FR3116065A1 - Detection of nucleic acids in a biological sample - Google Patents

Abstract

The present invention relates to an in vitro method for the release, detection and/or quantification of nucleic acids from a biological sample, comprising the following steps: a) lysis and protein denaturation of the sample by a protease, b) inactivation of the protease with an inhibitor of said protease, c) optionally amplification of the nucleic acids present in the lysate, and d) optionally detection and/or quantification of said nucleic acids.

Description

Detection of nucleic acids in a biological sample

FIELD OF THE INVENTION

The present invention relates to the field of technologies for detecting nucleic acids in a biological sample, in particular unpurified nucleic acids.

STATE OF THE ART

Nucleic acid detection techniques, with or without amplification, have been developed in many fields of biology.

In the vast majority of cases, these detection techniques (e.g. hybridization) preceded or not by amplification (e.g. PCR) require the release of nucleic acids by lysis followed by prior purification of the nucleic acids. This purification step complicates the implementation of the technique. In the past, there has therefore been interest in the development of direct detection techniques on the biological sample, without prior purification of the nucleic acids. The biological sample is subjected to a simple lysis step allowing the release of nucleic acids before amplification (optional) and detection and/or quantification of nucleic acids.

Nucleic acids can be released from a sample by heating at high temperature (eg: 95-98°C) and/or by chemical lysis (eg: sodium hydroxide), and/or by enzymatic lysis (eg: proteinase K).

Proteinase K is an enzyme of the protease family, widely used in nucleic acid extraction processes in biology and more particularly in molecular biology to release nucleic acids from histones, or from their envelope (viral capsid for example) , degrade the proteins in the medium that can digest and degrade the contaminants of nucleic acid preparation (DNase or RNase). Proteinase K degrades proteins by cutting peptide bonds at hydrophobic or aromatic amino acids. It is thus very often used in the preparation of nucleic acids, as a preliminary step to PCR techniques and in particular in the context of direct PCR where it makes it possible to clean the media.

“Direct PCR” is a technique for amplifying a target sequence (nucleic acids) directly from a sample, without prior purification, required for conventional PCR. This technique is faster than conventional PCR and is used in particular for gene detection and high-throughput screening. Direct PCR kits on samples generally include a lysis buffer specific to the treated sample and a PCR mix containing enzymes capable of amplifying nucleic acids from an unpurified extract.

In the case of PCR amplification with release of nucleic acids by lysis then purification of the nucleic acids, proteinase K is eliminated during the purification phase.

In the case of direct PCR amplification where there is no purification step, the proteinase K must be inactivated before the amplification step of these nucleic acids (for example: PCR, RT-PCR, qPCR, RT-qPCR, digital PCR), otherwise it would degrade the enzymes (e.g. taq polymerase or RT reverse transcriptase) needed to perform PCR amplification.

The conventional use of a heating step to inactivate proteinase K is known from the prior art, for example a temperature between 75° C. and 100° C. for a duration between 10 min and 1 h depending on the type of proteinase K. We will speak of inactivation by heating. But this heating step has drawbacks: it requires high-temperature heating equipment, generates a loss of time in the overall process, a loss of sensitivity (fragile nucleic acids, in particular RNAs, can be degraded by too strong heat), and metrological uncertainties when handling a large number of samples.

There is therefore still a need to develop a new method for inactivating the enzymes present in the lysis buffer of a sample, in particular for inactivating proteinase K, which would also make it possible not to alter, in particular in the case of an amplification of nucleic acids before the detection step, the functionality of the enzymes of the PCR amplification step (eg: Taq and RT).

The Applicant has precisely demonstrated that the use of a protease inactivating compound (eg: proteinase K) at the sample lysis step, made it possible to meet this need and could in particular be used before enzymatic amplification of the acids nuclei without prior purification (e.g. direct PCR).

Unexpectedly, the Applicant has in fact shown that it was possible to successfully combine inactivation of proteinase K by an inhibitor compound and enzymatic amplification of the nucleic acids present in the digestate (lysate) after inactivation, without altering the enzymes required for PCR amplification, and without loss of sensitivity compared to conventional heat inactivation.

This new method, according to the invention, of inactivation of the protease by an inhibiting compound present in the lysis buffer, has many advantages compared to the conventional method of heat inactivation:

• Time saving between 10 min and 1 hour (no more need for heating);
• Reduced cost: no more need for heating equipment; And
• No loss of nucleic acid by heating (no loss of sensitivity due to inactivation of proteinase K, unlike the sensitivity of certain RNAs at high temperatures).

The invention relates, according to a first aspect, to an in vitro method for the release, detection and/or quantification of nucleic acids from a biological sample, comprising the following steps:

1. lysis and protein denaturation of the sample by a protease,
2. inactivation of the protease by an inhibitor of said protease,
3. optionally amplification of the nucleic acids present in the lysate, and
4. optionally detection and/or quantification of said nucleic acids.

Another object of the invention is a kit or assembly for implementing an in vitro method for the release, detection and/or quantification of nucleic acids from a sample, comprising at least:

• reagents for the release of nucleic acids by lysis,
• a serine protease, preferably proteinase K,
• a serine protease inhibitor selected from the group consisting of phenylmethylsulfonyl fluoride (PMSF), aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF), and mixtures thereof, preferably phenylmethylsulfonyl fluoride (PMSF)
• optionally a proteinase K substrate, preferably bovine serum albumin; and/or a nonionic surfactant, and/or an RNase inhibitor, preferably an RNasine,
• optionally additionally a colorant,
• optionally additionally reagents for the amplification of said nucleic acids and/or the amplification of housekeeping genes (endogenous control) and/or genes of other control pathogens (exogenous control), and/or optionally additionally reagents for the detection of said nucleic acids

The invention also relates to the use of a protease inhibitor, in particular a serine protease inhibitor, preferably selected from the group consisting of phenylmethylsulfonyl fluoride (PMSF), aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF ), proteinase K inhibitor, and mixtures thereof, preferably phenylmethylsulfonyl fluoride (PMSF), to inactivate the protease present in a sample lysate intended in particular for amplification by PCR and/or detection and/or quantification of acids nuclei present in the sample.

Other characteristics, objects and advantages of the invention will emerge from the description which follows, which is purely illustrative and not limiting.

DETAILED DESCRIPTION OF THE INVENTION

A first object of the invention is therefore an in vitro method for the release, detection and/or quantification of nucleic acids from a biological sample, comprising the following steps:

a) lysis and protein denaturation of the sample by a protease,

b) inactivation of the protease by an inhibitor of said protease,

c) optionally amplifying the nucleic acids present in the lysate, and

d) optionally detection and/or quantification of the nucleic acids.

According to a first embodiment, the in vitro method of the invention comprises the following steps:

1. lysis and protein denaturation of the sample by a protease,
2. inactivation of the protease by an inhibitor of said protease, and
3. optionally detection and/or quantification of nucleic acids.

According to another particular and preferred embodiment, the in vitro method of the invention comprises the following steps:

a) lysis and protein denaturation of the sample by a protease,

b) inactivation of the protease by an inhibitor of said protease,

c) amplification of the nucleic acids present in the lysate, and

d) optionally detection and/or quantification of the amplified nucleic acids.

The in vitro method according to the invention comprises a step of releasing the nucleic acids from the sample, by simple lysis, without purification.

According to a particular mode, the method of the invention comprises a step c) of amplification by PCR, in particular by real-time direct PCR coupled with hydrolysis probes or coupled with HMR (high resolution melting) analysis or digital PCR or isothermal or any other nucleic acid amplification technique.

Direct PCR is a technique for amplifying these nucleic acids directly from a sample, without prior purification.

By “nucleic acids”, we mean in particular the genomic DNAs and RNAs (endogenous) of the subject and/or the DNAs and RNAs of pathogens having infected the subject (exogenous).

In particular, the nucleic acids detected (optionally amplified) according to the method of the invention are genomic nucleic acids of the subject tested, or nucleic acids of pathogens having infected the subject tested or an environmental medium, in particular DNAs and/or RNAs of bacteria, viruses or parasites. We will later speak of 'target nucleic acids' or 'target sequences', as opposed to 'control sequences', exogenous or endogenous for validation of the method.

According to a particular mode, these are nucleic acids of the subject tested, for example to detect and/or quantify mutations and/or other anomalies correlated with physiological or pathological disorders.

According to a particular mode, they are nucleic acids of pathogens having infected the subject, for example nucleic acids of viruses, bacteria, or parasites, to detect and/or quantify a bacterial or viral load.

'The biological sample' according to the invention may in particular be an environmental sample or a sample from an animal or human subject, in particular a sample of bodily fluid, a sample of tissue or an organ, or an eluate from a swab or any other medium for testing the environment.

According to a particular mode, the biological sample is a sample from a subject, in particular a sample of bodily fluid chosen from the group consisting of blood and its derivatives (e.g. serum, plasma, leukocyte fractions, in particular serum) , urine, saliva, faeces, mucous secretions, nasopharyngeal or oropharyngeal swabs, bronchoalveolar fluids, trans-tracheal aspirates, or a tissue, organ or biopsy sample, or an eluate from rag.

The “tested subject” according to the invention may be an animal or a human.

According to a particular mode, the subject is an animal chosen from the group consisting of ruminants, horses, birds, pigs, or pets.

According to another particular mode, the subject is a human.

It can be a healthy subject or a subject affected or likely to be affected by a physiological or pathological disorder.

According to a particular mode, it is a subject affected by a physiological or pathological disorder, such as a cancer, an inflammatory disease, an infectious disease, an autoimmune disease.

In particular, the subject tested is a subject infected or likely to be infected by a pathogen, in particular a virus or a bacterium.

As viruses, and without this list being exhaustive, mention may be made in particular of coronaviruses, such as SARS-COV-2, 229 ^E , OC43, HKU1, NL63 (human coronaviruses), as well as the bovine viral diarrhea virus or the african swine fever virus. Mention may also be made of other human respiratory viruses, such as influenza virus, parainfluenza virus, metapneumovirus, respiratory syncitial virus, rhinovirus, Adenovirus, Bocavirus, and Enterovirus.

As bacteria, and without this list being limiting, mention may in particular be made of human respiratory bacteria such as M.pneumniae , C.pneumoniae , B.pertussis , B.parapertussis , and L. pneumophila .

a) Step of lysis and protein denaturation of the sample by a protease

Proteases or peptidases have the function of cleaving proteins or peptides by hydrolyzing their peptide bonds.

According to a particular and preferred mode, a serine protease is used.

Serine proteases (EC 3.4.21) have in their active site a serine residue which plays an essential role in catalysis.

Among the serine proteases, mention may be made in particular of:

• Trypsin
• Chymotrypsin
• Proteinase K.

Thus, according to a particular embodiment of the invention, the protease used in step a) is chosen from the group consisting of serine proteases, in particular proteinase K, trypsin or chymotrypsin, preferably proteinase K.

According to a particular and preferred mode, a proteinase K is used in the step of lysis and denaturation of the sample to be tested.

Proteinase K is commonly used in molecular biology to digest proteins and remove contaminants from nucleic acid preparations. It is used to digest cells and extract nucleic acids (DNA or RNA) from mammalian cells or microorganisms.

The protease will usually be present in the lysis buffer.

The protease content, in particular proteinase K, will generally range from 0.01 mg/mL to 1 mg/mL, preferably from 0.04 mg/mL to 0.35 mg/mL of reaction medium (lysis buffer).

A person skilled in the art will adjust the protease content according to the nature of the sample and volume of the reaction medium.

The lysis buffer will generally include other ingredients, such as a pH regulating agent, for example a TRIS (tris(hydroxymethyl)aminomethane) buffer, and/or a reducing agent such as B mercapto-ethanol or dithiothreitol ( DTT) and/or a nucleic base analogue capable of inhibiting RNases such as vanadyl or vanadyl ribonucleoside complex (VRC).

Commercial buffer solutions dedicated to the preparation of nucleic acids, in particular for PCR amplification, can be used. Mention may be made in particular of the buffer solutions (pH 8.0) marketed by Fisher and Eurobio in particular.

The step of protein lysis and denaturation of the sample by a protease can be carried out at room temperature or under heat.

According to a particular mode, the lysis step is carried out without heating.

According to a particular and preferred mode, the lysis step is carried out with heating at a temperature ranging from 50° C. to 70° C., for a period ranging from 15 minutes to 1 hour.

At the end of this step, a lysate is obtained containing the nucleic acids to be amplified (optional) and detected.

Inactivation of protease, in particular proteinase K, is necessary especially in the case of amplification so that it does not interfere with the enzymes of the PCR amplification reaction.

b) Step of inactivation of the protease by an inhibitor of said protease

By “inhibitor” or “enzyme inhibitor” according to the invention, is meant in particular a compound which binds to an enzyme and which reduces its activity. The inhibitor can prevent the binding of the substrate to the active site by binding in its place, or cause a deformation of the enzyme rendering it inactive.

Binding of an inhibitor can prevent a substrate from binding to the active site of the enzyme and/or the enzyme from catalyzing its reaction. This inhibition may be reversible or irreversible. Irreversible inhibitors attach covalently to the enzyme and modify key amino acid residues necessary for enzyme activity. In contrast, reversible inhibitors bind non-covalently and different types of inhibition result depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both.

In the context of the present invention, a protease inhibitor is used.

The protease inhibitor will usually be present in the stop buffer, but it can be in a separate buffer.

According to a preferred mode, the protease inhibitor is in the stop buffer.

By 'stop buffer', otherwise known as 'inactivation buffer' , is meant according to the invention a buffer intended to inactivate the protease, that is to say to prevent its proteolytic activity at the end of the lysis step, and before the nucleic acid amplification step.

The protease inhibitor can be a chemical compound or a peptide compound.

According to a particular embodiment, it is a peptide compound.

As an example, mention may in particular be made of tetrapeptidyl chloromethyl ketone, otherwise known as Proteinase K inhibitor from Calbiochem. This peptide of formula MeOSuc-Ala-Ala-Pro-Phe-CH₂Cl acts as an irreversible inhibitor at the active site of proteinase K.

According to another particular and preferred mode, it is a chemical compound.

As examples of chemical compounds that can be used in the context of the invention, mention may be made in particular of compounds of the benzenesulfonyl fluoride type, their amino derivatives (e.g. benzenesulfonyl fluoride substituted, in particular on the phenyl ring, by an amino-(C1- C6) alkyl) and their salts, dialkylfluorophosphates (e.g. di(C1-C6) alkylfluorophosphate), and in particular:

• phenylmethylsulfonyl fluoride (PMSF), also called benzenesulfonyl fluoride, (CAS Number 329-98-6), which is a serine protease inhibitor which binds covalently to the active site of the protease, preventing its proteolytic activity;
• 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) and its alone, in particular in the form of its hydrochloride (CAS Number 30827-99-7), which is an irreversible serine protease inhibitor. It also inhibits trypsin, and chymotrypsin;
• diisopropylfluorophosphate (DFP);

preferably PMSF or AEBSF, more preferably PMSF.

Thus, according to a particular embodiment of the invention, the protease inhibitor used in step b) of the process of the invention is a serine protease inhibitor chosen from the group consisting of phenylmethylsulfonyl fluoride (PMSF), aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF), and mixtures thereof, preferably phenylmethylsulfonyl fluoride (PMSF).

By way of example, the PMSF from the company Merck or the AEBSF from the company Merck are used.

The concentration of protease inhibitor used in the method of the invention may range from 0.1 mM to 30 mM, in particular from 0.5 mM to 20 mM, and in particular from 0.5 mM to 15 mM.

A person skilled in the art will be able to adjust the concentration of protease inhibitor according to its nature and the nature and concentration of the target protease.

According to a particular embodiment, the protease is a proteinase K and the protease inhibitor is chosen from PMSF and AEBSF, preferably PMSF.

According to a particular mode, for a proteinase K concentration of 0.35mg/mL, a concentration ranging from 20mM to 60mM, preferably from 30mM to 50mM of PMSF and ranging from 6.5mg/mL to 13mg/mL of AEBSF.

This chemical inactivation step of the protease is generally carried out at room temperature.

Lure protein (optional)

According to a particular embodiment of the invention, the method of the invention further comprises bringing the lysate into contact with a decoy protein

By “decoy protein” according to the invention, is meant a substrate protein of the protease, making it possible to eliminate the residual proteolytic activity of the protease in the reaction medium.

As a substrate for the protease, in particular proteinase K, it is possible to use any protein that does not inhibit PCR, in particular plasma proteins, in particular albumin, preferably bovine serum albumin (BSA for Bovine Albumin Serum ), or collagens, for example porcine collagen.

Thus, according to a particular embodiment of the invention, the inactivation step b) further comprises bringing the lysate into contact with a substrate of said protease, in particular a plasma protein or a collagen, preferably a plasma protein such as than albumin, more preferably bovine serum albumin (BSA).

Preferably, a plasma protein is used, more preferably an albumin such as BSA.

The decoy protein concentration will generally range from 4mg/mL to 20mg/mL, preferably from 6mg/mL to 10mg/mL of reaction medium.

Those skilled in the art will know how to adjust the decoy protein concentration according to the concentration of the target protease.

The decoy protein can be present in the Stop Buffer (Inactivation Buffer) or in a separate buffer added before or after the Stop Buffer.

According to a preferred mode, the decoy protein is in the stop buffer.

Surfactant (optional)

According to a particular mode of the invention, the method of the invention further comprises bringing the lysate into contact with a surfactant chosen from the group consisting of nonionic surfactants, anionic surfactants, cationic surfactants, zwitterionic surfactants, and mixtures thereof.

This surfactant will facilitate the release of nucleic acids, before the amplification step, thus improving the sensitivity of the lysis process.

According to a particular embodiment, the surfactant is a nonionic surfactant.

As a nonionic surfactant, mention may be made in particular of octylphenoxypolyethoxyethanol (Triton ^TM X100), nonylphenol ethoxylate (Tergitol ^TM of the NP-40 type) or polysorbates, in particular polysorbate 20 otherwise called Polyethylene glycol sorbitan monolaurate (CAS Number: 9005-64-5). Mention may be made, for example, of polysorbate 20 marketed under the name ^TWEEN® 20 by the company Merck.

Thus, according to a particular mode of the invention, the inactivation step b) of the process of the invention further comprises bringing the lysate into contact with a nonionic surfactant, in particular an octylphenoxypolyethoxyethanol (Triton ^TM X 100 ), a nonylphenol ethoxylate (Tergitol ^TM type NP-40) or a polysorbate, such as polysorbate 20.

The content of surfactant used in the process according to the invention will generally range from 0.1% to 20% by weight relative to the weight of the reaction medium.

Those skilled in the art will know how to adapt the surfactant content according to its nature and the target sequence.

For example, for a polysorbate 20, a content of 5% to 20%, in particular 8% to 15% by weight relative to the weight of the reaction medium, may be used.

For an octylphenoxypolyethoxyethanol (Triton ^TM X 100), it is possible to use a content of 1% to 10%, in particular 2% to 5% by weight relative to the weight of the reaction medium.

For a nonylphenol ethoxylate (Tergitol ^TM of the NP-40 type), a content of 0.1% to 5%, in particular 0.2 to 1% by weight relative to the weight of the reaction medium, may be used.

The surfactant can be present in the Stop Buffer or in a separate buffer, used before, with or after the Stop Buffer.

Preferably, the surfactant is in the Stop Buffer.

RNA protectors (optional)

According to a particular embodiment of the invention, the method of the invention further comprises bringing the lysate into contact with an RNA protector.

By ‘RNA protector’, we mean an agent that prevents the degradation of RNAs. Mention may in particular be made of RNase inhibitors, in particular RNasine.

The RNasine content used in the process according to the invention will generally range from 0.05U/μl to 25U/μl by weight relative to the weight of the reaction medium.

The RNA protecting agent can be present in the Stop Buffer or in a separate buffer, used before, with or after the Stop Buffer.

Preferably, the RNA protecting agent is in the stop buffer.

According to a particular mode, step b) of inactivation also comprises bringing the lysate into contact with:

- a substrate of said protease, in particular a plasma protein or a collagen, preferably a plasma protein such as albumin, more preferably bovine serum albumin (BSA), and/or

- a surfactant chosen from the group consisting of nonionic, anionic, cationic, zwitterionic surfactants, and mixtures thereof, in particular a nonionic surfactant, in particular an octylphenoxypolyethoxyethanol, a nonylphenol ethoxylate, or a polysorbate such as polysorbate 20 , and or

- an RNase inhibitor, in particular RNasine.

Thus, the stop buffer according to the invention advantageously comprises:

• a protease inhibitor as described above, in particular to inactivate proteinase K,
• a decoy protein as described above, to eliminate the residual proteolytic activity of the protease in the reaction medium,
• a surfactant as described above, to facilitate ultimate release of the nucleic acids from their protein environment, and
• an RNase inhibitor as described above, in particular an RNasine, and
• advantageously additionally a dye, in particular a non-PCR inhibiting dye

The presence of a dye makes it possible to facilitate the implementation of the method of the invention.

According to a particular and preferred embodiment of the invention, the in vitro method for releasing, detecting and/or quantifying nucleic acids from a sample from a subject comprises the following steps:

1. lysis and protein denaturation of a sample chosen from the group consisting of a salivary sample, an oropharyngeal or nasopharyngeal sample, with a proteinase K, in particular implemented at a temperature of 50 to 60°C for 15 to 60 minutes,
2. inactivation of proteinase K by adding phenylmethylsulfonyl fluoride (PMSF), advantageously additionally bovine serum albumin (BSA), advantageously additionally a nonionic surfactant, and advantageously additionally an RNasine,
3. optionally amplification of the nucleic acids present in the lysate, and
4. optionally detection and/or quantification of nucleic acids.

c) Amplification step (optional) of the nucleic acids present in the lysate

This step of amplification of target sequences and control sequences is carried out by well-known PCR methods (e.g.: qPCR, RT-PCR, digital PCR, isothermal PCR, etc.), in the presence of enzymes (e.g.: Taq polymerase and RT -transcriptase).

By 'target sequences' or 'target nucleic acids' is meant genomic nucleic acids of the subject tested, or nucleic acids of pathogens having infected the subject tested, in particular DNAs and/or RNAs of bacteria, viruses, parasites or mushrooms.

According to a particular mode, these are nucleic acids of the subject tested, for example to detect and/or quantify mutations and/or other anomalies correlated with physiological or pathological disorders.

According to a particular mode, they are nucleic acids of pathogens having infected the subject, for example nucleic acids of viruses, bacteria or parasites, to detect and/or quantify a bacterial or viral load.

The amplification step may require the use of control, endogenous and/or exogenous sequences.

By “control sequences”, we mean in particular:

• endogenous sequences such as housekeeping genes, characterized by their intrinsic presence in all the cells of the sample in a constitutive way. Mention may be made in particular of the genes coding for highly conserved proteins such as the beta 2 microglobulin protein or the actin protein and in particular the beta form (we speak of 'endogenous control') and/or
• exogenous sequences such as genes of pathogens other than the one sought but very close in nature to the target; examples include mengovirus, also known as Columbia SK virus, mouse Elberfield virus and encephalomyocarditis virus, a non-pathogenic virus for humans serving as an exogenous control for RNA viral targets; for bacterial DNA targets, we can cite, for example, the sequence of a non-pathogenic microorganism such as Mycobacterium smegmatis or E.coli (we speak of 'exogenous control').

Endogenous controls validate the presence and integrity of the sample during all phases of the analytical process. They make it possible to validate the presence of cells, the correct handling and release of nucleic acids from the sample and to verify the absence of PCR inhibitors.

Exogenous controls are added to the sample during the analytical process and make it possible to validate the effectiveness and smooth running of the nucleic acid release and amplification phases.

A person skilled in the art will know how to adapt the method according to the invention (presence or absence of endogenous and/or exogenous control sequences) depending on the target and the purpose (detection and/or quantification method, diagnostic method, etc.).

According to a particular embodiment, the method of the invention allows co-amplification, in particular in the same well, of target sequences (e.g. target genomic sequence of the subject or sequence of a pathogen likely to have infected said subject), and control sequences.

Thus, the method according to the invention, preferably for direct PCR, comprises:

- a target sequence amplification system comprising sense and antisense primers for amplification of the “target sequences” described above and specific probes, advantageously labeled with fluorophores, for detecting the amplified target sequences; And

- optionally a system for amplification of endogenous (household genes) and exogenous (sequences of another pathogen) control sequences comprising sense and antisense primers for amplification of said control sequences and specific probes, advantageously labeled with fluorophores, of detection of said amplified control sequences.

Those skilled in the art will know how to choose the specific sense and antisense primers for the target sequences to be amplified, as well as the specific probes suitable for the detection of the amplified target and control sequences.

According to a particular mode, the method comprises:

- a system for the amplification of viral sequences of avian coronavirus (Infectious bronchitis virus, IBV) comprising sense and antisense primers for the amplification of said viral sequences and specific probes, advantageously labeled with fluorophores, for detecting the amplified target sequences; And

- optionally a system for amplifying exogenous control sequences (e.g. mengovirus sequences) comprising sense and antisense primers for amplifying said control sequences and specific probes, advantageously labeled with fluorophores, for detecting said amplified control sequences.

The kit for the implementation of such a method for detecting IBV may also comprise a reagent comprising the inactivated and calibrated exogenous virus (mengovirus) to be added constantly to each sample before the start of the method.

Commercially available reagents, primers and probes suitable for PCR amplification can be used, following the supplier's recommendations.

d) Step detection and/or quantification of nucleic acids, amplified or not

This detection and/or quantification step of the target sequences, whether amplified or not, is carried out by methods well known to those skilled in the art.

For direct detection (without amplification), mention may be made of hybridization methods well known to those skilled in the art (e.g. Northern blot, dotblot, etc.).

For detection with prior amplification (e.g. Taqman amplification, isothermal amplification, digital amplification), mention may be made of fluorescence detection methods well known to those skilled in the art.

set

The present invention also relates to a kit or assembly for implementing a method according to the invention, comprising at least:

• reagents for the release of nucleic acids by lysis;
• a serine protease, preferably proteinase K,
• a serine protease inhibitor selected from the group consisting of phenylmethylsulfonyl fluoride (PMSF), aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF), and mixtures thereof, preferably phenylmethylsulfonyl fluoride (PMSF),
• optionally additionally a proteinase K substrate, preferably bovine serum albumin (BSA) and/or a nonionic surfactant, and/or an RNase inhibitor, preferably an RNasine,
• optionally additionally a colorant,
• optionally reagents for the amplification of said nucleic acids and/or the amplification of housekeeping genes (endogenous control) and/or genes of other control pathogens (exogenous control), and/or
• optionally reagents for the detection of said nucleic acids.

Serine protease is usually present in the lysis buffer.

The serine protease inhibitor is usually present in the stop buffer.

The proteinase K substrate and the nonionic surfactant can be present in the stop buffer or in a separate buffer.

Thus, according to a particular embodiment, the kit or assembly according to the invention comprises at least:

• A lysis buffer comprising proteinase K,
• A stop buffer comprising:
  • a serine protease inhibitor preferably selected from the group consisting of phenylmethylsulfonyl fluoride (PMSF), aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF), and mixtures thereof, preferably phenylmethylsulfonyl fluoride (PMSF)
  • a proteinase K substrate, preferably bovine serum albumin (BSA)
  • a nonionic surfactant, preferably an octylphenoxypolyethoxyethanol, a nonylphenol ethoxylate, or a polysorbate such as polysorbate 20,
  • optionally additionally an RNase inhibitor, preferably an RNase and
  • optionally additionally a colorant.

According to a particular embodiment, the kit or assembly according to the invention comprises:

• A lysis buffer comprising a proteinase K (from 0.04mg/mL to 0.08mg/mL)
• A stop buffer comprising PMSF (20mM to 40mM) + BSA (3.25mg/mL to 6.5 mg/mL) + commercial buffer pH 8.0 + a nonionic surfactant such as Tween ^® 20 (5% to 10%) or alternatively Triton ^TM X-100 (1.25% to 2.5%) or Tergitol ^TM of the NP-40 type (0.25% to 0.5%), optionally an RNasine, and advantageously in addition a colorant.

A person skilled in the art will know how to adjust the contents of reagents, in particular the contents of serine protease, serine protease inhibitor, decoy protein and nonionic surfactant, depending on the nature of the sample and the nucleic acids to be detected. (genomics, pathogens).

The invention also relates to the use of a protease inhibitor, in particular a serine protease inhibitor, preferably selected from the group consisting of phenylmethylsulfonyl fluoride (PMSF), aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF ), proteinase K inhibitor, and mixtures thereof, preferably phenylmethylsulfonyl fluoride (PMSF), to inactivate the protease present in a sample lysate intended in particular for amplification and/or detection and/or quantification of the nucleic acids present in the sample.

FIGURES

qPCR results of PK inhibition test by heating and alternatively by a protease inhibitor (PMSF).

Evaluation of PCR amplification (fluorescence measurement) with and without PK inactivation.

Evaluation of a PCR amplification (fluorescence measurement) with or without PK, on a nasopharyngeal swab sample positive for SARS-Cov-2.

Evaluation of PCR amplification (fluorescence measurement) with inactivation by heating or chemical inactivation of PK, on a nasopharyngeal swab sample positive for SARS-Cov-2.

qPCR results of the PK inhibition test with PMSF in the presence or absence of BSA.

qPCR results of the PK inhibition test with PMSF, BSA and in the presence or absence of Tween 20.

qPCR results of the PK inhibition test with PMSF in the presence of reaction mixes of Taq and RT enzymes.

qPCR results of PK inhibition test with AEBSF.

The invention will be illustrated in the following non-limiting examples.

EXAMPLES

Example 1: Effect of a Proteinase K (PK) Inhibitor Compared to Conventional PK Inhibition by Heating

The technical effect of the efficiency of proteinase K (PK) inactivation by an inhibitor compound compared to conventional inactivation by heating has been demonstrated on a purified RNA model of avian coronavirus (Infectious bronchitis virus, IBV). The objective here was to demonstrate that the PK inhibitor indeed inactivated PK as well as conventional inactivation by heating at 95°C for 10 minutes can do.

For this we carried out a PCR amplification of the RNA of the purified IBV virus, according to different comparative conditions:

- without heating (Control),

- after heating at 95°C for 10 minutes (heating effect on RNA detection),

- after heating at 95°C for 10 min in the presence of PK (impact of heating on the inactivation of PK),

- with addition of 10 mM or 16 mM of a PK inhibitor, phenylmethylsulfonyl fluoride (PMSF), in the presence of PK (impact of the inhibitor on the inactivation of PK).

The results are presented in the and in Table 1 below:

Method cq RNA alone 24.82 RNA alone 95°C 10’ 25.10 RNA+ PK 95°C 28.71 RNA + PK + PMSF
10mM 28.61 RNA + PK + PMSF
10mM 26.55

The C _q (threshold cycle) corresponds to the intersection between an amplification curve and a threshold line. It corresponds to a relative measure of the concentration of the target in the PCR reaction.

“Cq” corresponds to a Nucleic Acid detection sensitivity value. Thus, the earlier the Cq, the more material there is (Nucleic Acid at the start) and therefore the greater the load of the pathogen in the patient tested. The Cq will therefore be one of the main data in molecular biology for establishing a relative quantification of the “infectious load”.

These results show that heating the RNA for 10 min does not induce loss of Cq, therefore no loss of material.

On the other hand, heating in the presence of PK induces a shift in the detection of RNA reflecting a loss of material, the PK therefore does not seem to be completely inactivated by heating at 95°C for 10 min.

The addition of a PK inhibitor (PMSF) at 10mM provides RNA detection equivalent to conventional heating inactivation. This indicates that the presence of PMSF at this concentration is sufficient to inactivate PK, as well as classical inactivation by heating. Moreover, when the concentration of PMSF is increased, it is observed that the detection is even closer to the control alone, indicating that the PK is inactivated even better than by conventional inactivation by heating.

The use of an inhibitor is therefore advantageous, in a condition of protein lysis and denaturation of a sample with a protease (e.g. PK), compared to conventional inactivation by heating.

Example 2: Necessary inactivation of PK before PCR amplification

Example 2-1: Amplification and Detection of Extracted IBV Viral RNA

The interest of inactivating PK before using it in PCR has been demonstrated here.

To do this, we used an IBV viral RNA already extracted, and added PK according to 3 operating conditions: PK inactivated by heating (comparative), or non-inactivated PK (control) or PK in the presence of 10mM PMSF (invention). Then we amplified this viral RNA by RT-qPCR.

The first sample contains a defined quantity of extracted IBV RNA to which PK has been added at a concentration of 0.3 mg/mL, inactivated by heating at 95° C. for 10 minutes (comparative). For the defined amount of IBV RNA, a 1/3000 dilution is used ^which comes out at a Cq of 24-25.

The second sample contains the same concentration of extracted IBV RNA, but the PK (same concentration) is not inactivated (control).

The third sample still contains the same concentration of RNA and PK, but this time the PK is inactivated by a stopping solution containing PMSF alone 10 mM (invention).

5 µL of each sample is added to the 15 µL of reaction mix (ID Gene™ Infectious Bronchitis Duplex from IDvet) and the RNA amplification by RT-qPCR is carried out according to the supplier's recommendations:

ID Gene™ Infectious Bronchitis Duplex from IDvet

Booster Program:

10 mins at 50°C 2 mins at 95°C 10 sec at 95°C 40 steps 30 sec at 60°C

*fluorescence acquisition. The results are presented in the .

It is observed that no amplification is detected in the case where Proteinase K is not inactivated (control), whereas the RNA is well detected in the two other cases (presence of a characteristic curve). The results clearly show us that in the absence of inactivation (by an inhibiting compound or by heating), proteinase K cannot in any case be used with a PCR technique because it degrades the enzymes necessary for carrying out the reaction.

Example 2-2: Amplification and detection of a SARS-Cov-2 viral RNA

We used supernatants from nasopharyngeal swabs that tested positive for the SARS-CoV-2 coronavirus and showed the interest of a chemical inactivation of the PK compared to an inactivation by heating.

Initially, we tested comparatively the conditions of heating or treatment with PK:

• Simple heating without proteinase K (PK) at 56°C 30min
• Simple heating without proteinase K (PK) at 95°C 10min;
• Addition of a lysis buffer with PK (0.04mg/mL) then incubation for 30min at 56°C
• Addition of a lysis buffer with PK then incubation for 30 min at 56°C at room temperature.

Then we amplified this viral RNA by RT-qPCR.

For the defined amount of SARS-Cov-2 viral RNA, a supernatant sample of nasopharyngeal swabs from a patient previously tested by purification and amplification and found to be positive at a Cq of 18-19 is used.

5 µL of each sample is added to the 8 µL of reaction mix and the RNA amplification by RT-qPCR is carried out according to the supplier's recommendations (see amplification program below).

For each amplification reaction, 0.4 µM of each of the primers and 0.2 µM of probe are used. The Master Mix and the Reverse Transcriptase (RT) are added according to the concentrations recommended by the supplier: Capital™ qRT-PCR Probe Mix 4X from Biotechrabbit (Ref: BR0502003):

• for 'without PK' conditions: direct sampling of 5µl after heating;
• for the conditions 'with PK': sampling of 20μl after heating with PK and addition of 5μl of stop buffer then sampling of 5μl and addition of 8μl of mix for PCR.

Booster Program:

10 mins at 50°C 2 mins at 95°C 10 sec at 95°C 40 steps 30 sec at 60°C

*fluorescence acquisition (fluorophore FAM= fluorescein).

The results are presented in the following table 4 and at .

Method Cq (FAM) Heating 56°C 30min without PK 23.1 Heating 56°C 30min with PK 19.9 Heating 95°C 10 min without PK 22.0 TA 30 min incubation with PK 19.5

It is observed that the addition of PK is of real interest to release earlier signals and therefore gain in sensitivity compared to simple heating.

In a second step, the conditions of inactivation of the PK were comparatively tested by heating or by treatment with a chemical inhibitor according to the invention (PMSF 4 mM), before the PCR amplification step on the protocol described above. .

The results are presented in the following table 5 and at .

Method Cq (FAM) Heating 56°C 30min with PK + chemical blocking 19.9 Heating 56°C 30min with PK + heat blocking 23.8 TA 30 min incubation with PK + chemical blocking 19.5 TA incubation 30 min with PK + heat blocking 22.7

It is observed that the use of a chemical blocking makes it possible to detect the signal earlier and therefore to be more sensitive in the detection of the samples.

Example 3: Effect of adding a PK decoy protein to the PK inhibitor

The objective here was to demonstrate that the addition of a "decoy" protein such as bovine serum albumin (BSA) makes it possible to inactivate the residual activity of the remaining PK, and thus to reduce the quantity of PMSF at add to completely inactivate PK activity.

For this, we amplified by PCR purified IBV RNA alone (expected detection control), purified RNA with 10 mM or 16 mM PMSF in the presence of PK (control inactivation by an inhibitor compound) and purified RNA with 10 mM PMSF or 16mM in the presence of PK + BSA at 4mg/ml or 8mg/ml.

The results are presented in the and the following table 6:

PMSF 10mm cq cq PMSF 16mM RNA alone 24.82 24.82 RNA alone RNA alone 95°C 10’ 25.1 25.1 RNA alone 95°C 10’ RNA + PK
PMSF 10mM 28.61 26.55 RNA + PK
PMSF 16mM RNA + PK
PMSF 10mM+
BSA 4mg/mL 25.41 25.38- 26.18 RNA + PK
PMSF 16mM+
BSA 4mg/mL RNA + PK
PMSF 10mM+
BSA 8mg/mL 25.01 25.09 RNA + PK
PMSF 16mM+
BSA 8mg/mL

In the presence of PMSF at 10mM with PK at 0.3mg/ml, the signal is not restored to 100% (Cq later) which means that the PK is not entirely inactivated. When the amount of PMSF is increased, it is observed that the delay of Cq becomes weaker, which indicates that the increase in PMSF makes it possible to better inactivate PK.

But too high concentrations of PMSF can interfere with the PCR enzymes and harm the PCR amplification step. The addition of a decoy protein (here BSA) restores the detection of the signal to a Cq equivalent to the control (RNA alone), which indicates that it makes it possible to eliminate the residual activities of the PK, thus protecting PCR enzymes. The more BSA is added, the greater the restoration of the signal.

These results show that it may be advantageous to combine a decoy protein such as BSA with a PK inhibitor such as PMSF, to obtain total inactivation of PK, at lower levels of PMSF.

Example 4: Effect of adding a nonionic surfactant to the PK inhibitor

The effect of adding a non-ionic surfactant such as polysorbate-20 (Tween 20) to the Stop Buffer was evaluated in this example. The objective was to show that it allowed better release of viral RNA and thus improve the lysis process, before the amplification step.

A dry swab of 25µl of nasopharyngeal swab supernatant positive for sars-cov2 was prepared and is then taken up in the lysis buffer (TE + PK 0.08 mg/mL) then heated at 56°C for 30min. A volume of 20 μl of the lysate thus obtained was inactivated with 5 μl of a stop buffer containing Tween 20 (PMSF 40 mM / BSA 6.5 mg/mL / Tween 20 10%) or a stop buffer of similar composition but without Tween 20 (PMSF 40 mM / BSA 6.5 mg/mL).

The results are presented in the .

It is observed that by adding Tween 20 in the stop buffer, there is a gain in sensitivity resulting in the obtaining of an earlier Cq in PCR.

These results show that the addition of a non-ionic surfactant in the stop buffer promotes the release of the viral RNA not yet extracted, and thus improves the sensitivity of the lysis process.

Example 5: Use of PK inactivated by PMSF with different Taq and RT PCR enzymes

Different Taq and RT (with their respective buffers) were tested. We amplified the extracted viral RNA alone (control) or in the presence of different doses of PK (0.12mg/mL and 0.24mg/mL) and constant doses of PMSF inhibitor (8mM) and BSA (1. 3mg/mL).

For each amplification reaction, 0.4 µM of each of the primers and 0.2 µM of probe are used. The Master Mix and the Reverse Transcriptase (RT) are added according to the concentrations recommended by the supplier(s).

In particular, we tested the following Taq and RT mixes:

Mix A: Capital™ qRT-PCR Probe Mix 4X from Biotechrabbit (Ref: BR0502003)

And Mix B: Lyoready 1-Step RT-qPCR Mix 2X from Meridian-Bioline (Ref: MDX024)

The results are presented in the .

PK is indeed inactivated since the signal observed with mixes A and B is equivalent to the signal without PK.

These results show that the use of a PK inactivated by PMSF does not alter the PCR amplification reactions in the presence of Taq and RT enzymes.

Those skilled in the art will know how to adjust the operating conditions (Taq, RT and inactivated PK levels) depending on the nature of the Taq and RT mixes marketed and the recommendations of the suppliers.

Example 6: Effect of AEBSF on PK Inactivation

PK is used at a typical concentration of 0.35 mg/mL.

Different concentrations of AEBSF were tested and a concentration of 1mg/mL was chosen to carry out the PCR amplification in the presence of extracted RNA (viral RNA IBV) to identify its possible inhibitory effect on the Taq and RT enzymes of the amplification PCR.

The results are shown in Table 7 below and in .

Method cq RNA alone 24.93 RNA + PMF BSA 24.82 RNA+ AEBSF
1mg/mL 27.23

10mM PMSF corresponds to 1.742 mg/mL based on the following calculation:

PMSF molar mass = 174.2 g/mol

0.01 mol/L * 174.2 g/mol = 1.742 g/L PMSF = 1.742 mg/mL

These results show that AEBSF, like PMSF, is another effective PK inhibitor compatible with PCR amplification (does not alter PCR enzymes).

Example 7: Implementation of a method according to the invention

7-1: Amplification and detection of IBV viral RNA

In this example, an attempt was made to detect avian coronavirus (Infectious bronchitis virus, IBV) nucleic acids in a nasopharyngeal swab. The process consisted of:

1/ Collect the swab supernatant to be tested and add the lysis buffer containing proteinase K (0.08mg/mL) inside

3/ Vortex 30s

4/ Centrifuge for a few seconds to bring all of the sample to the bottom of the tube

5/ Heat the sample for 30min at 56°C

6/ Vortex

7/ Centrifuge for a few seconds to bring all of the sample to the bottom of the tube

8/ Homogenize the stop solution (stop buffer comprising 40mM of PMSF, 6.5mg/mL of BSA and 10% of Tween 20 or 2.5% of Triton X-100 or 0.5% of Tergitol ^TM of type NP-40) delicately by suction-refoulement

9/ In a dilution pre-plate (or in eppendorf tubes), dispense 5µl of stop buffer per well of lysed sample

10/ Take 20µl of the lysate supernatant (after heating) and mix them delicately by suction/discharge with 5µl of stopping solution.

11/ In a plate or PCR strips, distribute the reaction mix according to the supplier's amplification protocol (ID Gene™ Infectious Bronchitis Duplex from IDvet)

12/ Add 5µl of the inactivated lysate

13/ Perform the PCR according to the supplier's recommendations.

7-2: Amplification and detection of SARS-Cov-2 viral RNA

In this example, we sought to detect nucleic acids of human coronavirus (SARS-Cov-2) in a nasopharyngeal swab. The process consisted of:

1/ Collect the swab supernatant to be tested and add the lysis buffer including proteinase K (0.04mg/ml in the presence of the sample)

3/ Vortex 30s

4/ Centrifuge for a few seconds to bring all of the sample to the bottom of the tube

5/ Heat the sample for 30min at 56°C

6/ Vortex

7/ Centrifuge for a few seconds to bring all of the sample to the bottom of the tube

8/ Homogenize the stop solution (stop buffer comprising 20mM of PMSF, 3.25mg/mL of BSA and 5% of Tween 20 or 1.25% of Triton X-100 or 0.25% of Tergitol ^TM type NP-40) delicately by suction-repression,

9/ In a dilution pre-plate (or in eppendorf tubes), dispense 5µl of stop buffer per well of lysed sample,

10/ Take 20µl of the lysate supernatant (after heating) and gently mix them by suction/discharge with 5µl of stop solution

11/ In a plate or PCR strips, distribute 8µl of a mix composed of 0.4 µM of each of the primers and 0.2 µM of probe. The Master Mix and the Reverse Transcriptase (RT) are added according to the concentrations recommended by the supplier: Capital™ qRT-PCR Probe Mix 4X from Biotechrabbit (Ref: BR0502003)

12/ Add 5µl of the inactivated lysate,

13/ Perform the PCR according to the supplier's recommendations.

Claims (11)

Processin vitrofor releasing, detecting and/or quantifying nucleic acids from a biological sample, comprising the following steps:

1. lysis and protein denaturation of the sample by a protease,
2. inactivation of the protease by an inhibitor of said protease,
3. optionally amplification of the nucleic acids present in the lysate, and
4. optionally detection and/or quantification of said nucleic acids.

Process according to Claim 1, characterized in that it comprises a stage c) of amplification of the said nucleic acids by PCR, in particular by direct PCR or by digital or isothermal PCR. Process according to Claim 1 or Claim 2, characterized in that the protease used in step a) of lysis is chosen from the group consisting of serine proteases, in particular proteinase K, trypsin or chymotrypsin, from preferably proteinase K. Process according to any one of Claims 1 to 3, characterized in that the protease inhibitor used in stage b) of inactivation is a serine protease inhibitor chosen from the group consisting of phenylmethylsulfonyl fluoride (PMSF ), aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF), and mixtures thereof, preferably phenylmethylsulfonyl fluoride (PMSF). Process according to any one of Claims 1 to 4, characterized in that step b) of inactivation also comprises bringing the lysate into contact with:
- a substrate of said protease, in particular a plasma protein or a collagen, preferably a plasma protein such as albumin, more preferably bovine serum albumin (BSA), and/or
- a surfactant chosen from the group consisting of nonionic, anionic, cationic, zwitterionic surfactants, and mixtures thereof, in particular a nonionic surfactant, in particular an octylphenoxypolyethoxyethanol, a nonylphenol ethoxylate, or a polysorbate such as polysorbate 20 , and or

• an RNase inhibitor, in particular RNasine.

Method according to any one of Claims 1 to 5, characterized in that the biological sample is a sample from a subject, in particular from an animal or from a human, in particular a sample of bodily fluid chosen from the group constituted by blood and its derivatives, urine, saliva, stools, mucous membrane secretions, nasopharyngeal or oropharyngeal swabs, broncho-alveolar fluids, trans-tracheal aspirations or a sample of tissue, organ or biopsy, or a swab eluate. Method according to any one of Claims 1 to 6, characterized in that the nucleic acids detected from the lysate are genomic nucleic acids of the subject tested, or nucleic acids of pathogens having infected the subject tested, in particular DNAs and /or RNAs of bacteria, viruses, parasites or fungi. Method according to any one of Claims 1 to 7, characterized in that it comprises the following steps:
a) lysis and protein denaturation of a sample chosen from the group consisting of a salivary sample, an oropharyngeal or nasopharyngeal sample, with a proteinase K, in particular carried out at a temperature of 50 to 60° C. for 15 to 60 minutes ,
b) inactivation of proteinase K by adding phenylmethylsulfonyl fluoride (PMSF), advantageously additionally a bovine serum albumin (BSA), advantageously additionally a nonionic surfactant, and advantageously additionally an RNasin ,
c) optionally amplifying the nucleic acids present in the lysate, and
d) optionally detection and/or quantification of the nucleic acids. Kit or assembly for implementing a method according to any one of Claims 1 to 8, characterized in that it comprises at least:

• reagents for the release of nucleic acids by lysis,
• a serine protease, preferably proteinase K;
• a serine protease inhibitor preferably selected from the group consisting of phenylmethylsulfonyl fluoride (PMSF), aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF), and mixtures thereof, preferably phenylmethylsulfonyl fluoride (PMSF);
• optionally further a proteinase K substrate, preferably bovine serum albumin (BSA); and/or a nonionic surfactant, and/or an RNase inhibitor, preferably an RNasine,
• optionally a colorant,
• optionally additionally reagents for the amplification of said nucleic acids and optionally the amplification of housekeeping genes (endogenous control) and/or genes of other control pathogens (exogenous control) and/or
• optionally additionally reagents for the detection of said nucleic acids.

Kit or assembly according to claim 9, characterized in that it comprises at least:

• A lysis buffer comprising proteinase K,
• A stop buffer comprising:
  • a serine protease inhibitor preferably selected from the group consisting of phenylmethylsulfonyl fluoride (PMSF), aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF), and mixtures thereof, preferably phenylmethylsulfonyl fluoride (PMSF)
  • a proteinase K substrate, preferably bovine serum albumin (BSA)
  • a nonionic surfactant, preferably an octylphenoxypolyethoxyethanol, a nonylphenol ethoxylate, or a polysorbate such as polysorbate 20,
  • optionally additionally an RNase inhibitor, preferably an RNase and
  • optionally additionally a colorant.

Use of a protease inhibitor, in particular a serine protease inhibitor, preferably selected from the group consisting of phenylmethylsulfonyl fluoride (PMSF), aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF), proteinase K inhibitor, and their mixtures, preferably phenylmethylsulfonyl fluoride (PMSF), to inactivate the protease present in a sample lysate intended in particular for amplification by PCR and/or detection and/or quantification of the nucleic acids present in the sample.