Palm-size and one-inch gel electrophoretic device for reliable and field-applicable analysis of recombinase polymerase amplification

2019 ◽  
Vol 11 (39) ◽  
pp. 4969-4976 ◽  
Author(s):  
Himankshi Rathore ◽  
Radhika Biyani ◽  
Hirotomo Kato ◽  
Yuzuru Takamura ◽  
Manish Biyani
Keyword(s):  
Nucleic Acid ◽  
Molecular Diagnostics ◽  
Gel Electrophoresis ◽  
Detection System ◽  
Recombinase Polymerase Amplification

A newly designed handheld one-inch gel electrophoresis-based detection system and recombinase polymerase amplification (RPA) can revolutionize nucleic acid-based molecular diagnostics for people in settings with poor healthcare infrastructure.

Development of acoustic wave biosensors for application in molecular diagnostics

2018 ◽  
Author(s):  
Αριστέα Γραμμουστιάνου
Keyword(s):  
Nucleic Acid ◽  
Acoustic Wave ◽  
Salmonella Typhimurium ◽  
Molecular Diagnostics ◽  
Peptide Nucleic Acid ◽  
Circulating Tumor Dna ◽  
Recombinase Polymerase Amplification ◽  
Tumor Dna

Από την ανάπτυξη του πρώτου βιοαισθητήρα από τον Leland C. Clark το 1962, η πρόοδος στον τομέα των βιοαισθητήρων είναι πρωτοφανής. Η αγορά των βιοαισθητήρων εκτιμάται ότι θα κοστίζει 27.06 δισεκατομμύρια δολάρια μέχρι το 2022. Επί του παρόντος, η επιστημονική έρευνα είναι επικεντρωμένη στην ανάπτυξη οικονομικών, μη επεμβατικών, ενσωματωμένων και φορητών βιοαισθητήρων που θα βρουν εφαρμογή στους τομείς της ταχείας διαγνωστικής, της ανάλυσης ποιότητας τροφίμων, του περιβαλλοντικού ελέγχου, της βιοάμυνας και της εγκληματολογίας. Η παρούσα διδακτορική έρευνα επικεντρώθηκε στην ανάπτυξη ακουστικών βιοαισθητήρων που μπορούν να βρουν εφαρμογή στη μοριακή διαγνωστική. Οι ακουστικοί βιοαισθητήρες επιτρέπουν την βιολογική ανίχνευση σε πραγματικό χρόνο και χωρίς την ανάγκη σήμανσης. Η έρευνα χρησιμοποίησε τα μοναδικά χαρακτηριστικά των ακουστικών βιοαισθητήρων για την ανάπτυξη βιοδοκιμών με εφαρμογή στους τομείς της ασφάλειας τροφίμων και της διαγνωστικής καρκίνου.Ο πρώτος στόχος της διατριβής ήταν η ακουστική ανίχνευση του τροφιμογενούς παθογόνου Salmonella Typhimurium. Δύο προσεγγίσεις ακολουθήθηκαν: η πρώτη χρησιμοποίησε έναν ακουστικό ανοσοαισθητήρα για την ανίχνευση ολόκληρων κυττάρων Salmonella Typhimurium. Ο βιοαισθητήρας πέτυχε την ανίχνευση των κυττάρων μέσα σε 30 λεπτά και με όριο ανίχνευσης τις 106 μονάδες σχηματισμού αποικίας/mL (106 cfu/mL). Η δεύτερη προσέγγιση χρησιμοποίησε έναν ακουστικό βιοαισθητήρα για την ενίσχυση και ανίχνευση της ενίσχυσης του γονιδίου invA της Salmonella Typhimurium επάνω στην ακουστική επιφάνεια. Για την ενίσχυση του γονιδίου χρησιμοποιήθηκε η ισόθερμη τεχνική recombinase polymerase amplification (RPA). Η ταυτόχρονη ανίχνευση των γονιδιακών αμπλικονίων πάνω στην επιφάνεια του βιοαισθητήρα έγινε με τη χρήση λιποσωμάτων που έδρασαν ως ενισχυτικοί ανιχνευτές. Η μεθοδολογία που παρουσιάστηκε για πρώτη φορά σε αυτή την έρευνα ήταν γρήγορη (ανίχνευση των γονιδιακών αμπλικονίων σε 5 μόνο λεπτά) και απλή (δε χρειάστηκαν μετα-ενισχυτικά βήματα επεξεργασίας των αμπλικονίων).Το δεύτερο μέρος της παρούσας διατριβής ασχολήθηκε με την ανάπτυξη ακουστικών βιοαισθητήρων για τη μοριακή διαγνωστική καρκίνου. Ένας DNA-βιοαισθητήρας σχεδιάστηκε για την ανίχνευση 4 microRNAs (miRNAS: miR-21, miR-155, miR-150, miR-107) που θεωρούνται δυναμικοί βιοδείκτες του καρκίνου του μαστού. Η ακουστική ανίχνευση πραγματοποιήθηκε με τη χρήση ενός μοναδικού ολιγονουκλεοτιδικού ιχνηθέτη. Μάλιστα, ο βιοαισθητήρας επέτρεψε τη γρήγορη, απλή και ταυτόχρονη ανίχνευση δύο miRNAs σε πραγματικό χρόνο και χωρίς τη χρήση μορίων επισήμανσης.Ο τελικός σκοπός της παρούσας δουλειάς ήταν η ανάπτυξη ενός ακουστικού βιοαισθητήρα για την ανίχνευση KRAS μεταλλάξεων, χαρακτηριστικών του ορθοκολικού καρκίνου, παρόντων σε circulating tumor DNA (ctDNA) μόρια. Η ανίχνευση των KRAS μεταλλάξεων έγινε μέσω της υβριδοποίησης των ctDNA μορίων σε έναν peptide nucleic acid (PNA) ανιχνευτή πάνω στην επιφάνεια του αισθητήρα. Χρησιμοποιήθηκαν ακουστικοί αισθητήρες υψηλούς θεμελιώδους συχνότητας και μία πατενταρισμένη τεχνολογία που συνδυάζει νουκλεοβάσεις με τροποιημένη αλδεΰδη (SMART nucleobases) και μοναδικούς PNA ιχνηθέτες που φέρουν μία «κενή» θέση στον σκελετό τους (DGL probes). Η τεχνολογία που αναπτύχθηκε πέτυχε την ακουστική ανίχνευση KRAS μεταλλάξεων παρόντων σε μόρια ctDNA εισάγοντας μία νέα εναλλακτική στις τρέχουσες μεθόδους διάγνωσης του ορθοκολικού καρκίνου.

scholarly journals A glucose meter interface for point-of-care gene circuit-based diagnostics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Evan Amalfitano ◽  
Margot Karlikow ◽  
Masoud Norouzi ◽  
Katariina Jaenes ◽  
Seray Cicek ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Synthetic Biology ◽  
Molecular Diagnostics ◽  
Point Of Care ◽  
Low Cost ◽  
Gene Circuit ◽  
Reporter Systems ◽  
Glucose Meter ◽  
Pandemic Response ◽  
Glucose Output

AbstractRecent advances in cell-free synthetic biology have given rise to gene circuit-based sensors with the potential to provide decentralized and low-cost molecular diagnostics. However, it remains a challenge to deliver this sensing capacity into the hands of users in a practical manner. Here, we leverage the glucose meter, one of the most widely available point-of-care sensing devices, to serve as a universal reader for these decentralized diagnostics. We describe a molecular translator that can convert the activation of conventional gene circuit-based sensors into a glucose output that can be read by off-the-shelf glucose meters. We show the development of new glucogenic reporter systems, multiplexed reporter outputs and detection of nucleic acid targets down to the low attomolar range. Using this glucose-meter interface, we demonstrate the detection of a small-molecule analyte; sample-to-result diagnostics for typhoid, paratyphoid A/B; and show the potential for pandemic response with nucleic acid sensors for SARS-CoV-2.

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

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.

Construction of a Novel Peptide Nucleic Acid Piezoelectric Gene Sensor Microarray Detection System

2005 ◽  
Vol 5 (8) ◽  
pp. 1266-1272 ◽  
Author(s):  
Ming Chen ◽  
Minghua Liu ◽  
Lili Yu ◽  
Guoru Cai ◽  
Qinghai Chen ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Peptide Nucleic Acid ◽  
Detection System ◽  
Microarray Detection

Properties of the single protein and two nucleic acids of tomato ring-spot virus

1977 ◽  
Vol 55 (8) ◽  
pp. 1028-1037 ◽  
Author(s):  
Wayne R. Allen ◽  
H. F. Dias
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Gel Electrophoresis ◽  
Average Molecular Weight ◽  
Virus Genome ◽  
Protein Subunit ◽  
Spot Virus ◽  
Single Protein ◽  
Middle Component ◽  
Ring Spot

Purified preparations of several isolates of tomato ring-spot virus were shown by rate-zonal centrifugation in sucrose and equilibrium centrifugation in CsCl to be composed of two individual nucleoprotein components. Acrylamide-gel electrophoresis showed that the lighter (middle) component contained a nucleic acid (RNA 2) that was distinct from the species (RNA 1) contained in the heavier (bottom) component. The bottom was more infectious than the middle component and infectivity was enhanced by mixing the components, indicating that the virus genome is divided between component types. Similar results were obtained from infectivity tests on the two nucleic acids. The nucleic acid contents of the middle and bottom components were about 40 and 41%, respectively. The average molecular weights of RNA 2 and RNA 1 from three virus isolates, as determined by acrylamide-gel electrophoresis, were 2.5 and 2.6 × 106, respectively. Molecular complexing between the RNA species during electrophoresis was prevented with the use of formamide. The single protein subunit from the same three isolates had an average molecular weight of about 58 000. Serological comparisons of five tomato ring-spot isolates associated with diseases of fruit trees and grapevines indicated that only the grape yellow vein strain was antigenically distinct. These and other properties indicate that this virus is similar to other members of the nepovirus group.

scholarly journals Scanning single-molecule counting system for Eprobe with highly simple and effective approach

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243319
Author(s):  
Takeshi Hanami ◽  
Tetsuya Tanabe ◽  
Takuya Hanashi ◽  
Mitsushiro Yamaguchi ◽  
Hidetaka Nakata ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Detection System ◽  
Signal To Noise Ratio ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Digital Measurement ◽  
Excitation Light ◽  
Target Nucleic Acid ◽  
Fluorescent Molecules

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

scholarly journals A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons

Biosensors ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 479
Author(s):  
Soumi Sukla ◽  
Prasenjit Mondal ◽  
Subhajit Biswas ◽  
Surajit Ghosh
Keyword(s):  
Nucleic Acid ◽  
Dengue Virus ◽  
Magnetic Beads ◽  
Molecular Beacon ◽  
Detection System ◽  
Denv Infection ◽  
Molecular Beacons ◽  
Nucleic Acid Amplification ◽  
Complementary Sequence ◽  
Reverse Transcription Pcr

Detecting dengue virus (DENV) infection in patients as early as possible makes the disease management convenient. Conventionally, DENV infection is diagnosed by ELISA-based methods, but sensitivity and specificity are major concerns. Reverse-transcription-PCR (RT-PCR)-based detection confirms the presence of DENV RNA; however, it is expensive, time-consuming, and skilled personnel are required. A fluorescence-based detection system that detects DENV RNA in patient’s serum directly, without any nucleic acid amplification step, has been developed. The method uses target-specific complementary sequence in the molecular beacon, which would specifically bind to the DENV RNA. The molecular beacons are approximately 40 bases long hairpin structures, with a fluorophore-quencher system attached at the terminal ends of the stem. These probes are biotinylated in the stem region, so that they can be immobilized on the streptavidin-tagged magnetic beads. These magnetic beads, coupled with biotinylated molecular beacons, are used for the detection of the target RNA in the serum by incubating the mixture. After incubation, beads are separated and re-suspended in a buffer. The measurement of fluorescence is taken in fluorometer after 15 min incubation at 50 °C. The whole work is carried out in a single tube. This rapid method can precisely detect dengue RNA within two hours, confirming ongoing DENV replication in the patient.

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