FR3123658A1 - METHOD FOR DETECTING A CORONAVIRUS IN REPLICATION - Google Patents

Abstract

The invention relates to an in vitro method for determining whether a sample contains a replicating coronavirus, said method comprising the detection of a Papain-like protease activity (PLpro) of coronavirus.

Description

METHOD FOR DETECTING A CORONAVIRUS IN REPLICATION

Field of invention

The invention relates to a rapid test for detecting the presence of a coronavirus in a sample.

State of the art

The international community is currently facing a pandemic of acute respiratory syndrome due to the coronavirus designated SARS-CoV-2. The diagnostic test by RT-PCR from nasopharyngeal swabs is the most widespread test for detecting the presence of this virus at the individual or population level. However, due to its technical characteristics (detection of the presence of RNA), this test cannot be used to detect a person in real time, whether they are symptomatic or not, but contagious. Other individual tests exist, based on immunochromatography techniques or on gene amplification techniques, but they do not provide an answer concerning the immediate contagiousness of the person.

Risk perception and risky behaviors become important variables in predicting the spread of the pandemic. Providing affordable, reliable and easy-to-use tools remains a challenge to assess the real risk at the individual level.

Systematic and high-performance rapid tests are essential to reduce the medical and economic impact of Covid-19.

In this context, the inventors now propose a rapid and reliable test for detecting the contagious risk.

More generally, the invention relates to an in vitro method for determining whether a sample contains a replicating coronavirus, in particular a SARS-Cov-2, said method comprising the detection of a Papain-like protease activity (PLpro) of coronavirus.

For this, the method preferably comprises bringing the sample into contact with at least one substrate peptide of the protease PLpro, preferably labeled in a detectable manner. Preferably, the activity of PLpro is revealed by a fluorescence test.

Another preferred object of the invention is an in vitro method for determining whether a human subject is infected with a coronavirus, in particular a SARS-Cov-2, and is contagious, said method comprising bringing a biological sample of said subject, such as a saliva sample, with at least one PLpro protease substrate peptide, preferably labeled detectably with a fluorophore, said peptide comprising or consisting of the sequence X ₁ LX ₂ GG, where X ₁ and X ₂ are any amino acids, identical or different, the peptide preferably consisting of the sequence RLRGG.

The test reveals the presence of PLpro in less than 30 minutes.

tricks

is a graph which shows the fluorescence assay of the activity of PLpro on the Z-RLRGG-AMC substrate, in comparison with the activity of PLpro on the ISG15-AMC substrate for a period of 60 minutes. The concentrations of the z-RLRGG-AMC peptide were increased respecting an enzyme/substrate ratio of 1/200.

is a graph which reports the measurement of the fluorescence emitted by the labeled substrate peptide in the presence of human saliva (3 samples) and of PLPro and of Triton X100, for a period of 30 minutes. Each saliva was tested in triplicate and the means and standard deviations are represented.

Claims (15)

An in vitro method for determining whether a sample contains a replicating coronavirus, said method comprising detecting papain-like protease activity (PLpro) of coronavirus. A method according to claim 1, comprising contacting the sample with at least one preferably detectably labeled PLpro protease substrate peptide. A method according to claim 2, wherein the substrate peptide comprises the sequence X ₁ LX ₂ GG, where X ₁ and X ₂ are any amino acids, the same or different. A method according to claim 3, wherein the substrate peptide consists of the sequence X ₁ LX ₂ GG, where X ₁ and X ₂ are any amino acids, the same or different. Process according to claim 3 or 4, wherein X ₂ is a basic amino acid, preferably arginine (R). A method according to claim 5, wherein the peptide consists of the sequence RLRGG. Method according to one of Claims 1 to 6, in which the detection of the PLpro activity is demonstrated by fluorescence measurement, the said method preferably comprising bringing the sample into contact with at least one substrate peptide of the PLpro protease carrying a fluorophore. Method according to claim 7, comprising bringing the sample into contact with at least one substrate peptide of the protease PLpro, said substrate peptide bearing a fluorophore at one end, and a protective group at the other. Method according to one of Claims 7 or 8, comprising bringing the sample into contact with at least one substrate peptide of the protease PLpro bearing a fluorophore, the fluorophore being an aminomethyl coumarin (AMC) group, preferably carried at the C-terminus of the substrate peptide. A method according to claim 9, wherein the substrate peptide is Z-RLRGG-(AMC), where (AMC) is aminomethyl coumarin, and Z is a carboxybenzyl group. Method according to one of Claims 1 to 6, in which the detection of the PLpro activity is demonstrated by colorimetric measurement. Method according to one of Claims 1 to 11, in which the coronavirus is a SARS-CoV-2. Method according to one of Claims 1 to 12, in which the sample is a sample of saliva. In vitro method for determining whether a human subject is infected with a coronavirus and is contagious, said method comprising the detection of a papain-like protease activity (PLpro) of coronavirus by bringing a biological sample of said subject into contact with at least a PLpro protease substrate peptide, preferably labeled in a detectable manner with a fluorophore, said peptide consisting of the sequence X ₁ LX ₂ GG, where X ₁ and X ₂ are any amino acids, identical or different, the peptide preferably consisting of the sequence RLRGG. A method according to claim 14, wherein the sample is a saliva sample.