WO2021210031A1

WO2021210031A1 - Method for detection of viruses and antibodies in biological samples

Info

Publication number: WO2021210031A1
Application number: PCT/IT2020/000034
Authority: WO
Inventors: Paolo Galiano; Milena Sinigaglia; Giovanni Di Cola
Original assignee: Alifax S.R.L.
Priority date: 2020-04-15
Filing date: 2020-04-15
Publication date: 2021-10-21

Abstract

Dual test which includes disposable cartridges, of the type developed with MEMS (Micro Electro-Mechanical Systems) technology, suitable for containing reagents and acquiring fluorescent signals, including at least one specific reaction mixture suitable for performing molecular biology tests and/or one specific reaction mixture for immunological analysis, which said tests are carried out through the use of a single instrument starting from biological samples isolated from virus infected or potentially infected patients.

Description

METHOD FOR DETECTION OF VIRUSES AND ANTIBODIES IN BIOLOGICAL SAMPLES

FIELD OF THE INVENTION The present invention relates to a test method for both the detection of viruses (pathogens) and the obtainment of an immunological response in biological samples from a subject positive or suspected to be positive for virus infections, wherein the test method is carried out using the same instrument.

The method uses a fluorescence detection system allowing to verify the presence of the viruses (by detecting their nucleic acids) and/or the presence of IgM and IgG antibodies directed against virus antigen in order to provide information on the immunological response status of the subject.

The invention allows the detection of viruses starting from an oral/nasopharyngeal swab and/or any biological sample which represents the elective site of contact with the viruses.

The invention also allows the detection of immunoglobulins of the IgG or IgM class from a drop of blood from the same subject.

The innovation of the invention consists in the possibility of using the same instrument to carry out both tests. This instrument is, at the same time, a thermal cycler and a fluorescence detector for certain excitation and emission wave frequencies.

STATE OF THE ART

Viruses are very small infectious agents, made up of a piece of genetic material, such as DNA or RNA, that’s enclosed in a coat of molecules (e.g. proteins). They are able to invade cells of the body and use components of those cells to help them multiplying, damaging or destroying infected cells.

In the last decades, humans have been facing unknown viruses for which the only available treatments are those capable only to relieve symptoms while the immune system fights off the virus. Antibiotics do not work for viral infections and the antiviral drugs are to be design according to the peculiar viral pathophysiology. Vaccines can help prevent infections, however the time to have a vaccine for new virus goes, depending on the type of vaccine, form 10 to 22 months, which is too long to prevent mortality of many people, especially those without access to medical care.

Recently, several viruses have jumped from animals to humans and triggered sizable outbreaks, claiming thousands of lives. One example is the viral strain that drove the 2014-2016 Ebola outbreak in West Africa which killed up to 90% of the people it infects, making it the most lethal member of the Ebola family.Nowadays, a new pandemy is currently driving outbreaks around the globe, posing a serious threat to public health as there aren’t still the effective drugs to eradicate it. SARS-CoV-2 is a new coronavims which causes a disease named “Coronavims Disease 2019” (COVID-19). It is correlated to mild flu-like symptoms, but it may result in severe acute respiratory syndrome, also leading to death in 3-4 % of cases [Situation Report - 46”, published by World Health Organization - WHO]

As the symptoms are unspecific and common, and there are many asymptomatic subjects, the spread of the infection is dramatically high and rapid. The vims is currently spreading worldwide representing therefore a global health emergency.

Unfortunately, there are currently no specific treatments and vaccines and the only existing cure is a symptomatic therapy. This is strongly impacting on the national health system, especially the intensive care unit, also affecting other critical admissions and emergencies. In this context, there is an increasing need for a Point of Care (PoC) device to achieve a prompt diagnosis to contain the infection spreading.

Currently, the main issues in the worldwide struggle to contain the novel coronavims (SARS-CoV-2) outbreak are represented by healthcare infrastructure and testing capacity. Indeed, among the immediate research needs given by the WHO there is the rapid Point of Care (PoC) diagnostics for use at the community level for the rapid identification on site of diseases and for containing the outbreaks.

Therefore, several assays that detect the SARS-CoV-2 have been and are currently under development, both in-house and commercially. Some of them may detect only the novel vims and some may detect also other strains (e.g. SARS-CoV) that are genetically similar. The worldwide research community efforts seem to mark progress on two fronts: immunoassays (manual ELISA, machine-based or lateral flow, rapid tests specific for SARS-CoV-2 antigen or antibodies) and molecular tests for developing in vitro diagnostics (IVDs) that detect SARS-CoV-2 nucleic acid as a matter of fact, Real Time PCR ( qPCR) is a sensitive and accurate method with the interesting possibility of multiplex targeting, greatly decreasing the cost per sample.

However, qPCR technique might be laborious and time-consuming, requiring skilled personnel and laboratory settings with expensive instruments. Therefore, the initial setup cost is significantly high for developing/underdeveloped countries.

Another disadvantage of qPCR when compared with rapid diagnostic tests is the requirement for a controlled, stable temperature during transport and storage of reagents, which steeply increases the costs and in extreme cases precludes the adoption of qPCR as a diagnosis tool.

In the past years, several technologies have emerged trying to solve this issue.

Among them, gelled and freeze-dried assays are particularly useful to streamline the use of molecular tests at the laboratory bench as well as introduce into a production line.

Nowadays, point-of-care (POC) diagnostic tests are being developed to facilitate diagnostics. For these novel platforms, miniaturization and multiplexing provide great potential to improve patient treatment through fast and accurate detection of cases of infections, either at physicians’ offices or at field clinics. Because the main goals are similar, it is not unexpected that qPCR and POC technologies are being merged to tackle world public health issues.

The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.

SUMMARY OF THE INVENTION

The present invention is set forth and characterized in the independent claims, while the dependent claims describe other characteristics of the invention or variants to the main inventive idea.

The present invention provides for a method for the detection of viruses and antibodies against viruses in a biological fluid sample. The method comprises a step of contacting the biological fluid sample with a first reaction mixture suitable to detect viral nucleic acid and/or with a second reaction mixture suitable to detect antibodies directed against viral antigen. The first reaction mixture and the second reaction mixture contain fluorescent probes configured to emit a detectable signal as a consequence of the presence of viral nucleic acid or antibodies respectively.

According to an aspect of the invention, the method further comprises a step to detect a detectable signal, wherein said virus nucleic acid or antibodies fluorescent signal detection is performed using a Real Time PCR ( qPCR) instrument.

The method thus provides an innovative solution that allows qPCR instruments to be suitable for the detection of immunoglobulin or antibodies.

Therefore, taking advantage of the flexibility and diffusion of qPCR instruments the invention offers to clinical laboratories a new approach to evaluate the immunological response status of a subject in fast and easy way.

In particular, the state of immune coverage is detected by the presence of IgM class antibodies, occurring during a first contact with the viral antigens (so called primary immunization) and by the presence of IgG class antibodies to confirm its protection status. In fact, IgG are firstly slightly produced during primary immunization while during a second infection with the same antigen their production occurs in larger amounts.

The presence of antibodies is detected in whole blood without the need of any treatment. Also crude serum and plasma may be used as starting sample.

According to another aspect of the invention, the fluorescent signal detection is performed by displacing the first reaction mixture and the second reaction mixture within wells of a suitable disposable cartridge. The cartridge may be of MEMS (Micro Electro-Mechanical Systems) technology.

According to a further aspect of the invention, the first reaction mixture and the second reaction mixture comprise a mixture of substances of which at least one substance is provided within the cartridge in lyophilized or gelled or dried form.

The invention also refers to a cartridge for use in the method above.

DETAILED DESCRIPTION OF THE INVENTION

We will now refer in detail to embodiments of the present invention. The description and phraseology or terminology shall not be understood as a limitation of the embodiment or of the invention per se.

Description of nucleic acids detection approach

The nucleic acids detection approach is based on One-Step Reverse transcriptase Real-Time polymerase chain reaction’ (One-Step RT-qPCR) to detect and/or identify RNA viruses and/or Real time PCR (qPCR) to detect and/or identify DNA viruses, starting from genetic material or nucleic acids extracted from positive biological samples or suspected to be positive for the presence of viruses.

The method involves the loading of RNA and/or DNA, or cDNA, into wells of a disposable cartridge containing the first reaction mixtures in freeze-dried or gelled or dried (or similar) form to test simultaneously several samples per cartridge depending on the number of wells in the cartridge.

For each sample, in addition to specific reagents for the identification of the viral genome, positive, negative and intemal/endogenous controls are provided, the purpose of which is to confirm the functioning of the test and the presence and quality of the starting sample. The method entails the insertion of the cartridge into an instrument to perform One-Step RT-qPCR and/or qPCR. The method allows to have an evidence about the presence/absence of the nucleic acid belonging to one or more RNA or DNA viral genome in about an hour from the start of the test.

The method provides, in a particular embodiment, to supply an automated extractor for the fast and effective extraction of RNA and/or DNA from fluids or biological samples such as blood, serum, plasma, urine, oral/nasopharyngeal swab, liquor, BAL, etc. or any sample suspected to be positive for the presence of viruses.

The strength of the developed method is the application of One-Step RT-qPCR (for RNA vims analysis) and qPCR (for DNA vims analysis) techniques in a ready to use cartridge with prefilled freeze-dried or gelled or dried (or similar) reagents. In a classic method, RNA requires its retro-transcription in cDNA, (complementary or copy DNA) (1st step) which can then be amplified in a Real Time PCR reaction (2nd step).

The invention refers also to a cartridge containing all the necessary reagents not only for the amplification reaction, but also for the retro-transcription of the RNA to cDNA. Furthermore, thanks to the presence of lyophilized/gelled/dried (or other similar methods) reagents in the cartridge, the healthcare professional does not need to prepare the reaction mix (enzyme for retro-transcription, enzyme for amplification, primers for retro-transcription, specific primers and probes, dNTPs, salts, buffers, etc.) and to load it into each well. Similarly, for the analysis of DNA vimses, since inside the cartridge wells are already present freeze-dried/gelled/dried (or similar) reagents for amplification, the user avoids reaction mix manual preparation and its dispensing in wells. The preparation and loading of the RT-qPCR and/or qPCR reaction mixture require some hands-on time and skills, thus inducing potential errors, which could introduce contamination with other samples or with substances that could inhibit amplification or degrade nucleic acid (RNA or DNA). Instead, the presence of dedicated reagents in a ready-to-use cartridge, implies that the execution of the molecular test will be greatly simplified and effective. Speed up of the diagnosis test execution, in turn, entails the possibility of prompt and tailored therapy for the patient, which is essential to reduce mortality.

The invention provides different panels for the detection of different types of RNA and/or DNA viruses, divided based on symptomatology they cause in infected patients. An example of a panel is assigned to identify Zika, Dengue, Chikungunya and/or other viruses that cause hemorrhagic fevers. Another example of a panel is suitable for the identification and discrimination of RNA viruses that cause acute respiratory syndromes such as SARS-CoV, SARS-CoV- 2, MERS-CoV, etc. Each panel comprises cartridges with a minimum of 6 to a maximum of 36 sets of primers and probes, which can be used with the One-Step RT-qPCR and/or qPCR method; each set produces an amplicon of a size between 50 and 500 bp and specifically detects and/or identifies RNA and/or DNA viruses responsible for diseases.

Each set is composed by:

- from one to eight "forward" primers, that is an oligonucleotide of length between 15 and 40 nucleotides, complementary to a portion of the cDNA or DNA strand.

- from one to eight "reverse" primers, that is an oligonucleotide of length between 15 and 40 nucleotides, complementary to a portion of cDNA or DNA on the opposite strand with respect to the strand on which the forward primer pairs. - from one to eight probes, that is an oligonucleotide of length between 15 and 45 nucleotides, complementary to a portion of cDNA or DNA of the same strand on which the forward primer (s) pairs or complementary to the opposite strand with respect to the strand on which the forward primer (s) pairs, at whose 5’ end a fluorophore is conjugated, and at the 3 'end a quencher is conjugated such as, for example, BHQ1 and BHQ2 or similar.

The fluorophore can be chosen from a group comprising Rodamine-X (ROXTM) Hexachlorofluorescein (HEcTM^ NED^M, Fluorescein (FAM^"™^ SYBR™ Green, VIC™, TAMRA, TET, JOE, Texas Red, Cy3, Cy5, Cy5.5 or their analogues. For each One-Step RT-qPCR or Real Time PCR reaction, within each single well, the sets can be combined to have a multiplex reaction, that means the generation of multiple amplicons at the same time. The amplification of each portion of cDNA or DNA is discriminable because the set of primers and relative probe is associated with a different fluorophore. The method provides that in each cartridge, for each RNA and/or DNA isolated from a sample, there are negative, positive and internal and/or endogenous controls comprising the following:

- at least one negative control. The absence of amplification by this set is an indication of the correct functioning of the test. - at least one positive control. The amplification starting from this set indicates the correct functioning of the cartridge and the One-Step RT- qPCR or Real Time PCR reaction and the presence/absence of possible reaction inhibitors. - at least an internal and/or an endogenous control. The presence of amplification by this set guarantees the presence and quality of the nucleic acids (RNA/DNA) within the sample and the absence of amplification inhibitors.

Each example is provided as an explanation of the invention and is not intended as a limitation of the same. For example, one or more illustrated or described features, since they are part of an embodiment, may be varied or adopted on, or in association with, other embodiments to produce further embodiments. It is understood that the present invention will be inclusive of such possible modifications and variations.

Before describing the embodiments, it is further clarified that the present description is not limited in its application to the constructional details as described in the following description using the attached examples. The present description can foresee other embodiments and can be realized or put into practice in various other ways.

Below are non-exhaustive examples of targets that make up panels for RNA and/or DNA viruses.

RNA viruses

“Tropical viruses” Panel

Chikungunya Zika Dengue West Nile Usutu

Tick-borne encephalitis virus Yellow fever Positive control Negative control

Internal and/or endogenous control “Hepatitis B virus” Panel Hepatitis B virus Positive control Negative control Internal and/or endogenous control “Hepatitis C virus” Panel Hepatitis C vims Positive control Negative control

Internal and/or endogenous control “MMnCoV” Panel SARS-CoV-2

Sarbecovimses (SARS-CoV-2, human SARS-CoV) MERS-CoV

Coronavims HKU 1 Coronavims NL63 Coronavims 229E Coronavims OC43 Positive control Negative control

Internal and/or endogenous control “Influenzae Panel”

Flu A Flu B RSV A RSV B Rhinovims

Parainfluenzae vims 1 Parainfluenzae vims 2 Parainfluenzae vims 3 Parainfluenzae vims 4 Enterovims

Human metapneumovims Positive control Negative control

Internal and/or endogenous control “Respiratory diseases (RNA viruses)” SARS-CoV-2

Sarbecovimses (SARS-CoV-2, human SARS-CoV)

MERS-CoV Coronavims HKU 1 Coronavims NL63 Coronavims 229E Coronavims OC43 Flu A Flu B RSV A RSV B Rhinovirus

Human metapneumovims Positive control Negative control

Internal and/or endogenous control

DNA viruses

“Respiratory diseases (DNA viruses)” Panel Adenovirus Bocavims 1 Bocavims 2 Bocavims 3 Bocavims 4 Positive control Negative control

Internal and/or endogenous control Description of the immunological approach

The detection of the immune status of the patient response in infected samples or suspected to be infected by RNA or DNA viruses is detected by an antibody (Ab)-antigen (Ag) reaction using a drop of blood, plasma or serum as a sample.

To obtain a fluorimetric reaction to be applied to the same cartridge and to the same instrument, a mixture of reagents is used allowing, in about 90 seconds, patient's immune status detection.

The basic reagent is eosin-boronate, the volume needed for the test may be placed in the cartridge well.

The recombinant IgM and IgG antigens are bound to a certain amount of sugar by placing them in two tubes or vials, IgM and IgG recombinand antigens are bound by amino group to glucose by a spontaneous reaction (Amadori rearrangement)

The mixture is mixed and left at 30-40 °C (in one preferable embodiment 37 °C) to react for 1-20 minutes, then a defined volume of blood, plasma or serum is added to react (in one preferable embodiment 5 minutes) during which the sugar - Ag-Ab complex is formed.

Some microliters of the sugar- Ag-Ab mixture are taken and injected into one of the wells of the cartridge containing the eosin-boronate reagent and the quenching reaction is recorded.

In a variant, the mixing of sugar-Ag-Ab with eosin-boronate reagent may be performed in a tube or vial and than moved into the cartridge well.

The cartridge is then inserted in the thermal cycler which with specific thermal ramps has improved mixing and optimized the reaction between a conjugate of boronate (in a form of embodiment eosin-boronate) and sugar-Ag-Ab complex.

Eosin-boronate has the peculiarity of being excited at a predetermined frequency and emits at a different predetermined emission frequency.

The detected fluorescence emission is a function of the affinity reaction between eosin-boronate and the sugar of the sugar-Ag-Ab complex and does not require any washing step to separate the excess amount of eosin-boronate.

The frequency of photometric emission by eosin-boronate and its detection within the thermal cycler, is therefore a function of the sugar-antigen-antibody complex eosin-boronate bond, capable of determining the patient's immunological situation with the detection of class immunoglobulins IgG or IgM. The fluorescence emission is directly proportional to the amount of the sugar- Ag-Ab complex that has formed and therefore gives us the measurement of the IgM and IgG present in the sample under examination.

The quenching reaction obtained in the detection of the fluorescence lasts about 90 seconds from the moment of beginning of the reaction.

The new invention therefore allows a rapid execution and an easy working procedure starting from a whole blood sampling without any treatment of the sample in the preliminary phase, or from serum or plasma.

The cartridge has a series of 6 wells useful for carrying out both the immunological and molecular biology tests which may be peculiar to the two tests.

The invention finds useful application in determining the patient's immune status in detecting the presence of immunoglobulins of the IgG or IgM class, which the detection of the virus obtained with classic oral/nasopharyngeal swab detected with the use of molecular biology technique.

The invention can also have a separate application between detection of the presence of antibodies and identification of the presence of viruses, with the application of dedicated cartridges to confirm the immune status, and of cartridges dedicated to molecular biology only.

The operational sequence of analysis of a subject at risk of viral infection that applies to any type of virus, sees as a first step the search for the presence or absence of antibodies against the virus in question, as said analysis is detected in about 2 minutes, and provides information on whether the immune status of the test subject has immunity acquired through the presence of IgG class immunoglobulins or if the patient is in the process of producing said acquired immunity with detection of IgM class immunoglobulins.

A sample comprising or suspected of comprising a virus of the same patient (such as, for example: oral/nasopharyngeal swab, blood, serum, plasma, urine, saliva, CSF etc.) can be processed to obtain nucleic acid to detect the viral genome(s) of interest, in the same instrument taking about an hour.

The invention therefore offers an important innovation also for mass screening to understand whether the immune status of the patients subjected to testing has an acquired immunization or not, and therefore detect their potential risk in infecting other people for the safety of every individual.

The dual test is therefore an innovation that uses a molecular biology test and an immunology test, applied to one mutual cartridge or more separate cartridges inserted in the same instrument which is a thermal cycler and a fluorescence detector for certain excitation and emission wave frequencies.

The dual test is performed on a drop of blood for the immunological test and on nucleic acid extracted from a sample obtained from a oral/nasopharyngeal swab and/or any other biological samples.

It is clear that modifications and/or additions of parts and steps may be made to the system and method to detect bacteria and other cells in a urine sample as described heretofore, without departing from the field and scope of the present invention.

In the following claims, the sole purpose of the references in brackets is to facilitate reading: they must not be considered as restrictive factors with regard to the field of protection claimed in the specific claims.

Claims

1. Method for the detection of viruses and antibodies against viruses in a biological fluid sample, said method comprising contacting said biological fluid sample with a first reaction mixture suitable to detect viruses nucleic acid and/or a second reaction mixture suitable to detect antibodies, said first reaction mixture and second reaction mixtures containing fluorescent probes configured to emit a detectable signal as a consequence of the presence of nucleic acid viruses or antibodies respectively, and detecting said detectable signal, wherein said viruses nucleic acid or antibodies fluorescent signal detection is performed by using a qPCR (Real Time Polymerase Chain Reaction) instrument.

2. Method according to claim 1, wherein said fluorescent signal detection is performed by displacing said first reaction mixture and said second reaction mixture within different wells of suitable disposable cartridge.

3. Method according to claim 2, wherein said disposable cartridge is developed with MEMS (Micro Electro-Mechanical Systems) technology.

4. Method according to claim 2 or 3, wherein said first reaction mixture and/or second reaction mixture comprise a mixture of substances of which at least one substance is provided within the cartridge in lyophilized or gelled or dried form.

5. Method according to claim 1, wherein the biological fluid sample used to extract virus RNA or DNA for virus nucleic acid detection is blood, serum, plasma, urine, oral/nasopharyngeal swab, liquor, BAL or other suitable biological fluid sample.

6. Method according to claim 5, wherein an automated or manual procedure provides the virus RNA or DNA extraction.

7. Method according to any of the previous claims, wherein said first reaction mixture contains reagents necessary to carry out One-Step Reverse transcriptase Real-Time PCR or Real Time PCR from RNA or DNA respectively.

8. Method according to any of the previous claims, wherein said first reaction mixture provide for a multiplex approach detecting up to three different acid nucleic sequences in each reaction mixture.

9. Method according to any of the previous claims, wherein said first reaction mixture produces amplicons of a size between 50 and 500 bp.

10. Method according to any of the previous claims, wherein the fluorescent probe of said first reaction mixture is a fluorophore chosen from a group consisting of: Rodamine-X (ROX^M), Hexachlorofluorescein (HEcTM^ NED™, Fluorescein (FAM™), SYBR™ Green, VIC™, TAMRA, TET, JOE, Texas Red, Cy3, Cy5, Cy5.5 or any fluorofore compatible with the method.

11. Method according to any of the previous claims from 1 to 4, wherein the biological fluid sample used to detect antibodies is blood or plasma or serum.

12. Method according to claim 11, wherein said biological fluid sample is crude.

13. Method according to claim 11 or 12, wherein said second reaction is suitable for detecting the presence of IgG or IgM class antibodies.

14. Method according to any of the previous claims from 11 to 13, wherein said second reaction contains recombinant viruses antigens suitable to form a complex antigen-antibody with IgG or IgM antibodies present in said fluid biological sample.

15. Method according to claim 14, wherein said antigen is human or synthetic antigen.

16. Method according to claim 14 or 15, wherein said antigen is bonded with a sugar.

17. Method according to claim 15 or 16, wherein said antigens are SARS- CoV-2 antigens.

18. Method according to any of the previous claims from 11 to 17, wherein said fluorescent probes of said second reaction mixture are boronate molecules, preferably, eosin-boronate molecules.

19. Method according to any of the previous claims from 14 to 18, wherein it provide to conjugate said antigens-antibodies complexes with said boronate molecules, preferably, eosin-boronate molecules, wherein the signal emitted by eosin-boronate molecules being directly proportional to the amount of the Antigens-Antibodies complexes that has formed and therefore giving the measurement of the IgM and IgG present in the fluid biological sample.

20. Cartridge of the disposable type suitable to be used in a qPCR instrument for the use in a method as in claims 1 to 19.

21. Cartridge according to claim 20, developed with MEMS (Micro Electro- Mechanical Systems) technology.

22. Cartridge according to claim 20 or 21, further provided with said first reaction mixture or said second reaction mixture, wherein said first reaction mixture and said second reaction mixture comprise a mixture of substances of which at least one substance is provided in lyophilized or gelled or dried form.

23. Cartridge according to any of the previous claims from 20 to 22, provided with a minimum of 6 to a maximum of 36 sets of primers and probes, which can be used with the One-Step RT-qPCR and/or qPCR method, where each set produces amplicons of a size between 50 and 500 bp.

24. Cartridge according to claim 23, wherein said primer and probe set includes: a) from one to eight forward primers, that is an oligonucleotide of length between 15 and 40 nucleotides, complementary to a portion of the cDNA or DNA strand b) from one to eight reverse primers, that is an oligonucleotide of length between 15 and 40 nucleotides, complementary to a portion of cDNA or DNA of the opposite strand with respect to the strand on which the forward primer pairs. c) from one to eight probes, that is an oligonucleotide of length between 15 and 45 nucleotides, complementary to a portion of cDNA or DNA of the same strand on which the forward primer(s) pairs or complementary to the opposite strand with respect to the strand on which the forward primer(s) binds, at whose 5’ end a fluorophore is conjugated, and at the 3 'end a quencher is conjugated.

25. Cartridge according to claim 23 or 24, wherein said sets can be combined so as to have a multiplex reaction, that means the generation of multiple amplicons at the same time, where said amplicons generated from the amplification of each portion of cDNA or DNA is discriminable since each said set of primers and probes is associated with a different fluorophore.

26. Cartridge according to any of the previous claims from 23 to 25, further comprising:

- at least one negative control as indication of the correct functioning of the cartridge.

- at least one positive control as indication the correct functioning of the One- Step RT-qPCR and/or qPCR reaction and the presence/absence of possible reaction inhibitors. - at least one internal and/or endogenous control as indication of the presence and quality of the nucleic acid and the absence of amplification inhibitors.

27. Cartridge according to any of the previous claims from 23 to 26, wherein the cartridge is suitable to identify viruses causing tropical disease comprising the following targets: Chikungunya, Zika, Dengue, West Nil, Usutu, Tick-borne encephalitis vims, Yellow fever, Positive control, Negative control, Internal and/or endogenous control.

28. Cartridge according to any of previous claims from 23 to 26, wherein the cartridge is suitable to identify vimses causing Hepatitis B disease comprising the following targets: Hepatitis B vims, Positive control, Negative control, Internal and/or endogenous control.

29. Cartridge according to any of the previous claims from 23 to 26, wherein the cartridge is suitable to identify vimses causing Hepatitis C disease comprising the following targets: Hepatitis C vims, Positive control, Negative control, Internal and/or endogenous control.

30. Cartridge according to any of the previous claims from 23 to 26, wherein the cartridge is suitable to identify MMnCoV comprising the following targets: SARS-CoV-2, Sarbecovimses (SARS-CoV-2, human SARS-CoV), MERS-CoV, Coronavims HKU1, Coronavims NL63, Coronavims 229E, Coronavims OC43, Positive control, Negative control, Internal and/or endogenous control.

31. Cartridge according to any of the previous claims from 23 to 26, wherein the cartridge is suitable to identify vimses causing Influenzae disease comprising the following targets: Flu A, Flu B, RSV A, RSV B, Rhinovims, Parainfluenzae vims 1, Parainfluenzae vims 2, Parainfluenzae vims 3, Parainfluenzae vims 4, Enterovims, Human metapneumovims, Positive control, Negative control, Internal and/or endogenous control.

32. Cartridge according to any of the previous claims from 23 to 26, wherein the cartridge is suitable to identify vimses causing respiratory disease comprising the following targets: Adenovims, Bocavims 1, Bocavims 2, Bocavims 3, Bocavims 4, Positive control, Negative control, Internal and/or endogenous control.

33. Cartridge according to any of the previous claims from 20 to 22, wherein said second reaction mixture is suitable for detecting the presence of IgG or IgM class antibodies.

34. Cartridge according to claim 33, wherein said second reaction contains recombinant viruses antigens suitable to form a complex antigen-antibody with IgG or IgM antibodies present in said fluid biological sample.

35. Cartridge according to claim 33, wherein said antigen is human or synthetic antigen.

36. Cartridge according to claim 34 or 35, wherein said antigen is bonded with a sugar.

37. Cartridge according to claim 35 or 36, wherein said antigens are SARS- CoV-2 antigens.

38. Cartridge according to any of the previous claims from 33 to 37, wherein said fluorescent probes of said second reaction mixture comprise boronate molecules, preferably, eosin-boronate molecules.

39. Cartridge according to any of the previous claims from 34 to 38, wherein for the detection of antigens-antibodies complexes with boronate, preferably, eosin-boronate molecules are used, wherein the signal emitted by eosin-boronate molecules is directly proportional to the amount of said antigens-antibodies complexes that has formed and provides the measurement of the IgM and IgG present in the fluid biological sample.

40. Use of a qPCR instrument for the detection of antibodies in a liquid biological sample by detecting a fluorescent signal emitted by at least one complex comprising at least one antibody, one antigen and one probe emitting fluorescent signal.