Diagnostic peptides in methods, kits, and systems for diagnosing viral infections

Abstract

This invention relates to diagnostic peptides for in vitro diagnostic testing of object infections, as well as methods, kits, and systems. A method of using the peptides of this invention includes the following steps: i) providing a body fluid sample; ii) contacting the body fluid sample with the peptide, said peptide comprising a fluorescent agent having an emission wavelength of 650-900 nm and a non-fluorescent agent having an absorption wavelength of 650-900 nm for quenching the emission of said fluorescent agent, and a cleavage site located between said fluorescent agent and the first non-fluorescent agent, said cleavage site being specifically targeted at a viral protease; iii) monitoring fluorescence from the peptide in step ii) in the 650-900 nm range, wherein an increase in fluorescence in the 650-900 nm range indicates the presence of a viral protease in the sample.

Description

Invention Field

[0001] This invention relates to a method for in vitro diagnosis of viral infection, a method for in vitro diagnosis of viral infection in a subject, a kit for detecting the presence of infection in a subject, and a diagnostic peptide in a system for detecting the presence of infection in a subject, wherein the method includes providing a body fluid sample. Background Technology

[0002] The clinical need for improved diagnostic methods for viral infections in patients is increasing. Rapid detection and identification of viral pathogens are crucial for limiting the spread and transmission of viral diseases and for monitoring treatment options. The events surrounding the SARS-related coronaviruses of the 2019 / 2020 global pandemic illustrate the need for such methods.

[0003] SARS is a viral respiratory illness caused by the SARS-related coronavirus (SARS-CoV). SARS was first reported in Asia in February 2003. In 2019 / 20, the SARS-CoV-19 pandemic caused more than 600,000 deaths worldwide.

[0004] The SARS coronavirus belongs to a group of viruses similar to those that cause the common cold. The SARS virus spreads through close person-to-person contact and is considered most easily transmitted through respiratory droplets produced when an infected person coughs or sneezes. Droplet transmission occurs when droplets from an infected person are propelled a short distance through the air (usually up to 3 feet) and settle on the mucous membranes of nearby people. The virus can also be spread when a person touches a surface contaminated with infectious droplets and then touches their mouth, nose, or eyes.

[0005] Real-time reverse transcriptase-PCR (RT-PCR) assays are currently favored for detecting coronaviruses due to their advantages as a specific and simple quantitative assay. In addition, real-time RT-PCR is more sensitive than conventional RT-PCR assays, which is of great help in the diagnosis of early infection. Therefore, real-time RT-PCR assays remain the main method for detecting various coronaviruses, including SARS-CoV-2

[14] .

[0006] Even so, improvements to real-time RT-PCR assays are still needed. Because RT-PCR methods are susceptible to contamination and require time-consuming sample processing and post-PCR analysis, the improved assay is based on TaqMan real-time RT-PCR, which can be easily implemented in routine diagnostic settings for HCoV detection. Furthermore, to further improve sensitivity, real-time quantitative RT-PCR assays for SARS-CoV can be performed using two TaqMan probes instead of one. This simple modification for quantification using dual TaqMan probes has wide applications in areas where ultrasensitivity is critical, where SARS-CoV detection is limited to one copy of RNA per reaction.

[0007] LAMP is a novel and highly efficient isothermal nucleic acid amplification method. However, result analysis is typically performed via time-consuming gel electrophoresis. If these methods rely on non-specific signal transduction schemes, such as fluorescent dyes intercalating into any double-stranded DNA amplicon, or if solution turbidity is caused by pyrophosphate release during polymerization, the possibility of unexpected signals originating from primer dimers or non-primer reactions cannot be ruled out.

[0008] In clinical testing, the lack of safe and stable external positive controls (EPCs) can be a serious problem for PCR / LAMP methods in diagnosing coronaviruses.

[0009] Meanwhile, the rapid mutational nature of coronaviruses highlights the necessity of accurately detecting genetically diverse coronaviruses.

[0010] There is a need for a rapid diagnostic method that can identify the presence of viral infection in bodily fluid samples and can be performed at the point of care. Invention Overview

[0012] This invention provides a diagnostic peptide for use in a method for diagnosing viral infections in vitro, said peptide being represented by formula (I):

[0013] [a]-[b]-[c] (I)

[0014] in:

[0015] [a] is a fluorescent agent with an emission wavelength of 650-900 nm.

[0016] [b] contains the amino acid sequence X aa1 ,X aa2 ,X aa3 peptides

[0017] Where X aa1 It is a hydrophobic or basic amino acid.

[0018] Where X aa2 Are they polar, neutral, or basic amino acids?

[0019] Where X aa3 Are they polar, neutral, or basic amino acids?

[0020] [c] is a non-fluorescent agent with an absorption wavelength of 650-900 nm, used to quench the emission of the fluorescent agent.

[0021] Where X aa1 ,X aa2 ,X aa3 The cleavage site represents a protease encoded by the virus, wherein cleavage of the cleavage site results in the release of the non-fluorescent agent from the peptide, wherein the release of the non-fluorescent agent indicates the presence of the virus, preferably wherein [b] represents a maximum of 30% by weight of the total mass of the diagnostic peptide based on [a]-[b]-[c].

[0022] In some jurisdictions, the present invention may be limited to a method for in vitro diagnosis of viral infections using the diagnostic peptide represented by formula (I):

[0023] [a]-[b]-[c] (I)

[0024] in:

[0025] [a] is a fluorescent agent with an emission wavelength of 650-900 nm.

[0026] [b] contains the amino acid sequence X aa1 ,X aa2 ,X aa3 peptides,

[0027] Where X aa1 It is a hydrophobic or basic amino acid.

[0028] Where X aa2 Are they polar, neutral, or basic amino acids?

[0029] Where X aa3 Are they polar, neutral, or basic amino acids?

[0030] [c] is a non-fluorescent agent with an absorption wavelength of 650-900 nm, used to quench the emission of the fluorescent agent.

[0031] Where X aa1 ,X aa2 ,X aa3 The cleavage site represents a protease encoded by the virus, wherein cleavage of the cleavage site results in the release of the non-fluorescent agent from the peptide, wherein the release of the non-fluorescent agent indicates the presence of the virus, preferably wherein [b] represents up to 50% by weight of the total mass of the diagnostic peptide based on [a]-[b]-[c].

[0032] Similarly, in some jurisdictions, the invention can be limited to the use of diagnostic peptides in the in vitro diagnosis of viral infections, wherein the peptides are represented by formula (I):

[0033] [a]-[b]-[c] (I)

[0034] in:

[0035] [a] is a fluorescent agent with an emission wavelength of 650-900 nm.

[0036] [b] contains the amino acid sequence X aa1 ,X aa2 ,X aa3 peptides,

[0037] Where X aa1 It is a hydrophobic or basic amino acid.

[0038] Where X aa2 Are they polar, neutral, or basic amino acids?

[0039] Where X aa3 Are they polar, neutral, or basic amino acids?

[0040] [c] is a non-fluorescent agent with an absorption wavelength of 650-900 nm, used to quench the emission of the fluorescent agent.

[0041] Where X aa1 ,X aa2 ,X aa3 The cleavage site represents a protease encoded by the virus, wherein cleavage of the cleavage site results in the release of the non-fluorescent agent from the peptide, wherein the release of the non-fluorescent agent indicates the presence of the virus, preferably wherein [b] represents up to 50% by weight of the total mass of the diagnostic peptide based on [a]-[b]-[c].

[0042] The present invention also relates to an in vitro method for diagnosing viral infection in a subject, the method comprising the following steps:

[0043] i) Contacting a body fluid / tissue sample with a peptide comprising a fluorescent agent having an emission wavelength of 650-900 nm and a non-fluorescent agent having an absorption wavelength of 650-900 nm to quench the emission of the fluorescent agent, and a cleavage site located between the fluorescent agent and the first non-fluorescent agent, the cleavage site being specifically targeted at a viral protease.

[0044] ii) Monitor the fluorescence of the peptide described in step ii) in the 650-900 nm range.

[0045] Increased fluorescence in the 650-900 nm range indicates the presence of viral proteases in the sample.

[0046] According to the present invention, the method defined herein enables rapid and specific detection of infection in a subject by analyzing bodily fluid / tissue samples. In the presence of a viral protease that identifies and cleaves the cleavage site, a first non-fluorescent agent is released from the reagent, resulting in a fluorescent signal that can be detected by a suitable detector. Surprisingly, it has been found that detecting the viral protease by monitoring a wavelength of 650-900 nm provides an improved detection limit compared to prior art methods. The method is simple to perform and suitable for point-of-care use.

[0047] The advantage of this method is that it can be performed on bodily fluid samples without requiring enrichment steps to accumulate cell counts. As a result, this method enables rapid detection of viral infections, for example, within minutes of reagent contact with the sample.

[0048] The method overcomes the problem of the lack of a safe and stable external positive control (EPC) in the PCR / LAMP method.

[0049] In other aspects, kits for diagnosing viral infections in subjects are provided, comprising:

[0050] a) A container containing the diagnostic peptide of formula (I),

[0051] b) A set of instructions for performing the diagnostic methods defined herein.

[0052] In other aspects, a system for diagnosing viral infections in subjects is provided, comprising:

[0053] a) A container used to receive samples.

[0054] b) A container containing the diagnostic peptide of formula (I),

[0055] c) An apparatus adapted to receive the container and monitor the fluorescence signal emitted from the peptide when the peptide comes into contact with the body fluid sample.

[0056] This invention also relates to diagnostic peptides represented by formula (Ib):

[0057] [a]-[connector 1]-[b]-[-connector 2]-[c](Ib)

[0058] in:

[0059] [a] is a fluorescent agent with an emission wavelength of 650-900 nm.

[0060] [b] contains 3-10 amino acids and has the amino acid sequence X. aa1 ,X aa2 ,X aa3 peptides,

[0061] Where X aa1 Is it histidine, arginine, or lysine?

[0062] Where X aa2 It's glutamine.

[0063] Where X aa3 Are they polar, neutral, or basic amino acids?

[0064] [c] is a non-fluorescent agent with an absorption wavelength of 650-900 nm, used to quench the emission of the fluorescent agent.

[0065] [Linker 1] and [Linker 2] are independently selected from optionally substituted hydrocarbon groups and non-protein hydrolyzed hydrocarbon groups.

[0066] Where X aa1 ,X aa2 ,X aa3 The cleavage site represents the protease encoded by the virus.

[0067] The cleavage of the cleavage site results in the release of a non-fluorescent agent from the peptide, wherein the release of the non-fluorescent agent indicates the presence of a virus belonging to the Coronaviridae family, preferably wherein the virus is SARS-CoV-2 (SARS-CoV-19).

[0068] The present invention also relates to methods, uses, kits, and systems for using diagnostic peptides as defined herein.

[0069] Description of the implementation plan

[0070] As used herein, the term "object" means an animal or human individual at risk of infection or suspected of being infected. The terms "patient," "object," "individual," etc., are used interchangeably herein and refer to any animal or human suitable for the methods described herein. In some non-limiting embodiments, the patient, object, or individual is a human being.

[0071] As used herein, the term "body fluid sample" refers to biological material isolated from an object. The steps for obtaining the sample are not part of this invention.

[0072] As used in this article, the term "viral protease" refers to a peptidase encoded by viral RNA.

[0073] As used herein, the terms “protease” and “peptidase” refer to polypeptides encoded by viral RNA that are capable of cleaving peptide bonds (C(O)NH) at cleavage sites (amino acid motifs) in proteins.

[0074] As used herein, the term "peptide" refers to an oligomer containing at least 3 amino acids. Preferably, the peptide contains no more than 20 amino acids. More preferably, the peptide contains 4 to 20 amino acids, and more preferably 6 to 15 amino acids. In one embodiment, the peptide preferably contains 3 to 10 amino acids. The amino acids used can be any amino acid, preferably selected from naturally occurring amino acids or synthetic amino acids, particularly derivatives of natural amino acids. Preferably, the peptide includes a "non-natural amino acid motif" containing a cleavage site for a viral protease. As used herein, a "non-natural amino acid motif" refers to a sequence that is not naturally occurring, i.e., a genetically engineered sequence and a sequence derived from molecular modeling.

[0075] As used herein, the terms “monitoring,” “measuring / measurement,” “detecting / detection,” or “diagnostic” mean assessing the presence, absence, quantity, or amount (which may be an effective amount) of a given substance in a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise assessing the value or classification of a subject’s clinical parameters.

[0076] The term "virus-encoded protease" refers to a protease that is translated from the genetic information (RNA) of a viral genome.

[0077] As used in this article, the term "infection" refers to a clinically relevant viral load. An infection may be symptomatic or asymptomatic in the individual. A clinically relevant viral load may result in symptomatic or asymptomatic individuals.

[0078] In a first aspect, a diagnostic peptide is provided for use in a method for diagnosing viral infection in vitro, said peptide being represented by formula (I):

[0079] [a]-[b]-[c] (I)

[0080] in:

[0081] [a] is a fluorescent agent with an emission wavelength of 650-900 nm.

[0082] [b] contains the amino acid sequence X aa1 ,X aa2 ,X aa3 peptides

[0083] Where X aa1 It is a hydrophobic or basic amino acid.

[0084] Where X aa2 Are they polar, neutral, or basic amino acids?

[0085] Where Xaa3 Are they polar, neutral, or basic amino acids?

[0086] [c] is a non-fluorescent agent with an absorption wavelength of 650-900 nm, used to quench the emission of the fluorescent agent.

[0087] Where X aa1 ,X aa2 ,X aa3 The cleavage site represents the protease encoded by the virus, wherein cleavage of the cleavage site results in the release of a non-fluorescent agent from the peptide, wherein the release of the non-fluorescent agent indicates the presence of the virus, preferably wherein [b] represents the total mass based on [a]-[b]-[c], up to 50% by weight of the total mass of the diagnostic peptide.

[0088] The term "hydrophobic amino acid" refers to an amino acid that primarily possesses side chains that impart a tendency to associate in an aqueous environment. The terms "hydrophobic" and "hydrophobicity" are well known to those skilled in the art, as known from the manual 'IUPAC Gold Book', IUPAC. Compendium of Chemical Terminology, 2nd Edition, compiled by A.D. C. Naught and A. Wilkinson, Blackwell Scientific Publications, Oxford (1997). https: / / doi.org / 10.1351 / goldbook.HT06964 Examples of hydrophobic amino acids are alanine, leucine, valine, ortholeucine, orthovaline, isoleucine, isovaline, alloisoleucine, phenylalanine, proline, methionine, and tryptophan.

[0089] The term "polar or neutral amino acid" refers to an amino acid that can form one or more hydrogen bonds. Examples of polar or neutral amino acids are serine, threonine, cysteine, asparagine, glutamine, and tyrosine.

[0090] The term "basic amino acid" refers to an amino acid that has a basic side chain at neutral pH. Examples of basic amino acids are arginine, lysine, and histidine.

[0091] Xaa1 is a hydrophobic or basic amino acid. Preferably, Xaa1 is selected from: alanine, leucine, valine, ortholeucine, orthovaline, isoleucine, isovaline, alloisoleucine, phenylalanine, histidine, arginine, and lysine; more preferably, it is selected from: histidine, lysine, and arginine. Preferably, Xaa1 is histidine. Xaa2 is a polar, neutral, or basic amino acid. Preferably, Xaa2 is selected from: histidine, asparagine, and glutamine; more preferably, Xaa2 is selected from histidine and glutamine. Preferably, Xaa2 is glutamine.

[0092] Xaa3 is a polar, neutral, or basic amino acid. Preferably, Xaa3 is selected from serine, threonine, and glycine. More preferably, Xaa3 is selected from serine and threonine, and even more preferably, Xaa3 is serine.

[0093] Preferably, the amino acid sequence X aa1 ,X aa2 ,X aa3 It has a sequence selected from the following: LQS, HQS, FHT, NleQS, NvaQS, VLQS (SEQ ID NO:1), VLNS (SEQ ID NO:2), RQS or KQS.

[0094] The peptide preferably contains 4 to 10 amino acids, more preferably 5 to 9 amino acids.

[0095] Preferably, the cleavage site consists of 3 to 8 amino acids, more preferably 4 to 7 amino acids. The cleavage site is preferably composed of multiple amino acids, for example, at least 2, more preferably at least 3, more preferably at least 4, and even more preferably at least 5.

[0096] Preferably, the total mass is based on [a]-[b]-[c], where [b] represents up to 50% of the total mass of the diagnostic peptide by weight. More preferably, [b] represents up to 45% of the total mass of the diagnostic peptide by weight based on [a]-[b]-[c], even more preferably up to 40% of the total mass of the diagnostic peptide by weight, even more preferably up to 35% of the total mass of the diagnostic peptide by weight, even more preferably up to 30% of the total mass of the diagnostic peptide by weight, and even more preferably up to 25% of the total mass of the diagnostic peptide by weight.

[0097] Preferably, [b] represents 5% to 50% of the total mass of the diagnostic peptides based on [a]-[b]-[c], more preferably 7% to 45% by weight, even more preferably 10% to 40% by weight, and even more preferably 15% to 35% by weight.

[0098] Preferably, the increase in fluorescence is defined as a relative increase, which is obtained by subtracting the fluorescence measured in the presence of both the diagnostic peptide and the sample (F1) from the fluorescence of the diagnostic peptide in the absence of the sample (F2). Preferably, the relative increase is expressed as the proportion of the fluorescence of the diagnostic peptide in the absence of the sample (F2) according to the following equation:

[0099] Relative fluorescence percentage = [(F1-F2) / F2]*100

[0100] Preferably, the amino acid sequence X aa1 ,X aa2 ,X aa3 It has other amino acid, hydrocarbon moiety and / or non-protein hydrolyzed hydrocarbon linker attached to the side.

[0101] In a preferred embodiment, the hydrocarbon linker is selected from: β-alanine, 4-aminobutyryl, 2-(aminoethoxy)acetyl, 3-(2-aminoethoxy)propyl, 5-aminopentanoyl, 6-aminohexyl, 8-amino-3,6-dioxaoctyl and 12-amino-4,7,10-trioxadodecyl, preferably 6-aminohexyl.

[0102] According to one implementation scheme, the diagnostic peptide is represented by formula (Ia):

[0103] [a]-[connector 1]-[b]-[-connector 2]-[c] (Ia)

[0104] The linkers are independently selected from optionally substituted hydrocarbon groups and non-protein hydrolyzed hydrocarbon groups.

[0105] In one aspect of the invention, the diagnostic peptide is represented by formula (Ib):

[0106] [a]-[connector 1]-[b]-[-connector 2]-[c] (Ib)

[0107] in:

[0108] [a] is a fluorescent agent with an emission wavelength of 650-900 nm.

[0109] [b] contains 3-10 amino acids and has the amino acid sequence X. aa1 ,X aa2 ,X aa3 peptides,

[0110] Xaa1 is histidine, arginine, or lysine.

[0111] Xaa2 is glutamine.

[0112] Xaa3 is a polar, neutral, or basic amino acid.

[0113] [c] is a non-fluorescent agent with an absorption wavelength of 650-900 nm, used to quench the emission of the fluorescent agent.

[0114] [Linker 1] and [Linker 2] are independently selected from optionally substituted hydrocarbon groups and non-protein hydrolyzed hydrocarbon groups.

[0115] Where X aa1 ,X aa2 ,X aa3 The cleavage site represents a protease encoded by the virus, wherein cleavage of the cleavage site results in the release of a non-fluorescent agent from the peptide, wherein the release of the non-fluorescent agent indicates the presence of a virus (preferably a virus belonging to the Coronaviridae family). More preferably, the virus is SARS-CoV-2 (SARS-CoV-19).

[0116] The peptide of formula (Ib) can also be defined by its use in in vitro methods for diagnosing SARS-CoV-2 (SARS-CoV-19) infection.

[0117] Fluorescent agents with an emission wavelength of 650-900 nm are preferably anthocyanin moiety (dyes). Preferably, non-fluorescent agents with an emission wavelength of 650-900 nm are anthocyanin moiety (dyes).

[0118] Preferably, the fluorescent agent is a cyanogen dye with an emission wavelength of 650-900 nm, and the non-fluorescent agent is a cyanogen dye with an absorption wavelength of 650-900 nm.

[0119]

[0120] In the implementation scheme, the first fluorescent agent is a cyanogenide dye having the general formula shown in Formula II, wherein R 1 Selected from H, halogenated and Where R 17 Selected from: carboxyl, amino, and sulfanato groups; X is selected from: O, S, NH, and N-alkyl groups; R 2 R 3 R 9 R 10 Each is independently selected from H and hydrocarbon groups; R 4 R 5 R 11 R 12 Each is independently selected from: H, hydrocarbon group, and sulfonyl group, or atoms bonded to them to form an aromatic ring; R 6 R 7 R 13 R 14 Each is independently selected from H and hydrocarbon groups, R 8 and R 15 Each group is independently selected from hydrocarbon groups, (CH2)qFG, or (CH2).P LN, where R 8 and R 15 At least one of them is (CH2)qFG, where q is an integer from 1 to 20, and FG is a functional group that does not react directly with carboxyl, hydroxyl, amino, or thiol groups, where p is an integer from 1 to 20, and LN is a linker group that reacts with carboxyl, hydroxyl, amino, or thiol groups; R 16 It is an H or hydrocarbon group.

[0121] Preferably, the fluorescent agent is a reagent, wherein R 1 yes Where X is O, and R 17 It is SO3Na; R 2 R 3 R 9 R 10 It is a hydrocarbon group, preferably methyl; R 4 and R 11 It is H, and R 5 and R 12 It is H or sulfonate; R 6 R 7 R 13 R 14 It is H; R8 is (CH2)qFG, where q is 4 and FG is sulfonyl; R 15 It is (CH2) P LN, where p is 5 and LN is a carboxyl group, R 16 It is H.

[0122] Even more preferably, the fluorescent agent is a reagent, wherein R 1 yes Where X is O and R 17 It is SO3Na; R 2 R 3 R 9 R 10 It is methyl; R 4 and R 11 It is H, and R 5 and R 12 It is sulfonated; R 6 R 7 R 13 R 14 It is H; R8 is (CH2)qFG, where q is 4 and FG is sulfonyl; R 15 It is (CH2) P LN, where p is 5 and LN is a carboxyl group, R 16 It is H. Preferably, the fluorescent agent is a reagent corresponding to Formula III.

[0123]

[0124] Non-fluorescent agents with absorption wavelengths of 650-900 nm are compounds that have little or no intrinsic fluorescence and can effectively quench fluorescence from neighboring fluorophores with little background. In an embodiment, the non-fluorescent agent is a cyanide molecule. Cyanide molecules, also known as cyanide dyes, comprise compounds having two substituted or unsubstituted nitrogen-containing heterocycles linked by a polyacetylenic chain.

[0125] In a preferred embodiment, the non-fluorescent agent is a reagent, wherein R 1 It is chlorinated, R 2 R 3 R 9 R 10 It is methyl; R 4 It is H and R 5 It is an N-hydrocarbon group, preferably N[(CH2)3SO3Na]2; R 11 and R 12 Formation of an aromatic ring monosubstituted with a sulfonyl group; R 6 R 7 R 13 R 14 It is H; R 8 It is (CH2)qFG, where q is 3 and FG is sulfonyl; R 15 It is (CH2) P LN, where p is 5 and LN is a carboxyl group; R 16 It is H.

[0126] In another embodiment, the fluorescent agent and the non-fluorescent agent are the same reagent, preferably wherein R 1 yes Where X is O and R 17 It is SO3Na, R 2 R 3 R 9 R 10 It is a hydrocarbon group, preferably a methyl group, R 4 R 5 R 11 R 12 It is H, R 6 R 7 R 13 R 14 It is H, R 8 It is (CH2)qFG, where q is 4 and FG is sulfonyl, R 15 It is (CH2)PLN, where p is 5 and LN is a carboxyl group, R 16 It is H.

[0127] The non-fluorescent agent may also be a quenching component such as BHQ3, (Biosearch) QC-1 (Li-COR.com), TF7QWS, TF8QWS (AAT Bio), or particles containing such compounds (e.g., gold nanoparticles and iron nanoparticles). In embodiments, the peptide substrate is nanoparticles containing peptides as defined herein. In a preferred embodiment, the non-fluorescent agent is QC-1 (Li-COR.com).

[0128] Examples of fluorescent agents that can be used with this invention include, but are not limited to, Alexa. 660, Alexa 680, Alexa 700, Alexa 750 ATTO 680, ATTO 700, DY-647, DY-650, DY-673, DY-675, DY-676, DY-680, DY-681, DY-682, DY-690, DY-700, DY-701 , DY-730, DY-731, DY-732, DY-734, DY-750, DY-751, DY-752, DY-776, DY-781, DY-782, DY-831, La Jolla blue, Cy5, Cy5.5, Cy7, IR 800CW, IR 38. IR 800RS, IR 700DX, IR 680 and TF7WS (AATBio), etc. "Alexa Fluor" dye is available from Molecular Peptides Inc., Eugene, OR, USA (www.peptides.com). "ATTO" dye is available from ATTO-tec GmbH, Siegen, Germany (www.atto-tec.com). "DY" dye is available from Dyomics GmbH, Jena, Germany (www.dyomics.com). La Jolla Blue is available from Hyperion Inc. "Cy" dye is available from Amersham Biosciences, Piscataway, NJ, USA (www.amersham.com). "Infrared dyes" can be obtained from Bioscience,Inc.Lincoln,NE,USA( www.licor.com )get.

[0129] In a preferred embodiment, the fluorescent agent is IR dye 800CW, Cy7, or TF7WS, preferably Cy7. Preferably, Cy7 has a structure according to Formula IV:

[0130]

[0131] Where R 18 It is a linker group that reacts with a carboxyl, hydroxyl, amino, or thiol group. Cy7 is available, for example, from ClickChemistryTools; [https: / / clickchemistrytools.com / product / cy7-nhs-ester / ].

[0132] Preferably, the peptide has the structure ([a]-[b]-[c]). m,p Where m is an integer from 1 to 8 and p is + (positive) or – (negative). Preferably, the peptide has the structure ([a]-[b]-[c]). m,p , where m is an integer from 1 to 8 and p is negative, more preferably m is an integer from 2 to 7 and p is negative, even more preferably m is an integer from 3 to 6 and p is negative, and most preferably m is an integer from 3 to 6 and p is negative.

[0133] Preferably, the peptide has the structure ([a]-[b]-[c]). m,p , where m is an integer from 1 to 8 and p is positive, more preferably, m is an integer from 2 to 7 and p is positive, even more preferably, m is an integer from 3 to 6 and p is positive, and most preferably, m is an integer from 3 to 6 and p is positive.

[0134] Preferably, the cleavage site consists of 3 to 8 amino acids, more preferably 4 to 7 amino acids. The cleavage site preferably contains multiple amino acids, for example, at least two, preferably at least three, more preferably at least four, and even more preferably at least five. Preferably, the cleavage site has an XYZ structure, wherein X is at least one amino acid, Y is part of a molecular structure consisting of at least two amino acids, and Z is at least one amino acid. X and Z can be any amino acid. Preferably, Y comprises a dipeptide composed of an aliphatic hydrophobic amino acid and an aromatic or cyclic amino acid, or a basic amino acid and a hydrophilic amino acid. The aliphatic hydrophobic amino acid is preferably selected from glycine, alanine, leucine, valine, and their derivatives. The aromatic or cyclic amino acid is preferably selected from phenylalanine, tyrosine, tryptophan, proline, and their derivatives. The basic amino acid is preferably lysine, and the hydrophilic amino acid is preferably threonine or serine. Preferably, the cleavage site contains non-natural amino acids. Non-natural amino acids are known to those skilled in the art.

[0135] Preferably, the peptide comprises a cell-penetrating portion. The cell-penetrating portion comprises an amino acid sequence consisting of X1, X2, X3, X4, X5, X6, X7, X8, and X9, wherein X1 is A, L, G, or R; X2 is W, A, L, G, or R; X3 is R or K; X4 is R, K, L, or S; X5 is R, K, L; X6 is R, K, L; X7 is R, K, L; X8 is A, V, R, K, L, S, or Q; and X9 is A, V, R, K, L, S, Q, W, F, or Y. Preferably, the cell-penetrating portion is a nine-arginine peptide, wherein for all X1, X2, X3, X4, X5, X6, X7, X8, and X9, X is R.

[0136] As used herein, a “cell-penetrating peptide” is a molecule whose core is a peptide. However, other chemical groups may be covalently linked to the peptide core to enhance the overall stability of the molecule and / or provide it with additional properties (such as targeting capabilities). For example, the cell-penetrating peptide according to the invention may also contain one or more groups covalently linked to the C-terminus selected from: cysteamide, cysteine, thiol, amide, carboxyl, optionally substituted straight-chain or branched C1-C6 alkyl, primary or secondary amine, osidic derivative, lipid, phospholipid, fatty acid, cholesterol, polyethylene glycol, nuclear localization signal (NLS), and / or targeting molecules. Optionally or additionally, the cell-penetrating peptide may also contain one or more chemical entities covalently linked to the N-terminus selected from: acetyl, fatty acid, cholesterol, polyethylene glycol, nuclear localization signal, and / or targeting molecules. If necessary, for example, with the addition of cholesterol to the N-terminus, the peptide bridge may be used to bind non-peptide molecules.

[0137] Preferably, [a] is a cyanogen moiety having an emission wavelength of 700-850 nm, [b] is a peptide containing a sequence selected from: LQS, HQS, RQS, KQS, FHT, NleQS, NvaQS, VLQS (SEQ ID NO:1), VLNS (SEQ ID NO:2), more preferably selected from HQS, RQS or KQS, [linker 1] and [linker 2] are both 3-(2-aminoethoxy)propyl, and [c] is a cyanogen moiety having an absorption wavelength of 700-850 nm.

[0138] Preferably, the object is a human or an animal. Preferably, the object is a human.

[0139] Preferably, the object is an animal. Preferably, the object is an animal and belongs to a species selected from the following: domestic pig (Susscrofa domesticus), domestic chicken (Gallus Gallus domesticus), domestic cat (Felis catus), domestic dog (Canis lupus familiaris), cattle (Bos Taurus), sheep (Ovis aries), goats ((Capra) and domestic goats (aegagurus hircus), preferably selected from the following species: domestic pig, domestic chicken, domestic cat, domestic dog, cattle, more preferably selected from the following species: domestic pig, domestic chicken, domestic cat, even more preferably selected from species of domestic pig and domestic chicken, most preferably the species domestic pig.

[0140] Preferably, the viral infection is caused by a virus belonging to the Picornaviridae, Coronaviridae, Adenoviridae, or Coronaviridae families. Preferably, the virus is selected from the following Picornaviridae family: rhinovirus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E virus.

[0141] Preferably, the rhinovirus is an enterovirus.

[0142] Preferably, the virus is selected from the following Adenoviridae viruses: atadenovirus, avian adenovirus, ichtadenovirus, mammalian adenovirus, siadenovirus, and combinations thereof.

[0143] Preferably, the virus is selected from the following genera: alpha coronavirus, beta coronavirus, gamma coronavirus, and delta coronavirus. Preferably, the coronavirus family is alpha coronavirus or beta coronavirus.

[0144] Preferably, the virus belongs to the genus Coronavirus and is selected from the following species: α-coronavirus 1 (TGEV, feline coronavirus, canine coronavirus), human coronavirus 229E, human coronavirus NL63, long-winged bat coronavirus 1, long-winged bat coronavirus HKU8, porcine epidemic diarrhea virus, horseshoe bat coronavirus HKU2, and yellow bat coronavirus 512. Preferably, the virus is porcine epidemic diarrhea virus. Preferably, the virus is porcine epidemic diarrhea virus and the target species is domestic pig.

[0145] Preferably, the virus belongs to the genus *β-coronavirus* and is selected from the following species: β-coronavirus 1 (bovine coronavirus, human coronavirus OC43), hedgehog coronavirus 1, human coronavirus HKU1, Middle East respiratory syndrome-related coronavirus, mouse coronavirus, *Pterygota* bat coronavirus HKU5, *Drosophila* bat coronavirus HKU9, severe acute respiratory syndrome-related coronavirus (SARS-CoV, SARS-CoV-2), and *Pterygota* coronavirus HKU4. Preferably, the virus is a severe acute respiratory syndrome-related coronavirus (SARS-CoV, SARS-CoV-2), more preferably, it is a severe acute respiratory syndrome-related coronavirus (SARS-CoV-2). Preferably, the virus is a severe acute respiratory syndrome-related coronavirus (SARS-CoV, SARS-CoV-2), and the target is a human.

[0146] Preferably, the virus belongs to the genus *Gammacoronavirus* and is selected from avian coronaviruses and beluga coronavirus SW1. Preferably, the virus is an avian coronavirus. Preferably, the virus is an avian coronavirus and the target species is domestic chicken.

[0147] Preferably, the virus belongs to the genus *δ-coronavirus* and is selected from *Nightingale Coronavirus HKU11* and *Pig Coronavirus HKU15*. Preferably, the virus is *Pig Coronavirus HKU15*. Preferably, the virus is *Pig Coronavirus HKU15* and the target species is domestic pig.

[0148] Preferably, the viral protease is encoded by viral RNA. The viral protease is preferably selected from hepatitis-encoded serine proteases or metalloproteinases (NS2 and NS3), rhinovirus genome-encoded cysteine ​​proteases (3C and 2A), 3C protease, coronavirus genome-encoded cysteine ​​protease (3CL), adenovirus genome-encoded serine-centered neutral protease, and retrovirus-encoded aspartic protease. Preferably, the viral protease is a 3C-like (3CL) protease. Preferably, the coronavirus protease is a cysteine ​​protease encoded by the hSARS coronavirus genome, or the SARS coronavirus 19 main protease (EC number 3.4.22.69, BRENDA database release 2021.1 (January 2021)).

[0149] Preferably, the protease is a cysteine ​​protease encoded by the SARS genome, wherein the SARS protease is the main protease of SARS coronavirus 19 (EC number 3.4.22.69, BRENDA database release 2021.1 (January 2021)). The main protease operates at no fewer than 11 cleavage sites on the large polyprotein 1ab (replicaase 1ab, ~790 kDa); the cleavage sites are Leu-Gln=Ser-Ala-Gl (where = is an easily cleaved peptide bond).

[0150] Preferably, the virus-encoded protease is a protease with at least 50% homology to the amino acid sequence of a 3C-like protease [Severe Acute Respiratory Syndrome Coronavirus 2], preferably at least 55%, more preferably at least 60%, even more preferably 70%, more preferably at least 75%, even more preferably 80%, even more than 85%, even more preferably at least 90%, even more preferably at least 95%, preferably at least 97%, and most preferably at least 98%, said 3C-like protease having accession number YP_009725301, version number YP_009725301.1, 18-JUL-2020 NCBI Protein Database GenPept [2021-03-26]; available from https: / / www.ncbi.nlm.nih.gov / protein / 1802476809.

[0151] Preferably, the virus-encoded protease is a protease that shares at least 50% homology with the amino acid sequence of a 3C-like protease (3CL pro; R1A_SARS) at positions 3241-3546, preferably at least 55%, more preferably at least 60%, even more preferably 70%, more preferably at least 75%, even more preferably 80%, even more than 85%, even more preferably at least 90%, even more preferably at least 95%, preferably at least 97%, and most preferably at least 98%, said 3C-like protease having accession number P0C6U8 (Uniprot), integrated into UniProtKB / Swiss-Prot: Sequence Update: June 10, 2008, Last Modified: February 10, 2021, Version 104 [2021-03-26]; available from https: / / www.uniprot.org / uniprot / P0C6U8.

[0152] Preferably, the virus-encoded protease has at least 50% homology with the amino acid sequence of 3C protease [human rhinovirus A1], preferably at least 55%, more preferably at least 60%, even more preferably 70%, more preferably at least 75%, even more preferably 80%, even more than 85%, even more preferably at least 90%, even more preferably at least 95%, preferably at least 97%, and most preferably at least 98%, said 3C protease has accession number YP_009508983.1, version number YP_009508983.1, 24-AUG-2018 NCBI Protein Database GenPept [2021-03-26]; https: / / www.ncbi.nlm.nih.gov / protein / YP_009508983.

[0153] Preferably, the virus-encoded protease has at least 50% homology with the amino acid sequence of the PEDV main protease [swine epidemic diarrhea virus], preferably at least 55%, more preferably at least 60%, even more preferably 70%, more preferably at least 75%, even more preferably 80%, even more than 85%, even more preferably at least 90%, even more preferably at least 95%, preferably at least 97%, and most preferably at least 98%, said PEDV main protease has accession number 4XFQ_A, PDB database, stored on 28-12-2014, version 1.0 2016-1-20 [2021-03-26]; https: / / www.rcsb.org / structure / 4XFQ .

[0154] Homology is typically measured using sequence analysis software such as the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705; MEGAlign (DNAStar, Inc., 1228 S. Park St., Madison, Wisconsin 53705); and MacVector (Oxford Molecular Group, 2105 S. Bascom Avenue, 200 Campbell, CA 95008), BLAST, which is available via the National Center for Biotechnology Information (NCBI) [Internet]. Bethesda (MD): National Center for Biotechnology Information, National Library of Medicine (US). Information);

[2021] – https: / / www.ncbi.nlm.nih.gov / (Madden, TL, Tatusov, RL & Zhang, J. (1996) "Applications of network BLAST server" Meth. Enzymol. 266: 131-141). Such software matches similar sequences by assigning homology to various substitutions, deletions and other modifications. Conserved substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid, glutamic acid, asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Substitution can also be based on conserved hydrophobicity or hydrophilicity (Kyte and Doolittle, J.Mol.BioL 157:105-132, 1982), or on the ability to present similar peptide secondary structures (Chou and Fasman, Adv.Enzymol.47:45-148, 1978).

[0155] Preferably, the virus-encoded protease has at least 50% sequence similarity to the amino acid sequence of the 3C-like protease [Acute Acute Respiratory Syndrome Coronavirus 2], preferably at least 55%, more preferably at least 60%, even more preferably 70%, more preferably at least 75%, even more preferably 80%, even more than 85%, even more preferably at least 90%, even more preferably at least 95%, preferably at least 97%, and most preferably at least 98%, said 3C-like protease having accession number YP_009725301, version number YP_009725301.1, 18-JUL-2020 NCBI Protein Database GenPept [2021-03-26]; available from https: / / www.ncbi.nlm.nih.gov / protein / 1802476809.

[0156] Preferably, the virus-encoded protease is a protease having at least 50% sequence similarity at positions 3241-3546 to the amino acid sequence of a 3C-like protease (3CL pro; R1A_SARS), preferably at least 55%, more preferably at least 60%, even more preferably 70%, even more preferably at least 75%, even more preferably 80%, even more than 85%, even more preferably at least 90%, even more preferably at least 95%, preferably at least 97%, and most preferably at least 98%, said 3C-like protease having accession number P0C6U8 (Uniprot), which is integrated into UniProtKB / Swiss-Prot: Sequence Update: June 10, 2008, Last Modified: February 10, 2021, Version 104 [2021-03-26]; available from https: / / www.uniprot.org / uniprot / P0C6U8.

[0157] Preferably, the virus-encoded protease has at least 50% sequence similarity to the amino acid sequence of the 3C protease [human rhinovirus A1], preferably at least 55%, more preferably at least 60%, even more preferably 70%, more preferably at least 75%, even more preferably 80%, even more than 85%, even more preferably at least 90%, even more preferably at least 95%, preferably at least 97%, and most preferably at least 98%, said 3C protease has accession number YP_009508983.1, version number YP_009508983.1, 24-AUG-2018 NCBI Protein Database GenPept [2021-03-26]; https: / / www.ncbi.nlm.nih.gov / protein / YP_009508983.

[0158] Preferably, the virus-encoded protease has at least 50% sequence similarity to the amino acid sequence of the PEDV main protease [swine epidemic diarrhea virus], preferably at least 55%, more preferably at least 60%, even more preferably 70%, more preferably at least 75%, even more preferably 80%, even more than 85%, even more preferably at least 90%, even more preferably at least 95%, preferably at least 97%, and most preferably at least 98%, said PEDV main protease having accession number 4XFQ_A, PDB database, stored on 28-12-2014, version 1.0 2016-1-20 [2021-03-26]; https: / / www.rcsb.org / structure / 4XFQ .

[0159] The term "% sequence similarity" refers to the percentage of amino acid residues with similar physicochemical properties that are allowed to be conservatively substituted within a defined alignment length in a given alignment between two amino acid sequences. The percentage of similarity can be readily determined by those skilled in the art, for example, according to well-known methods, such as Curr Protoc Bioinformatics. 2013; 43:3.5.1–3.5.9.

[0160] Preferably, the method is more specific to viral infection than to host inflammatory response. Preferably, the method is selective for specific viruses or viral families.

[0161] Preferably, the viral infections are selected from: meningitis, gastrointestinal infections, summer influenza, hand-foot-mouth disease, poliovirus 3C poliovirus, common cold, allergic asthma exacerbation, encephalitis, hepatitis A (chronic jaundice), equine encephalitis, smallpox, rubella (German measles), respiratory infections, pediatric bronchiolitis, viral pneumonia, acute respiratory syndrome, swine diseases, yellow fever, hepatitis C, diseases of pigs, cattle and sheep, acute upper respiratory tract, eye and intestinal infections, herpes (systemic and local) infections, rabbit hemorrhagic diseases, potato diseases and plant diseases.

[0162] Preferably, the viral protease is a 3C protease and the infection is selected from meningitis, gastrointestinal infection, summer influenza, hand-foot-mouth disease, poliovirus 3C polio, common cold, allergic asthma attack and encephalitis.

[0163] Preferably, the viral protease is a 3C-like protease and the infection is selected from pediatric bronchiolitis, viral pneumonia and acute respiratory syndrome (SARS), COVID-19, porcine epidemic diarrhea virus, feline infectious peritonitis, canine coronavirus infection, and calf enteritis.

[0164] Preferably, the viral protease is a serine protease and the infection is selected from the following: smallpox, equine encephalitis, respiratory infections, yellow fever, hepatitis C, and diseases of pigs, cattle, and sheep.

[0165] Viral proteases are preferably proteases involved in the protein hydrolysis of viral polypeptides.

[0166] Preferably, the method detects infection, wherein the virus (pathogen) is selected from: the Picornaviridae, Coronaviridae, Adenoviridae, Coronaviridae, and combinations thereof; most preferably, the method detects infection wherein the virus (pathogen) is a Coronaviridae virus. Even more preferably, the method detects infection wherein the virus (pathogen) is SARS, preferably SARS-CoV and / or SARS-CoV-2, even more preferably SARS-CoV-2 (2019nCoV). The peptide defined herein has the advantage of enabling rapid detection of virus-encoded proteases in patient samples.

[0167] The embodiments described for the diagnostic peptides, with appropriate modifications, are applicable to aspects of the present invention concerning methods / uses of peptides and kits / systems comprising said peptides.

[0168] In another aspect of the invention, a method for viral infection of an in vitro diagnostic subject is described, the method comprising the following steps:

[0169] i) Contacting a body fluid / tissue sample with a peptide comprising a fluorescent agent having an emission wavelength of 650-900 nm, a non-fluorescent agent having an absorption wavelength of 650-900 nm for quenching the emission of the fluorescent agent, and a cleavage site located between the fluorescent agent and the first non-fluorescent agent, the cleavage site being specifically targeted at a viral protease.

[0170] ii) Monitor the fluorescence of the peptide from step i) in the 650-900 nm range.

[0171] Increased fluorescence in the 650-900 nm range indicates the presence of virus in the sample.

[0172] Preferably, the body fluid sample is blood, saliva, sputum, bronchoalveolar fluid, tissue biopsy section, and / or tears. Preferably, the body fluid sample is saliva.

[0173] The sample may be present on a swab or other collection device. Preferably, the sample is a bodily fluid / tissue sample. The collection device is preferably a swab.

[0174] Preferably, the method includes the step of contacting the collection device containing the body fluid / tissue sample with a buffer solution in the container.

[0175] Optionally, the collection device is a container, preferably a vial or bottle for receiving saliva or sputum.

[0176] Optionally, the method includes the step of centrifuging the sample to separate solid matter and / or precipitated biological material from the supernatant. Preferably, the centrifugal force is in the range of 1,000 g to 25,000 g, more preferably in the range of 3,000 g to 20,000 g, and more preferably in the range of 3,700 g to 18,000 g. The centrifugation time is preferably in the range of 1 minute to 30 minutes, in the range of 2 to 20 minutes, and more preferably in the range of 3 to 10 minutes. The supernatant or aggregate produced by centrifugation is preferably in contact with a reagent. In a preferred embodiment, the supernatant from centrifugation is preferably in contact with a reagent. In another preferred embodiment, the aggregate produced by centrifugation is preferably in contact with a reagent.

[0177] Optionally, after adding the reagent to the sample, the mixture is stirred. It is preferred to stir the mixture by pipetting, vortexing, or shaking.

[0178] In a particularly preferred embodiment, no cell enrichment step is performed on the sample before it comes into contact with the reagent. "No cell enrichment step" means that the sample is not incubated at 37°C for several hours to increase the viral load. In other words, the sample can be directly transferred from the collection device to the container in which the reagent is added.

[0179] The sample is preferably diluted in a assay buffer, preferably in the range of 1:10 to 1:10000, more preferably 1:100 to 1:1000.

[0180] Preferably, the assay buffer can be maintained in a pH range of 5-9, more preferably about 6 to about 8, and more preferably about 6.5 to about 7.5. Preferably, the buffer includes HEPES, PIPES, Tris-hydrochloride (Tris-HCl), or MOPS.

[0181] Preferably, the sample is in contact with the lysis agent. More preferably, the buffer solution contains a detergent capable of lysing cellular material in the body fluid sample. Preferably, the buffer solution contains one or more nonionic detergents selected from: N-octyl-D-glucopyranoside, N-octyl-D-maltodextrin, ZWITTERGENT. 3.14. Deoxycholate; n-Dodecanoyl sucrose; n-Dodecyl-D-glucopyranoside; n-Dodecyl-D-maltoside; n-Octyl-D-glucopyranoside; n-Octyl-p-D-maltoside; n-Octyl-p-D-thioglucopyranoside; n-Decanoyl sucrose; n-Decano-p-D-maltoside; n-Decano-p-D-thioglucopyranoside; n-Heptyl-D-glucopyranoside; n-Heptyl-p-D-thioglucopyranoside; n-Hexyl-D-glucopyranoside; n-Nonyl-p-D-glucopyranoside; n-Octayl sucrose; n-Octyl-D-glucopyranoside; n-Undecyl-D-maltoside; APO-10; APO-12; Big CHAP; Big CHAP, deoxy; 35; d2E5; d2E6; Ci2E8; Ci2E9; cyclohexyl-n-ethyl-p-D-maltose; cyclohexyl-n-hexyl-p-D-maltose; cyclohexyl-n-methyl-D-maltose; digitalis saponins; ELUGENT TM ; C-100; X-080; X-100;HECAMEG;MEGA-10;MEGA-8;MEGA-9;NOGA;NP-40; F-127; X-100; Xl 14; 20; or Mixtures of 80 or higher.

[0182] The buffer solution preferably contains an ionic detergent selected from the following: BATC, cetyltrimethylammonium bromide, chenodeoxycholic acid, cholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholic acid, glycolithocholic acid, lauroyl sarcosine, taurine chenodeoxycholic acid, tauronic acid, taurine dehydrocholic acid, tauronic lithocholic acid, tauroursodeoxycholic acid, TOPPA, and mixtures thereof.

[0183] Preferably, the buffer solution contains a zwitterionic detergent selected from the following: aminosulfobetaine, CHAPS, CHAPSO, carboxybetaine, and methylbetaine.

[0184] Preferably, the buffer solution comprises an anionic detergent selected from the following: SDS, N-lauryl sarcosine, sodium deoxycholate, alkyl-aryl sulfonates, long-chain (fatty) alcohol sulfates, olefin sulfates and sulfonates, α-olefin sulfates and sulfonates, sulfated glyceryl monoesters, sulfated ethers, sulfosuccinates, alkane sulfonates, phosphate esters, alkyl hydroxyethyl sulfonates, sucrose esters, and mixtures thereof.

[0185] The step of monitoring the increase in fluorescence in step iii) is preferably performed in a detector suitable for receiving a container containing the sample and reagents. The detector preferably provides readings in relative fluorescence units (RFU). The user can compare the readings with a control, and the relative increase in RFU between the control and the sample indicates viral infection.

[0186] Preferably, the virus is as described above. Preferably, the virus belongs to the family Picornaviridae or Coronaviridae.

[0187] The viral protease is preferably selected from: hepatitis-encoded serine proteases or metalloproteinases (NS2 and NS3), rhinovirus genome-encoded cysteine ​​proteases (3C and 2A), coronavirus genome-encoded cysteine ​​proteases (3CL), adenovirus genome-encoded serine-centered neutral proteases, and retrovirus-encoded aspartic proteases. Preferably, the coronavirus protease is a cysteine ​​protease encoded by the hSARS coronavirus genome or the SARS coronavirus 19 main protease (EC number 3.4.22.69). More preferably, the viral protease is a 3C-like (3CL) protease.

[0188] Preferably, a method for viral infection of in vitro diagnostic subjects is described herein, the method comprising the following steps:

[0189] i) Contact a saliva sample with a peptide comprising a fluorescent agent having an emission wavelength of 650-900 nm, a non-fluorescent agent having an absorption wavelength of 650-900 nm for quenching the emission of the fluorescent agent, and a cleavage site located between the fluorescent agent and the first non-fluorescent agent, the cleavage site being specifically targeted at a 3C-like protease.

[0190] ii) Monitor the fluorescence of the peptide from step i) in the 650-900 nm range.

[0191] The increase in fluorescence in the 650-900 nm range indicates viral infections selected from the following: pediatric bronchiolitis, viral pneumonia and acute respiratory syndrome (SARS), COVID-19, porcine epidemic diarrhea virus, feline infectious peritonitis, canine coronavirus infection, and calf enteritis. Preferably, the viral infection is acute respiratory syndrome (SARS) or COVID-19, and more preferably, COVID-19.

[0192] In other aspects, a kit for diagnosing viral infections in subjects is provided, comprising:

[0193] a) A container containing the diagnostic peptide of formula (I),

[0194] b) A set of instructions for performing the diagnostic methods defined herein.

[0195] In another aspect, a system for diagnosing viral infections in subjects is provided, comprising:

[0196] a) A container used to receive samples.

[0197] b) A container containing the diagnostic peptide of formula (I),

[0198] c) A device suitable for receiving a container and monitoring the fluorescence signal emitted from the peptide when it comes into contact with a body fluid sample.

[0199] The preferred embodiments of the in vitro methods for diagnosing viral infections described herein have been appropriately modified for use with the kits described herein.

[0200] On the other hand, a system for diagnosing viral infections in subjects is provided, which includes:

[0201] a) A container used to receive samples.

[0202] b) A container containing the diagnostic peptide of formula (I),

[0203] c) A device suitable for receiving a container and monitoring the fluorescence signal emitted from the peptide when it comes into contact with a body fluid sample.

[0204] The preferred embodiment of the method for detecting viral infection described herein has been appropriately modified for use in the system described herein.

[0205] Preferably, the device is a near-infrared spectrophotometer. Preferably, the device is a point-of-care device.

[0206] The invention has been described above with reference to several exemplary embodiments. Modifications and alternative implementations of some parts or elements are possible and are included within the scope of protection defined in the appended claims. Example

[0207] Example 1 - Detection of SARS-CoV-19

[0208] Samples containing SARS-CoV-19 virus were contacted with a fluorescent quenching peptide (TVRLQSGF), which comprises a fluorescent agent with an emission wavelength of 650-900 nm and a non-fluorescent agent with an absorption wavelength of 650-900 nm, as well as a cleavage site specifically targeting the coronavirus (Ex 1.1). The negative control (scrambled) peptide was FQVLRS (Ex 1.2).

[0209] Measurements were taken at 0, 1, 2, and 4 hours after reagent contact using a handheld fluorometer (DeNiro NIR Fluorimeter, DetactDiagnostics BV, Netherlands). The RFU of the control sample (buffer) was subtracted from each measurement.

[0210] An increase in RFU or samples was detected, but no increase was detected in the negative control.

[0211] Example 2

[0212] The synthetic peptides were purchased from CRB (Billingham, UK). The fluorescent agent was 10Cy7 (Cy7 DBCO, from ClickChemistry Tools, Arizona, USA), and the non-fluorescent agent was QC-1 (QC-1NHSester, LICOR, Nebraska, USA).

[0213] The peptide stock solution in ultrapure water was added to the recombinant SARS-CoV-2 main protease (Mysourcebio, California, USA) to give a final concentration of 5 μM peptide. The concentration of the SARS-CoV-2 main protease varied between 0.2–2 μM. Buffer 30 used consisted of 20 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and pH 7.3. Fluorescence was monitored at 0, 5, 15, 30, and 45 minutes after adding the peptide solution. Fluorescence was monitored using a DeNIRO NIR detector (DetactDiagnostics, Groningen, Netherlands).

[0214] As controls, subtilisin (Merck, Netherlands) and elastase (Sigma Aldrich, Netherlands) were used. The concentration and buffer of the control protease were the same as those used in the experiment with the SARS-CoV-2 main protease.

[0215] Calculate the relative activity using the following formula:

[0216]

[0217] The results are shown in Table 1 below:

[0218] Table 1

[0219]

[0220]

[0221] The ratio of active units of the SARS-CoV-2 main protease to elastase provides a measure of a compound's specificity for a particular protease. The ratio of active units is shown in the figure below:

[0222] Table 2

[0223]

[0224] Example 3 – Mass Spectrometry Characterization

[0225] Peptides 1–5 were analyzed using RP-HPLC-MS. Mass spectra were obtained on an Agilent Technologies 6530 Precision Mass Q-TOF LC / MS equipped with an Agilent Infinity 1260 / 1290 HPLC system. RP-HPLC was performed using an RP-aqueous C30 analytical column, 5 μM, 150 × 2.0 mm [Phenomenex]. The mobile phases were A) 10 mM ammonium formate / 2% v / v MeCN, pH 6.2, and B) 10 mM ammonium formate / 90% v / v MeCN, pH 6.2.

[0226] The gradient used was 0.2 mL-min, 25 minutes.

[0227] Each peptide solution in Table 3 was incubated at 0.1 mg / mL with SARS-CoV-2 main protease (10 μM).

[0228] Table 3

[0229]

[0230]

[0231] Cleavage of peptide 1 occurred between QS. Cleavage of peptide 2 occurred between QS. Cleavage of peptide 3 was not observed.

[0232] A solution of each peptide in Table 4 at 0.1 mg / mL was incubated with elastase (2 μM). The results are shown in Table 4.

[0233] Table 4

[0234]

[0235] Example 4 – Clinical Data

[0236] Clinical samples that had been characterized as positive or negative for SARS-CoV-2 by PCR were used (Corman et al., Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. EuroSurveill. 2020; 25(3): pii=2000045)(StreekLab, Haarlem, The Netherlands). Buffer A was PBS, pH 7.2.

[0237] The substrate stock solution was prepared by adding 1 mL of buffer A to a final concentration of 0.1 mg / mL (26 μM).

[0238] Blank (peptide solution only)

[0239] Add 50 μL of peptide stock solution to 200 μL of buffer A and vortex.

[0240] 'Blank' was measured on DeNIRO.

[0241] Sample measurement

[0242] Add 100 μL of buffer A to 500 μL of Eppendorf, vortex the sample vial, remove 100 μL of sample and add it to the Eppendorf. Add 50 μL of peptide stock solution (final concentration 20 μM). Vortex the Eppendorf for 2 × 5 seconds and immediately measure fluorescence on a DeNIRO. Then place the Eppendorf at room temperature and repeat the measurement at 5, 10, 15, 30, and 60 minutes. The results are shown in Table 5. A relative activity >10% indicates the presence of SARS-CoV-2(+), and a relative activity <10% indicates the absence of SARS-CoV-2(-).

[0243] Table 5

[0244]

[0245] Example 5 – SARS-CoV-2 Test

[0246] Peptides 1 and 2, as described in Example 2, and peptide 4 (Cy7-Ahx-[PEG1]-FHT-[PEG1]K[(QC1)-CONH2) were tested in nasopharyngeal (NP) samples from individuals suspected of being infected with SARS-CoV-2. The samples were previously used to run PCR tests to detect the presence of infection. The samples were NP swabs in M4 virus transfer medium. All samples were stored at 4°C for a maximum of 48 hours prior to analysis.

[0247] Confirmatory PCR was performed on all samples according to the method described in Corman et al., Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020; 25(3): pii=2000045. https: / / doi.org / 10.2807 / 1560-7917.ES.2020.25.3.2000045. Positive samples had CT values ​​ranging from 16 to 33.8. One sample with a CT > 35 was not included.

[0248] Peptide stock solutions were prepared by dissolving peptides 1, 2, and 4 in buffer A (PBS, pH 7.2) to a final concentration of 61.86 μM.

[0249] Samples were monitored using a DeNIRO near-infrared fluorometer (DetactDiagnostics 5 BV, Netherlands).

[0250] Negative control: peptide solution only

[0251] 1. Add 81 μL of peptide stock solution (1, 2, or 4) to 169 μL of buffer A, vortex, and measure the “blank” in DeNiro.

[0252] Sample measurement [Total volume of sample + buffer + substrate in Eppendorf solution = 250 μL]

[0253] 1. Add 69 μL of buffer A to 0.5 mL of Eppendorf solution.

[0254] 2. Briefly vortex the Copan swab bottle to remove 100 μL of sample solution and add Eppendorf solution.

[0255] 3. Add 81 μL of CoviTact peptide stock solution to Eppendorf (final concentration of 20 μM peptide).

[0256] 4. Vortex 2×5 seconds

[0257] 5. Measure immediately at [0 minutes] and at 5 minutes.

[0258] The results are presented in Table 6.

[0259] Table 6

[0260]

[0261] Table 7 shows the positive and negative predictive values, which are calculated using the following formula:

[0262] Positive predictive value = 100 * TP / (TP + FP)

[0263] Negative predictive value = 100 * TN / (TN + FN)

[0264] Total Consistency Percentage = 100 * (TP + TN) / Total Number

[0265] Table 7

[0266]

[0267]

[0268] Peptide 2 according to the invention shows a high (approximately 90%) correlation with the presence / absence of confirmed infection.

[0269] Table 8 compares the peptide content of the peptides according to the present invention with those of the prior art.

[0270] Table 8

[0271]

[0272] - Not observed; + Observed. sequence list <110> Oriquino G Ltd. <120> Diagnostic peptides in methods, kits, and systems for diagnosing viral infections <130> P6092836PCT <150> EP20191741.6 <151> 2020-08-19 <150> EP20166722.7 <151> 2020-03-30 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 1 Val Leu Gln Ser 1 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 2 Val Leu Asn Ser 1 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 3 Ala Ala Phe Ala Ala 1 5

Claims

1. The use of the peptide represented by the following formula in the preparation of a kit for in vitro detection of SARS-CoV-2 virus in saliva samples: [a]-[Connector 1]- HQS-[Connector 2]-[c] in: [a] is a fluorescent agent with an emission wavelength of 650-900 nm. [Linker 1] and [Linker 2] are independently selected from hydrocarbon groups and substituted hydrocarbon groups. HQS stands for histidine-glutamine-serine peptide. [c] is a non-fluorescent agent with an absorption wavelength of 650-900 nm, which is capable of quenching the emission of the fluorescent agent.

2. The use as claimed in claim 1, wherein the hydrocarbon group and the substituted hydrocarbon group are selected from β-alanine, 4-aminobutyryl, 2-(aminoethoxy)acetyl, 3-(2-aminoethoxy)propyl, 5-aminopentanoyl, 6-aminohexyl, 8-amino-3,6-dioxaoctyl and 12-amino-4,7,10-trioxadodecyl.

3. The use as described in claim 1 or 2, wherein the kit further comprises a assay buffer for diluting the sample.

4. The use as described in claim 3, wherein the assay buffer is formulated to achieve a dilution factor ranging from 1:10 to 1:10000.

5. The use as described in claim 4, wherein the dilution ratio ranges from 1:100 to 1:1000.

6. The use as described in claim 1, wherein the kit further comprises a lysis agent.

7. A system for in vitro detection of SARS-CoV-2 virus in saliva samples, comprising: a) A container for receiving saliva samples. b) A container containing a peptide represented by the following formula: [a]-[connector 1]-HQS-[connector 2]-[c], in: [a] is a fluorescent agent with an emission wavelength of 650-900 nm. Linker 1 and linker 2 are independently selected from hydrocarbon groups and substituted hydrocarbon groups. HQS stands for histidine-glutamine-serine peptide. [c] is a non-fluorescent agent with an absorption wavelength of 650-900 nm, which is capable of quenching the emission of the fluorescent agent; c) An apparatus adapted to receive the container and monitor the fluorescence signal emitted from the peptide when the peptide comes into contact with the saliva sample.

8. Peptides represented by the following formula: [a]-[Connector 1]-HQS-[Connector 2]-[c] in: [a] is a fluorescent agent with an emission wavelength of 650-900 nm. HQS stands for histidine-glutamine-serine peptide. [c] is a non-fluorescent agent with an absorption wavelength of 650-900 nm, used to quench the emission of the fluorescent agent. [Linker 1] and [Linker 2] are independently selected from hydrocarbon groups and substituted hydrocarbon groups.

9. The peptide of claim 8, wherein the hydrocarbon group and the substituted hydrocarbon group are selected from β-alanine, 4-aminobutyryl, 2-(aminoethoxy)acetyl, 3-(2-aminoethoxy)propyl, 5-aminopentanoyl, 6-aminohexyl, 8-amino-3,6-dioxaoctyl and 12-amino-4,7,10-trioxadodecyl.

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