed by restriction digestion to check the insert release. The positive clones were induced by IPTG. The induced culture fractions were checked at different hours and the induction was high at the 4th hour onwards. The expressed proteins were purified and confirmed by using hyperimmune serum against FAV4 by western blot analysis and the protein size of 50kda was obtained. The purified recombinant FAV4 protein was used as a serodiagnostic agent using enzyme linked immunosorbent assay and latex agglutination test.Sindrom hidroperikarda i hepatitisa nova je bolest peradi uzrokovana serotipom 4 ptičjeg adenovirusa. Virus je bio umnožen u primarnoj staničnoj kulturi podrijetlom od jetrenoga tkiva pilećega zametka. Citopatski učinak javio se nakon treće pasaže. DNK je bila izdvojena iz zaražene kulture i rabljena kao kalup za umnožavanje dijela gena heksona (700 bp) uz upotrebu specifičnih početnica. Umnoženi odsječak bio je kloniran u vektoru pProEX HT b, a proizašli proizvod prebačen u DH5α stanice. Rekombinantni klonovi bili su analizirani lančanom reakcijom polimerazom i izdvajanjem plazmida nakon čega je pomoću restrikcijske digestije provjerena uspješnost postupka. Pozitivni klonovi bili su inducirani pomoću IPTG-a. Frakcije induciranih stanica bile su provjeravane svakog sata, a indukcija je bila velika nakon četiri sata. Proizvedene bjelančevine bile su pročišćene i identificirane uporabom hiperimunoga seruma za FAV4 Western blot analizom te se pokazalo da je proizvedena bjelančevina veličine 50 kda. Pročišćena rekombinantna bjelančevina FAV4 bila je rabljena kao antigen u imunoenzimnom testu i lateks aglutinacijskom testu

Brzi lateks aglutinacijski test za serološki dokaz serotipa 4 ptičjeg adenovirusa upotrebom rekombinantnog antigena

Hydropericardium hepatitis syndrome (HPS) is a newly emerging disease of poultry, which is caused by fowl adenovirus serotype 4. The virus was propagated in a primary chicken embryo liver culture. The cytopathic effect was observed from the third passage onwards. DNA was isolated from the infected culture and used as a template for amplification of a partial hexon gene (700 bp) using hexon gene specific primers. The amplified product was cloned into pProEX HT b vector and the ligated product was transformed into DH5α cells. The recombinant clones were analyzed by colony PCR and plasmid isolation, followed by restriction digestion to check the insert release. The positive clones were induced by IPTG. The induced culture fractions were checked at different hours and the induction was high at the 4th hour onwards. The expressed proteins were purified and confirmed by using hyperimmune serum against FAV4 by western blot analysis and the protein size of 50kda was obtained. The purified recombinant FAV4 protein was used as a serodiagnostic agent using enzyme linked immunosorbent assay and latex agglutination test.Sindrom hidroperikarda i hepatitisa nova je bolest peradi uzrokovana serotipom 4 ptičjeg adenovirusa. Virus je bio umnožen u primarnoj staničnoj kulturi podrijetlom od jetrenoga tkiva pilećega zametka. Citopatski učinak javio se nakon treće pasaže. DNK je bila izdvojena iz zaražene kulture i rabljena kao kalup za umnožavanje dijela gena heksona (700 bp) uz upotrebu specifičnih početnica. Umnoženi odsječak bio je kloniran u vektoru pProEX HT b, a proizašli proizvod prebačen u DH5α stanice. Rekombinantni klonovi bili su analizirani lančanom reakcijom polimerazom i izdvajanjem plazmida nakon čega je pomoću restrikcijske digestije provjerena uspješnost postupka. Pozitivni klonovi bili su inducirani pomoću IPTG-a. Frakcije induciranih stanica bile su provjeravane svakog sata, a indukcija je bila velika nakon četiri sata. Proizvedene bjelančevine bile su pročišćene i identificirane uporabom hiperimunoga seruma za FAV4 Western blot analizom te se pokazalo da je proizvedena bjelančevina veličine 50 kda. Pročišćena rekombinantna bjelančevina FAV4 bila je rabljena kao antigen u imunoenzimnom testu i lateks aglutinacijskom testu

Analytical affinity chromatography-on-a-chip for selective capture and sensitive detection of protein and polynucleotide biomarkers

Affinity Chromatography is a powerful technique which has been applied to the highly selective purification of several biomolecules from complex mixtures. This technique is currently a core technology in the industrial purification of disruptive biopharmaceuticals such as monoclonal antibodies. The use of high affinity ligands, together with densely functionalized three-dimensional solid-phase supports, confers a remarkable analytical potential, making it a current standard for the quantification of several compounds in certified laboratories, ranging from health biomarkers to environmental contaminants. Aiming at extending the application of affinity chromatography to a portable setup, we report the miniaturization of this system down to nL-scale, by trapping Q-sepharose or protein-A agarose beads in microfluidic channels with total volumes ranging from 60 to 210 nL. This versatile and simple platform combined the high surface area and robust surface chemistry provided by the chromatographic media with the high degree of fluidic control, portability, improved reaction kinetics and low reagent expenditure inherent to microfluidics. Furthermore, the microfluidic structures are simple in terms of microfabrication and can be sequentially operated using standard pipette tips and a negative pressure source at the outlet (Figure A). This system was tested within the scope of prostate cancer diagnostics for the capture of protein and polynucleotide biomarkers. Along these lines, prostate specific antigen (PSA) was selectively captured from unprocessed human serum and a 23 bp polynucleotide (ssDNA analogous to micro RNA MIR145) in fetal bovine serum as model matrix, by coupling a monoclonal anti-PSA IgG2a with protein-A beads or a complementary ssDNA strand with Q-sepharose beads, respectively. The assay schematics are described in Figure A. Clinically relevant sensitivities below 10 ng/mL PSA (Figure B) and 10 pM polynucleotide were achieved using a horseradish peroxidase-labelled reporter and measuring chemiluminescence directly on the bead surface. The results demonstrate a high potential for the miniaturization of analytical affinity chromatography, providing good sensitivities in a portable setup, particularly considering the amenability of integrating miniaturized thin-film sensors for optical transduction, as previously demonstrated by our group. Please click Additional Files below to see the full abstract

Electrochemical Genosensing of E. coli Based on Padlock Probes and Rolling Circle Amplification

Isothermal amplification techniques are emerging nowadays for the rapid and accurate detection of pathogenic bacteria in low resource settings, where many infectious diseases are endemic, and the lack of reliable power supply, trained personnel and specialized facilities pose critical barriers for timely diagnosis. This work addresses the detection of E. coli based on DNA isothermal amplification performed on magnetic particles (MPs) followed by electrochemical genosensing on disposable electrodes by square-wave voltammetry. In this approach, the bacterial DNA is preconcentrated using a target-specific magnetic probe and then amplified on the MPs by rolling circle amplification (RCA). Two different electrochemical readout methods for the RCA amplicons are tested. The first one relied on the labelling of the magnetic RCA product with a digoxigenin probe followed by the incubation with antiDIG-HRP antibody as electrochemical reporter. In the second case, the direct detection with an HRP-probe was performed. This latter strategy showed an improved analytical performance, while simultaneously avoiding the use of thermocyclers or bulky bench top equipment

Growth behaviour and mechanical properties of PLL/HA multilayer films studied by AFM

Scanning- and colloidal-probe atomic force microscopy were used to study the mechanical properties of poly(L-lysine)/hyaluronan (PLL/HA)n films as a function of indentation velocity and the number of polymer deposition steps n. The film thickness was determined by two independent AFM-based methods: scratch-and-scan and newly developed full-indentation. The advantages and disadvantages of both methods are highlighted, and error minimization techniques in elasticity measurements are addressed. It was found that the film thickness increases linearly with the bilayer number n, ranging between 400 and 7500 nm for n = 12 and 96, respectively. The apparent Young’s modulus E ranges between 15 and 40 kPa and does not depend on the indenter size or the film bilayer number n. Stress relaxation measurements show that PLL/HA films have a viscoelastic behaviour, regardless of their thickness. If indentation is performed several times at the same lateral position on the film, a viscous/plastic deformation takes place

Efficient DNA-assisted synthesis of trans-membrane gold nanowires

Whereas electric circuits and surface-based (bio)chemical sensors are mostly constructed in-plane due to ease of manufacturing, 3D microscale and nanoscale structures allow denser integration of electronic components and improved mass transport of the analyte to (bio)chemical sensor surfaces. This work reports the first out-of-plane metallic nanowire formation based on stretching of DNA through a porous membrane. We use rolling circle amplification (RCA) to generate long single-stranded DNA concatemers with one end anchored to the surface. The DNA strands are stretched through the pores in the membrane during liquid removal by forced convection. Because the liquid–air interface movement across the membrane occurs in every pore, DNA stretching across the membrane is highly efficient. The stretched DNA molecules are transformed into trans-membrane gold nanowires through gold nanoparticle hybridization and gold enhancement chemistry. A 50 fM oligonucleotide concentration, a value two orders of magnitude lower than previously reported for flat surface-based nanowire formation, was sufficient for nanowire formation. We observed nanowires in up to 2.7% of the membrane pores, leading to an across-membrane electrical conductivity reduction from open circuit to o20 Ω. The simple electrical read-out offers a high signal-to-noise ratio and can also be extended for use as a biosensor due to the high specificity and scope for multiplexing offered by RCA.Correction in: Microsystems &amp; Nanoengineering (2018) 4:9 DOI: 10.1038/s41378-018-0012-7, WOS: 000434457800001QC 20180601</p

Real-Time QCM Measurements of Rolling Circle Amplification Products

We demonstrate real-time experimental evidence for mass underestimation, to analyse high molecular weight biomolecules while using Quartz Crystal Microbalance (QCM) platform. For this, we present an experimental evidence with an assay consisting of an aptamer-antibody sandwich model with norovirus-like particles (NoVLPs). We combine this with the proximity ligation assay to generate bulky rolling circle products on a gold spot centralized quartz resonator surface. Real-time monitoring of the assay build-up reflects that bulky NoVLPs and amplified oligonucleotides generate fluctuating signals during measurement using a microfluidic QCM sensor. Such an understanding of mass underestimation for heavier molecules becomes indispensable to get deeper insights into molecular interactions in biotechnology studied using QCM devices