WO2021220192A4

WO2021220192A4 - Method and portable device for detection of nucleic sequences in suspected coronavirus samples

Info

Publication number: WO2021220192A4
Application number: PCT/IB2021/053530
Authority: WO
Inventors: Alcino Orfeu DE LEÃO E FLORES; Gonçalo Maria REIMÃO PINTO DE FRANÇA DORIA
Original assignee: Stab Vida - Investigação E Serviços Em Ciências Biológicas, Lda
Priority date: 2020-04-30
Filing date: 2021-04-28
Publication date: 2022-02-03
Also published as: EP4142945A2; WO2021220192A2; WO2021220192A3; CA3175648A1; AU2021265579A1; JP2023524117A; CN115836133A; IL297281A; BR112022021086A2

Abstract

Present invention refers to method and portable device(1) for detection and identification of specific sequences of nucleic acids in different samples, by utilisation of reverse transcription technique and/or isothermal amplification, using specific oligonucleotide primers of target region(s) to detect, whereby the amplification of the same is conducted in indirect and non- specific form, or direct and specific, using discriminatory amplification reagents, which signal, colourimetric/fluorescent, is recorded by device (1) where reaction is carried out. Device is controlled by a mobile application(9), also recording data acquired by device (1) analyses, stores it in remote server in cloud (10), which analyses global set of all data allowing global analysis of for example, evolution of epidemics. Invention is useful for detection and identification of relevant nucleic sequences for different types of diagnostic in different clinical, pharmaceutical, veterinary, food, environmental, biotechnological, biosafety areas, particularly useful in quick, low-cost diagnostics, and at point of care, of SARS-CoV-2 virus.

Claims

48 AMENDED CLAIMS received by the International Bureau on 20 December 2021 (20.12.2021)

1. An integrated system for the detection and identification of specific nucleic acid sequences characterized in that the specific nucleic acids sequences belong to the virus of the Coronaviridae family, particularly the species of the severe acute respiratory syndrome, SARS-Cov-2, in suspected coronavirus samples, said integrated system comprising: i) a portable device (1) comprising a reaction container (2), a battery (4), a temperature sensor (6), wherein in the portable device (1) further comprises: a) one or more spectrophotometric sensors (7) configured to differentially quantify the emission of photons in different wavelengths between 315 to 1400 nm, preferably with a resolution less than or equal to 40nm; b) one or more illumination sources (3) coupled to light filters; c) one heat source (8) that is constituted by one or more resistors, Peltier module, or any other heat source which reaches at least 100°C and at minimum 4°C above room temperature; d) one microcontroller (5) with integrated Wi-Fi and/or Bluetooth containing a firmware; e) said reaction container (2) contains a suspected coronavirus sample and reverse transcription and/or isothermal amplification reagents for real-time discrimination of the amplification comprising one or more of the SEQ ID No. 12 to 109;

(ii) a mobile application (9) for carry out the recorded and generated signals of the portable device (1) transmitted via Bluetooth and/or Wi-Fi; (iii) a remote server in a cloud (10) for storing and processing the signals recorded and transmitted by the portable device (1).

2. The integrated system according to claim 1 characterised in that the mobile application (9) of the portable device (1) is compatible with Android or iOS, or any other operating system of a mobile device.

3. The integrated system according to the preceding claims characterised in that the samples come from the higher or lower respiratory tract, nasopharyngeal aspirate, or nasal wash, from faeces, saliva, samples of human or animal urine, environmental samples, food samples.

4. The integrated system according to the preceding claims, characterised in that the suspected coronavirus sample is RNA or DNA extracted and purified from samples presumably containing target nucleic acids, such as oral swabs, nasopharyngeal or nasal, blood, urine, saliva, sputum, faeces, environmental samples or food, or from contact surface, among others, or with the direct addition without extraction and purification of RNA or DNA from these same samples.

5. The integrated system according to the preceding claims, characterised in that the one or more spectrophotometric sensors (7) are of the photovoltaic, photodiode, photoresist or phototransistor type, and may be coupled to light filters, for example, a band-pass filter.

6. The integrated system according to the preceding claims, characterised in that the one or more sources of illumination (3) are incandescent, light-emitting diode or laser.

7. The integrated system according to the preceding claims, characterised in that the temperature sensor (6) is of the thermistor, thermoresistance, thermocouple type, or based on a semiconductor.

8. A method of using the integrated system defined in the claims 1-7 based on the reaction from reverse transcription and/or isothermal amplification of one or more genomic regions, with real-time discrimination of the amplified fragments in suspected coronavirus samples, characterised in that it comprises the following steps: a) Collection and processing of a sample to be analysed; b) Addition of collected sample to the reaction container (2) containing the reaction mixture with the reagents for the reverse transcription and/or isothermal amplification reaction comprising one or more of the SEQ ID No. 12 to 109; c) Addition of reaction container (2) containing the sample to be analysed to the portable device (1), followed by thermostatic heating of the container (2) and subsequent reverse transcription and/or isothermal amplification reaction with recording of the discriminating signals of isothermal amplification in the portable device (1); d) Transmission of the referred recorded signals by one or more spectrophotometric sensors (7) of the portable device (1) to the remote server in a cloud (10) by means of Bluetooth or Wi-Fi and/or to the mobile application (9; e) Analysis of signals recorded by the portable device (1) in the mobile application (9) for determination of the final results, positive or negative, wherein positive indicates the presence of the nucleic acid sequences that are the target in the sample f) Analysis of the result of step e) wherein when the final result is positive for a variation in the fluorescence intensity in the wavelengths of the emission of fluorophore higher than 10% of the initial fluorescence signal and occurs in less than 1600 seconds; g) Recording of the final result maintained in the remote server in a cloud (10) said final result is processed by the mobile application (9) and transmitted to the referred server in the cloud (10) or the signals recorded and generated by the portable device (1) are processed by the mobile application (9) and transmitted to a remote server in the cloud (10).

9. The method according to claim 8, characterised in that the samples come from the higher or lower respiratory tract, nasopharyngeal aspirate, or nasal wash, from faeces, saliva, samples of human or animal urine, environmental samples, food samples.

10. The method according to claims 8-9, characterised in that the reverse transcription and/or isothermal amplification reaction is carried out with the addition of a RNA or DNA sample extracted and purified from samples presumably containing target nucleic acids, such as oral swabs, nasopharynx or nasal, blood, urine, saliva, sputum, faeces, environmental or food samples, or from contact surfaces, among others, or with the direct addition without extraction and purification of the RNA or DNA from these same samples, to the reaction amplification mixture. 52

11. The method according to claims 8-10, characterised in that the reverse transcription reaction is carried out using a polymerase DNA enzyme with reverse transcriptase activity, such as HIV-1, M-MLV, AMV, among others.

12. The method according to claims 8-11, characterised in that the reaction mixture is a combination of oligonucleotide primers, dNTPs, thermostable polymerase DNA enzyme with strand displacement activity and/or polymerase DNA enzyme with reverse transcriptase activity, buffer for the activity of the polymerase DNA enzymes and reagent for real-time discrimination of the amplified fragments.

13. The method according to claims 8-12, characterised in that the polymerase DNA enzyme with reverse transcriptase activity is reversibly coupled to an aptamer which inhibits the activities thereof at temperatures lower than 40°C.

14. The method according to claims 8-13, characterised in that the oligonucleotide primer sequences comprise one or more of SEQ. ID No. 12 to 95.

15. The method according to claims 8-14, characterised in that the reverse transcription reaction and/or isothermal amplification is carried out at a temperature of 37-70°C, for at least 10 minutes up to 120 minutes.

16. The method according to claims 8-15, characterised in that the reverse transcription reaction and/or isothermal amplification is carried out at a preferred temperature of 65°C for 30 minutes. 53

17. The method according to claims 8-16, characterised in that the real-time discrimination of the amplified fragments is carried out in indirect or direct manner.

18. The method according to claim 17, characterised in that the real-time indirect discrimination of the amplified fragments of DNA is carried out using a colourimetric pH indicator reagent, such as, phenol red, among others, or an intercalating DNA reagent, such as SYBR™ green.

19. The method according to claim 17, characterised in that the real-time direct discrimination of the amplified fragments of DNA is carried out using one or more specific oligonucleotide probes conjugated with a fluorophore, and/or a quencher, or a combination of both.

20. The method according to claim 19, characterised in that the fluorophore is FAM, HEX, ROX or Cy5, or similar, and the quencher being DABCYL, BHQ-1, BHQ-2, BHQ-3, or similar.

21. The method according to claim 19, characterised in that the oligonucleotide probes belong to SEQ. ID No. 96 to 109.

22. The method according to claims 8-21, characterised in that the mobile application (9) for control of the portable device (1) is compatible with Android or iOS, or any other operating system of a mobile device.