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Identifier

000434216

Title

Investigation of real-time colorimetric LAMP method for the rapid detection of Influenza A and SARS-CoV-2

Alternative Title

Μελέτη της χρωματομετρικής μεθόδου LAMP σε πραγματικό χρόνο για την ταχεία ανίχνευση των ιών της γρίπης και του SARS-CoV-2

Author

Χατζηιωαννίδου, Στυλιανή Κ.

Thesis advisor

Γκιζελή, Ηλέκτρα
Παπαδάκης, Γιώργος

Abstract

The demand for efficient diagnostic test for detection of pathogens has been increasing lately. This need has led to the development of effective diagnostic tests, with the usage of nucleic acid molecular detection methods. Most of the methods that are used, require expensive and bench-top laboratory equipment. The ultimate goal of diagnostic tests is to be applied at point of care systems. Creating rapid, affordable and accurate diagnostic test is very important for the point of care. Loop-mediated isothermal amplification method (LAMP) can contribute to the development of rapid test for the detection of pathogens. The objective of this study was the enhancement, the reduction of the detection time of the real-time quantitative colorimetric LAMP method and the application of it in clinical samples of Influenza A and SARS-CoV-2. To achieve the improvement of the method, two different parameters were evaluated: the colorimetric dye and the polymerase enzyme. Also, a portable new device (IRIS) was used for the development of real-time quantitative colorimetric LAMP. The colorimetric dye that was used for the detection of LAMP products was the first element that was evaluated. Three different dyes were used for the detection, Hydroxyl-Naphthol-Blue (HNB), phenol red and crystal violet. Each dye change color in a different way when the DNA amplification takes place. More specifically, the color change of HNB dye depends on the concentration of the Mg2+ in the solution, phenol red dye changes color with the change of pH and of the crystal violet changes color when it binds to DNA products. Comparing the sensitivity in color changes of each dye, it was confirmed that they didn’t have any difference in their performance as far as it concerns the sensitivity in color change. All the dyes could change color just as rapidly when there was the production of amplicons. The second parameter that was evaluated was the performance of the polymerase enzyme. Six different polymerase enzymes were used for the experiments, Bst 2.0, Bst 2.0 with master mix, Bst 3.0 from NEB, Bst 2.0 from Jena Bioscience, Bst from SBS Genetech Co and Bsm from Thermo Scientific. The best time-to-positive results in LAMP reaction was with Bst polymerase from SBS Genetech Co. This polymerase was able to reduce the time-to-positive results of the experiment at approximately 14 min, defining it the fastest amplification of all the other polymerases. All the experiments were performed with the addition of Influenza A and SARS-CoV-2 as template. The template of Influenza A was DNA and of SARS-CoV-2 was RNA. The method was able to define the limit of detection of both targets. Real-time quantitative colorimetric LAMP was able to detect up to 1-10 copies per μL in the reaction. Moreover, during validation studies, the results showed 97.4% and 100% agreement with qRT-PCR for SARS-CoV-2 RNA detection extracted from positive and negative patients’ samples (89), respectively. Furthermore, the method has the ability to operate with crude clinical samples like saliva. Finally, accomplishing the reduction of the detection time in real-time quantitative colorimetric LAMP, can provide a rapid and accurate solution for point-of care diagnostic methods.

Language

English

Subject

Real time detection

Ανίχνευση νουκλεϊκών οξέων

Ισοθερμική αντίδραση

Ιός SARS-CoV-2

Ιός γρίπης

Χρωματομετρική μέθοδος

Issue date

2020-11-27

Collection