The first portable thermal cycler for Real-time PCR (qPCR), known as Q3-plus (Q3), is a patented device developed by STMicroelectronics, a world leader in semiconductor technology, where I am carrying out my thesis internship. The point of strength of the system (consisting of the instrument itself, an intuitive software and a Lab-on-a-Chip, LOC, disposable) is the possibility of using it for diagnostic purposes even in environments without laboratory equipment and qualified personnel, which make it ideal for Point-of-Care contexts. These features can be extremely useful in addressing an emergency, such as the COVID-19 pandemic, that the world has had to face in the last year: my group has therefore worked to develop assays for the detection of the SARS-CoV- 2 that can be user-friendly, fast and effective. First, the goal was the development of a rapid assay, based on the Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) for virus detection. The test is based on a LOC in which all the reagents needed to carry out the reaction are lyophilized: using this assay and the portable thermal cycler, the final user is able to quickly test patients suspected of being positive for the virus with only the sample of the patient himself needed, to be used to rehydrate the LOC, without the need for advanced laboratory equipment. Unlike other diagnostic kits already on the market, based on DNA controls, this assay is based on RNA positive controls, which allow to evaluate the functioning of reverse transcriptase, an enzyme involved in pRT-PCR, during the course of the reaction itself. The work I carried out primarily involved the choice of an in vitro transcription kit suitable for transcribing the target sequences of the genome (indicated by the CDC), the optimization of the reaction mix for qRT-PCR and the lyophilization and evaluation of the conservation of the obtained lyophilized LOCs. Moreover, as part of this project, we have explored further potential of Q3: although, as mentioned before, the main application of the tool is the execution of analyses based on qPCR, we have considered the possibility of going beyond this use by combining the advantages of Q3 with those of the revolutionary CRISPR-Cas technology. Working with a partner, again in the context of the pandemic emergency, we worked on the development of an alternative COVID-19 detection method, based not on the golden standard of qPCR, but on CRISPR. My contribution in this context was from a biological point of view, so I worked to make the chemical protocol provided by our partner suitable for the Q3 device. In addition, optical implementations of the instrument have been developed with the aim of increasing its detection capability. Using this as a starting point, however, we are already working to go beyond this, not only by implementing Q3, but by creating an ad hoc LOC that allows to have, in a portable instrument, an integrated sample-to-answer flow of detection of the virus: starting from a swab and extracting the RNA of the virus, in less than an hour the result of the test will be available, without the need for a specialized operator or an equipped structure. This project is part of the next generation application objectives of ST's instrument, which will go beyond its mere use for the detection of COVID-19: in fact, by exploiting the versatility of CRISPR technology, capable of detecting any nucleic acid sequence by simply modifying the specificity of the enzyme, other viral sequences can be detected quickly, guaranteeing an early diagnosis even for patients who live in disadvantaged contexts also lacking specialized structures.
Il primo termociclatore portatile per Real-time PCR (qPCR), noto come Q3-plus (Q3), è un dispositivo brevettato sviluppato da STMicroelectronics, leader mondiale nella tecnologia dei semiconduttori, dove sto svolgendo il mio internato di tesi. Il punto di forza del sistema (costituito dallo strumento in sé, da un software intuitivo e da un Lab-on-a-Chip, LOC, monouso) è la possibilità di utilizzarlo a scopo diagnostico anche in ambienti privi di attrezzature di laboratorio e personale qualificato, che lo rendono ideale per contesti Point-of-Care. Queste caratteristiche possono essere utili per affrontare un'emergenza, come la pandemia di COVID-19, che il mondo ha dovuto fronteggiare nell'ultimo anno: il mio gruppo ha dunque lavorato per sviluppare saggi per la rilevazione del genoma del SARS-CoV-2 che possano essere user-friendly, rapidi ed efficaci. Per prima cosa, l’impegno è stato rivolto allo sviluppo di un assay rapido, basato sulla Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) per il rilevamento del virus. Il test è basato su un LOC in cui tutti i reagenti per svolgere la reazione sono liofilizzati: sfruttando questo saggio e il termociclatore portatile, l’utente finale è in grado di testare rapidamente pazienti sospettati di essere positivi al virus avendo a disposizione solamente il campione del paziente stesso, da usare per reidratare il LOC, senza la necessità di un’avanzata strumentazione di laboratorio. A differenza di altri kits diagnostici già presenti sul mercato, basati su controlli a DNA, questo saggio è basato su controlli positivi a RNA, che permettono di valutare il funzionamento della trascrittasi inversa, enzima coinvolto nella pRT-PCR, durante lo svolgimento della reazione stessa. Il lavoro da me svolto ha previsto la scelta di un kit di trascrizione in vitro adatto a trascrivere le sequenze target del genoma, l’ottimizzazione della mix di reazione per la qRT-PCR e del protocollo di liofilizzazione e la valutazione della conservazione dei LOC liofilizzati ottenuti. Inoltre, nell’ambito di questo progetto, abbiamo esplorato ulteriori potenzialità del Q3: abbiamo considerato la possibilità di combinare i vantaggi del Q3 con quelli della rivoluzionaria tecnologia CRISPR-Cas. Collaborando con un partner, sempre nel contesto dell’emergenza pandemica, abbiamo lavorato allo sviluppo di un metodo di rilevamento del COVID-19 alternativo, basato non sul golden standard della qPCR, ma sulla CRISPR. Il mio contributo in questo contesto è stato dal punto di vista biologico, per cui ho lavorato per rendere il protocollo chimico fornito dal nostro partner adatto al dispositivo Q3. Inoltre, sono state messe a punto implementazioni ottiche dello strumento con lo scopo di aumentare la sua capacità di rilevamento. Utilizzando questo come punto di partenza, si vuole anmdare oltre, non solo implementando il Q3, ma creando un LOC ad hoc che permetta di avere, in uno strumento portatile, un interno flusso sample-to-answer di rilevamento del virus: partendo da uno swab ed estraendo l’RNA del virus, in meno di un’ora si arriverà direttamente al risultato, senza la necessità né di un operatore specializzato, né di una struttura attrezzata. Questo progetto fa parte degli obiettivi di applicazione di prossima generazione dello strumento di ST, che potranno andare oltre il suo mero utilizzo per il rilevamento di COVID-19: infatti, sfruttando la versatilità della tecnologia CRISPR, in grado di rilevare qualunque sequenza di acido nucleico andando a modificare in modo semplice la specificità dell’enzima, altre sequenze virali potranno essere rilevate in modo rapido, garantendo una diagnosi precoce anche a pazienti che si trovano a vivere in contesti svantaggiati e in cui strutture specializzate sono assenti.
Development of assays for the detection of SARS-CoV-2 based on CRISPR-Cas and Real-Time PCR technologies in a compact device.
MESSORI, ANITA
2020/2021
Abstract
The first portable thermal cycler for Real-time PCR (qPCR), known as Q3-plus (Q3), is a patented device developed by STMicroelectronics, a world leader in semiconductor technology, where I am carrying out my thesis internship. The point of strength of the system (consisting of the instrument itself, an intuitive software and a Lab-on-a-Chip, LOC, disposable) is the possibility of using it for diagnostic purposes even in environments without laboratory equipment and qualified personnel, which make it ideal for Point-of-Care contexts. These features can be extremely useful in addressing an emergency, such as the COVID-19 pandemic, that the world has had to face in the last year: my group has therefore worked to develop assays for the detection of the SARS-CoV- 2 that can be user-friendly, fast and effective. First, the goal was the development of a rapid assay, based on the Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) for virus detection. The test is based on a LOC in which all the reagents needed to carry out the reaction are lyophilized: using this assay and the portable thermal cycler, the final user is able to quickly test patients suspected of being positive for the virus with only the sample of the patient himself needed, to be used to rehydrate the LOC, without the need for advanced laboratory equipment. Unlike other diagnostic kits already on the market, based on DNA controls, this assay is based on RNA positive controls, which allow to evaluate the functioning of reverse transcriptase, an enzyme involved in pRT-PCR, during the course of the reaction itself. The work I carried out primarily involved the choice of an in vitro transcription kit suitable for transcribing the target sequences of the genome (indicated by the CDC), the optimization of the reaction mix for qRT-PCR and the lyophilization and evaluation of the conservation of the obtained lyophilized LOCs. Moreover, as part of this project, we have explored further potential of Q3: although, as mentioned before, the main application of the tool is the execution of analyses based on qPCR, we have considered the possibility of going beyond this use by combining the advantages of Q3 with those of the revolutionary CRISPR-Cas technology. Working with a partner, again in the context of the pandemic emergency, we worked on the development of an alternative COVID-19 detection method, based not on the golden standard of qPCR, but on CRISPR. My contribution in this context was from a biological point of view, so I worked to make the chemical protocol provided by our partner suitable for the Q3 device. In addition, optical implementations of the instrument have been developed with the aim of increasing its detection capability. Using this as a starting point, however, we are already working to go beyond this, not only by implementing Q3, but by creating an ad hoc LOC that allows to have, in a portable instrument, an integrated sample-to-answer flow of detection of the virus: starting from a swab and extracting the RNA of the virus, in less than an hour the result of the test will be available, without the need for a specialized operator or an equipped structure. This project is part of the next generation application objectives of ST's instrument, which will go beyond its mere use for the detection of COVID-19: in fact, by exploiting the versatility of CRISPR technology, capable of detecting any nucleic acid sequence by simply modifying the specificity of the enzyme, other viral sequences can be detected quickly, guaranteeing an early diagnosis even for patients who live in disadvantaged contexts also lacking specialized structures.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/14074