Multiplex PCR method for detecting microorganisms and its use - Patent Application 20070122997

Abstract

The present invention relates to a method for amplifying at least two different nucleic acid sequences present in a sample using primer pair (1) and primer pair (2). In particular, the first primer of primer pair (2) also contains the sequence of the first primer of primer pair (1) at the (5') position, and the second primer of primer pair (2) also contains the sequence of the second primer of primer pair (1) at the (5') position. The present invention also relates to a kit and a detection kit capable of carrying out the method and their use.

Description

[Technical Field]

[0001] The present invention relates to the field of detection and amplification of at least one nucleic acid of interest, particularly in infection diagnosis. Accordingly, the present invention relates to a method for amplifying at least two different nucleic acid sequences present in a sample using primer pair 1, which amplifies a first nucleic acid sequence (A), and at least one hybrid or chimeric primer pair 2, which amplifies a second nucleic acid sequence (B). In particular, the first primer, such as the sense primer of primer pair 2, further comprises the sequence of the first primer of primer pair 1 at its 5' end, and the second primer of primer pair 2 further comprises the sequence of the first or second primer of primer pair 1 at its 5' end. The present invention also relates to primers, kits, and uses thereof that can be used to carry out the method. [Background technology]

[0002] In recent years, infection diagnosis has undergone significant development, particularly through the use of molecular biology methods such as genome amplification or polymerase chain reaction (PCR). PCR is increasingly replacing traditional serological and microbiological methods such as bacterial culture or ELISA testing. PCR offers several advantages: it is rapid, sensitive, and specific. Although it remains relatively expensive and therefore reserved for challenging or specialized analytical laboratories, the specificity and sensitivity of PCR diagnosis will eventually replace traditional analysis in the first place.

[0003] Standard PCR allows the amplification of a nucleic acid of interest using a specific primer pair. For example, in the case of suspected E. coli infection, a primer pair that allows amplification of E. coli-specific nucleic acids can be used. If the source of the infection is unknown, a primer pair specific to each pathogen intended to be detected must be used. In a PCR approach called "simplex," a single primer pair is used, and the PCR reaction must be repeated as many times as necessary to identify the number of viruses, bacteria, or yeasts intended to be identified as the cause of the infection. In a PCR approach called "multiplex," all primer pairs are added in one single reaction, allowing all desired pathogens to be identified simultaneously.

[0004] Multiplex PCR is a PCR method that allows multiple different nucleic acid sequences to be simultaneously identified from the same biological sample.This allows multiple targeted amplifications to be performed in one identical reaction.Therefore, multiplex PCR is particularly suitable for infectious disease diagnosis.During infectious disease diagnosis, different primer pairs are used to target multiple bacteria, viruses, or yeasts in the same PCR reaction in the presence of a target biological sample.

[0005] However, the primer pairs used in multiplex PCR need to be significantly optimized so that all primers can function at the same temperature during PCR, in particular to prevent the phenomenon of cross-reactivity.

[0006] For example, in the case of community-acquired pneumonia, multiplex PCR can reveal at least one pathogen in 77% of hospitalized patients compared with 39% by culture (NJ Gadsby, CD Russell, MP McHugh, Comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia. Clin Infect Dis, 2016). In the case of diarrhea, it is possible to detect up to three times more pathogens in stool than conventional methods (CR Stensvold, HV Nielsen, Comparison of microscopy and PCR for detection of intestinal parasites in Danish patients supports an incentive for molecular screening platforms. J Clin Microbiol, 2012). Thus, one single test can indicate the presence of multiple infectious agents in a sample in less than two hours. Today, panels for meningitis, sexually transmitted diseases, respiratory infections, and infectious enteritis are already being proposed to clinicians.

[0007] Nevertheless, multiplex PCR is limited to detecting a predetermined amount of pathogens, and the panel contains a primer pair that targets at least one fragment of a specific gene of the pathogen.In order to expand the detection panel, a detection kit may contain a large number of primers, so that it is possible to detect a large number of microorganisms.Therefore, the more it is desired to increase the number of pathogens targeted in the panel, the more the number of primer pairs tends to increase.In a general method, pathogens are detected by primer pairs, and the amplicon is specific to the pathogen.

[0008] However, high concentrations of different primers in one single test can lead to harmful, contradictory effects. Therefore, primer competition occurs, resulting in primer inhibition. Furthermore, due to the use of multiple primers, the possibility of non-specific amplification is much higher. Therefore, excessive multiplexing leads to very low detection sensitivity.

[0009] It is also known that adding a non-target complementary sequence to the 5' end of the primer can improve the effectiveness of PCR amplification, as shown in a 2007 study by I. Afonina et al. (Primers with 5' flaps improve real-time PCR.) However, these additions do not address the problems of nonspecific amplification or loss of sensitivity associated with the high primer concentrations typically used during multiplex PCR.

[0010] The present invention aims to solve the problems and overcome the drawbacks of the prior art, in particular to prevent or limit the deterioration of sensitivity of multiplex PCR, which may result in the absence or insufficient amplification of targeted nucleic acids due to the phenomenon of competition between primers when the primers are present in the same test in too large an amount, as well as to suppress or limit the deterioration of sensitivity of multiplex PCR associated with non-specific amplification, which consumes amplification reagents in an undesirable manner. Summary of the Invention

[0011] Thus, according to a first aspect, the present invention relates to a method for amplifying at least two different nucleic acid sequences of interest present in a sample using a primer pair 1 capable of amplifying a first nucleic acid sequence of interest (A) and at least one hybrid primer pair 2 capable of amplifying a second nucleic acid sequence of interest (B), said method comprising: a. contacting the sample with a primer pair 1 capable of amplifying a first nucleic acid sequence (A) and a primer pair 2 capable of amplifying a second nucleic acid sequence (B), ■ Primer pair 1 is formed from a first primer and a second primer capable of amplifying a nucleic acid corresponding to a first nucleic acid sequence (A), ■ a contacting step in which primer pair 2 is formed from a first primer and a second primer capable of amplifying a nucleic acid corresponding to a second nucleic acid sequence (B); b. adding selected reagents and conditions that allow PCR amplification; c. amplifying the first and second nucleic acid sequences (A and B) by performing an amplification reaction based on the specific transcription of the nucleic acid sequence (A) and the nucleic acid sequence (B) present in the sample; The sequence of the first primer of primer pair 2 further comprises the sequence of the first primer of primer pair 1 at its 5' end, and the sequence of the second primer of primer pair 2 further comprises the sequence of the second primer of primer pair 1 at its 5' end.

[0012] According to a variant, the sequence of the first primer of primer pair 2 (called chimeric or hybrid) further comprises at its 5' end the sequence of the first or second primer of primer pair 1, and the sequence of the second primer of primer pair 2 further comprises at its 5' end the sequence of the first or second primer of primer pair 1.

[0013] The concentration of primer pair 2 is preferably equal to or less than the concentration of primer pair 1, and more preferably less than that.

[0014] The method according to the invention may also include a third primer pair 3, a fourth primer pair 4, etc., each comprising a first primer (the sequence of which further comprises at its 5' end the sequence of the first or second primer of primer pair 1), and a second primer (the sequence of which further comprises at its 5' end the sequence of the first or second primer of primer pair 1).

[0015] According to another variant, the second primer targeting the first nucleic acid sequence (A) can also contain, in its 5' region, the sequence of the first primer targeting this same nucleic acid (A).

[0016] For example, the first oligonucleotide of primer pair 1, 2, 3, or 4 can be a sense or antisense primer, and the second oligonucleotide of primer pair 1, 2, 3, or 4 can be an antisense or sense primer.

[0017] Preferably, the method according to the invention is a multiplex PCR and performs differential quantitative amplification, i.e. it allows targets not to be amplified in the same way, resulting in differences in the amount produced for the same number of cycles between targets, which PCR according to the invention is also referred to in the remainder of the specification as relay PCR or PCR according to the invention.

[0018] PCR according to the invention makes it possible in particular to detect and identify microorganisms such as bacteria, viruses, yeasts, etc. that are involved in infectious diseases. Diseases can be detected in subjects such as animals or humans, or in plants.

[0019] According to a second aspect, the present invention further relates to primers and kits comprising the specific primers, detection kits capable of carrying out the method of the invention, and their use for detecting microorganisms present in the case of infectious diseases.

[0020] The present invention will now be described in detail with reference to the drawings described below, which are merely illustrative of some embodiments of the invention and are not limiting. [Brief explanation of the drawings]

[0021] [Figure 1] 1 illustrates the principle of relay PCR according to the present invention. [Figure 2A]A standard duplex PCR test (multiplex with two targets) is shown, containing primer pair 1 targeting Staphylococcus aureus and primer pair 2 targeting Escherichia coli. The ratio of primer pair 1:primer pair 2 is 1:1. Black: Amplification curve targeting a specific gene in Escherichia coli. Grey: Amplification curve targeting a specific gene in Staphylococcus aureus. [Figure 2B] Figure 1 shows a relay PCR test according to the present invention, comprising primer pair 1 targeting Staphylococcus aureus and primer pair 2 targeting Escherichia coli. The ratio of primer pair 1:primer pair 2 is 10:1. Black: amplification curve targeting specific genes in Escherichia coli. Grey: amplification curve targeting specific genes in Staphylococcus aureus. [Figure 2C] Figure 1 shows a relay PCR test according to the present invention, comprising primer pair 1 targeting Staphylococcus aureus and primer pair 2 targeting Escherichia coli. The ratio of primer pair 1:primer pair 2 is 1:1. Black: amplification curve targeting a specific gene in Escherichia coli. Grey: amplification curve targeting a specific gene in Staphylococcus aureus. [Figure 3A] A standard duplex PCR test is shown, including primer pair 1 targeting Klebsiella pneumoniae and primer pair 2 targeting Staphylococcus aureus. The ratio of primer pair 1:primer pair 2 is 1:1. Black: Amplification curve targeting specific genes in Klebsiella pneumoniae. Grey: Amplification curve targeting specific genes in Staphylococcus aureus. [Figure 3B]Figure 1 shows a relay PCR test according to the present invention, comprising primer pair 1 targeting Klebsiella pneumoniae and primer pair 2 targeting Staphylococcus aureus. The ratio of primer pair 1:primer pair 2 is 10:1. Black: amplification curves targeting specific genes in Klebsiella pneumoniae. Grey: amplification curves targeting specific genes in Staphylococcus aureus. [Figure 3C] Figure 1 shows a relay PCR test according to the present invention, comprising primer pair 1 targeting Klebsiella pneumoniae and primer pair 2 targeting Staphylococcus aureus. The ratio of primer pair 1:primer pair 2 is 1:1. Black: amplification curves targeting specific genes in Klebsiella pneumoniae. Grey: amplification curves targeting specific genes in Staphylococcus aureus. [Figure 4]The structures of primer pairs used in standard PCR, which can amplify a target sequence, e.g., A, are shown, including a sense primer with sequence FW1 and an antisense primer with sequence RT1. The structures of hybrid or chimeric primer pairs used in relay PCR, which can amplify a target sequence, e.g., B, are also shown. The hybrid or chimeric primer pair includes a sense primer with sequence FW1+FW2 and an antisense primer with sequence RT1+RT2. Sequence FW2 can hybridize to a region of target sequence B, where sequence FW1 corresponds to the sequence of the sense primer in primer pair 1 present at a higher concentration in the mixture. The sequences FW1 and FW2 are adjacent to form a single sense primer with a length of 12 to 140 nucleotides. The hybrid or chimeric primer pair also includes an antisense primer with sequence RT1+RT2, where sequence RT2 is complementary to a region of target sequence B, where sequence RT1 corresponds to the sequence of the antisense primer in primer pair 1 present at a higher concentration in the mixture. The sequences FW1 and FW2 are adjacent to form a single primer with a length of 12 to 140 nucleotides. Due to the effect of the second PCR cycle, primer pair 1, which is present at a higher concentration in the mixture, amplifies the amplicon generated by primer pair 2 in the first cycle, and thus can also amplify target sequence B. Therefore, primer pair 1 can amplify not only target sequence A but also target sequence B from the second PCR cycle. [Figure 5] The sequence of the bacterial gene 16S is shown. The sequence contains nine variable regions specific to each species and ten conserved regions between species adjacent to the variable regions. The variable regions are numbered V1 to V9. The conserved regions allow, according to the knowledge of those skilled in the art, to define the nucleotide sequences of sense and antisense primers that can hybridize on these conserved regions surrounding at least one variable region of interest (e.g., V1 to V2). [Figure 6]These are agarose gels showing PCR results using the standard duplex method (multiplex with two targets) or the duplex relay method of the present invention. Under all conditions, 100 copies of the Klebsiella pneumoniae genome were added. Line 1: Amplicon of the 5' region of the 16S gene (amplicon covering variable regions V1 to V2) obtained using a high concentration of primers targeting conserved regions adjacent to the variable region (control PCR). Line 2: Amplicon of the central region of the 16S gene (amplicon covering variable regions V4 to V6) obtained using a high concentration of primers targeting conserved regions adjacent to the variable region (control PCR). Line 3: Amplicon in standard multiplex PCR of the 5' region (V1 to V2) and the central region (V4 to V6) of the 16S gene using two primer pairs targeting each variable region, with high primer concentration. 4: Amplicon of two regions of the 16S gene in the method of the present invention in a duplex relay, where the first primer pair targets the 5' region (V1-V2) and the second hybrid or chimeric primer pair contains sequences that can target not only the central region (V4-V6) but also the sequence of the first primer pair, and the primers are used at high (first primer pair) and low (second primer pair) concentrations, respectively. Arrows indicate the amplification or lack of amplification of the amplicon of interest (in this case, regions V1-V2 of the 16S gene). [Figure 7] Agarose gels showing PCR results using the standard triplex method (multiplex with three targets) or the method of the present invention (triplex relay). Under all conditions, 100 copies of the Klebsiella pneumoniae genome were added. 1: Amplicons obtained using standard triplex PCR, with primers at 1 mM each. 2: Amplicons obtained using triplex relay PCR, using chimeric or hybrid primers at a low concentration of 0.04 mM. Triple relay amplicons V4-V6 and V7-V9 are slightly larger than their counterparts in standard triplex PCR due to the increased length of the chimeric primers after polymerization. [Figure 8] This agarose gel shows the results of a multiplex relay PCR using a single primer at a high concentration. Under all conditions, 100 copies of the Klebsiella pneumoniae genome were added. 1: Amplicon obtained by duplex relay PCR using a high concentration of primer pair 1 targeting the V1-V2 region of the 16S gene and a low concentration of hybrid or chimeric primer pair 2 targeting the V7-V9 region of the 16S gene. 2: Amplicon obtained by duplex relay PCR using primer pair 1 targeting the V1-V2 region of the 16S gene and a low concentration of hybrid or chimeric primer pair 2 targeting the V7-V9 region of the 16S gene, consisting of a high concentration of sense primer and a low concentration of hybrid or chimeric antisense primer containing the sense primer sequence at its 5' end. Therefore, the antisense primer of the first pair and the sense and antisense primers of the second pair are hybrid or chimeric primers. Only the sense primer of primer pair 1 is present at a high concentration.

[0022] definition Within the meaning of the present invention, a "patient" refers to a subject that may be a human or an animal, preferably a human or a mammal. The subject is preferably a human patient, regardless of age or sex. Neonates, infants, and children are also included.

[0023] Within the meaning of the present invention, "diagnosis" means a test aimed at detecting and identifying one or more pathologies / pathologies, in particular at detecting and identifying infectious diseases in a subject or plant, so that the subject or plant can be treated accordingly.

[0024] Within the meaning of the present invention, a "primer pair" or "oligonucleotide pair" means two oligonucleotides that make it possible to amplify a target sequence within the context of PCR.

[0025] Within the meaning of the present invention, a "primer" or "oligonucleotide" refers to a short segment of nucleic acid of one to several tens of nucleotides. Such primers are capable of hybridizing with a complementary sequence. In the context of the present invention, they are capable of hybridizing with a fragment of a target sequence. The target sequence can be a nucleic acid sequence (DNA or RNA).

[0026] Within the meaning of the present invention, "sequence FW1" means the sequence of an oligonucleotide of one to several tens of nucleotides, in particular the sequence of a sense primer that can hybridize with the complementary sequence of the target sequence.

[0027] Within the meaning of the present invention, "sequence RT1" means the sequence of an oligonucleotide of one to several tens of nucleotides, in particular the sequence of an antisense primer capable of hybridizing with the complementary sequence of the sequence of interest.

[0028] Within the meaning of the present invention, "primer pair capable of amplifying a nucleic acid sequence of interest" refers to a primer pair comprising a sense primer and an antisense primer capable of amplifying a specific sequence of interest by PCR. For example, the nucleic acid sequence of interest may be a sequence present in the variable region of the bacterial gene 16S. The primers are designed to target the sequence of interest according to the general knowledge of those skilled in the art.

[0029] Within the meaning of the present invention, "amplification" refers to the amplification of a target sequence during a PCR reaction, thus increasing the copy number of the target sequence. The primer hybridizes with a previously denatured complementary sequence by chain extension under the action of a polymerase. The reaction proceeds in multiple successive cycles, allowing the copy number of the amplicon to increase.

[0030] Within the meaning of the present invention, an "amplicon" refers to a nucleic acid fragment that is amplified during PCR and corresponds to the sequence separated by the two primers used. An amplicon is thus a copy of the target sequence.

[0031] Within the meaning of the present invention, a "hybrid primer" or "chimeric primer" refers to a primer that contains a nucleic acid sequence capable of hybridizing with the complementary strand of a target sequence and a nucleic acid sequence at its 5' that corresponds to the sequence of a primer belonging to another primer pair, for example, the sequence of a sense or antisense primer of another primer pair. Within the context of the present invention, primer pairs 2, 3, and 4 are formed from hybrid or chimeric primers. According to a variant of the present invention, primer pair 1 contains one "standard" primer and one hybrid or chimeric primer. For example, the first oligonucleotide of primer pair 2 contains a sequence capable of hybridizing with a specific nucleic acid sequence (target B) and a nucleic acid sequence at its 5' that corresponds to the sequence of the first oligonucleotide of primer pair 1 that can hybridize with another specific nucleic acid sequence (target A).

[0032] Within the meaning of the present invention, "concentration ratio" means the ratio of the concentration of primer pair 1 to a second primer pair, such as primer pair 2. For example, a ratio of 1:1 corresponds to identical concentrations of the first and second pair, while a ratio of 10:1 corresponds to a 10-fold greater concentration of the first pair compared to the second pair. DETAILED DESCRIPTION OF THE INVENTION

[0033] Therefore, the present invention relates to a new PCR method called multiplex PCR. "Standard" multiplex PCR is known, which is capable of identifying a large number of target sequences. This type of standard multiplex PCR requires the presence of high concentrations of each primer pair targeting a specific nucleic acid. Therefore, in standard multiplex PCR, each primer pair is present at the same high concentration in the multiplex PCR reaction. Reaction conditions begin to deteriorate when there are more than four or five different primer pairs. Furthermore, the emergence of competition between primers leads to inhibition of specific primer hybridization. Furthermore, the use of multiple primers can lead to nonspecific amplification. This results in very low detection sensitivity for standard multiplex PCR.

[0034] The present invention relates to a new multiplex PCR method called multiplex relay PCR (or relay PCR), in which one single primer pair or one single primer is used at a high concentration, and all other primers are at a very low concentration. This method can maintain very high sensitivity for all targeted nucleic acids.

[0035] It is also possible to promote the amplification of some nucleic acids over others (quantitative asymmetric amplification or differential quantitative amplification), in particular the amplification of the nucleic acid of target A over others. Thus, the nucleic acid that is preferentially amplified is present in greater abundance. In diagnostics, preferring some nucleic acids has medical advantages due to the fact that some nucleic acids provide more information than others.

[0036] The present invention therefore relates to a method for amplifying at least two different nucleic acid sequences of interest present in a sample using a primer pair 1 capable of amplifying a first nucleic acid sequence of interest (A) and at least one primer pair 2 capable of amplifying a second nucleic acid sequence of interest (B), said method comprising: a. contacting the sample with a primer pair 1 capable of amplifying a first nucleic acid sequence (A) and a primer pair 2 capable of amplifying a second nucleic acid sequence (B), i. primer pair 1 is formed from a first primer and a second primer capable of amplifying a nucleic acid corresponding to a first nucleic acid sequence (A); ii. contacting, wherein primer pair 2 is formed from a first primer and a second primer capable of amplifying a nucleic acid corresponding to a second nucleic acid sequence (B); b. adding selected reagents and conditions that allow PCR amplification; c. amplifying the first and second nucleic acid sequences (A and B); The sequence of the first primer of primer pair 2 further comprises the sequence of the first primer of primer pair 1 at its 5' end, and the sequence of the second primer of primer pair 2 further comprises the sequence of the second primer of primer pair 1 at its 5' end.

[0037] During step c, the amplification of the first and second nucleic acid sequences (A and B) is carried out by an amplification reaction based on the polymerization (extension) of the nucleic acid sequence (A) and the nucleic acid sequence (B) present in the sample.

[0038] According to a variant, the sequence of the first primer of primer pair 2 further comprises at its 5' end the sequence of the first or second primer of primer pair 1, and the sequence of the second primer of primer pair 2 further comprises at its 5' end the sequence of the first or second primer of primer pair 1.

[0039] Preferably, the sequence of the first primer of primer pair 2 and the sequence of the first or second primer of primer pair 1 are adjacent, and the sequence of the second primer of primer pair 2 and the sequence of the first or second primer of primer pair 1 are adjacent. According to a variant, the sequences are not adjacent, but are separated by a few nucleotides, by a spacer (or "linker") of 1 to 12 nucleotides in size.

[0040] According to another variant, the method according to the invention comprises the sequence of the second primer of primer pair 1 further comprises and is flanked 5' by the sequence of the first primer of said primer pair 1; - the sequence of the first primer of primer pair 2 further comprises the sequence of the first primer of primer pair 1 at its 5' end, and the sequence of the second primer of primer pair 2 further comprises the sequence of the first primer of primer pair 1 at its 5' end, and the sequences are adjacent to each other.

[0041] According to another variant, the sequence of the second primer of primer pair 1 further comprises at its 5' end the sequence of the first primer of said primer pair 1, and / or the sequence of the first primer of primer pair 2 further comprises at its 5' end the sequence of the first primer of primer pair 1, and the sequence of the second primer of primer pair 2 further comprises at its 5' end the sequence of the first primer of primer pair 1, said sequences being separated by a number of nucleotides by a spacer (or "linker") of 3 to 12 nucleotides in size.

[0042] According to a preferred embodiment of the present invention, the method uses: at least one primer pair 1 capable of amplifying target sequence A, a sense primer of sequence FW1 that is complementary to a fragment of target sequence A, the primer having a size of 6 to 70 nucleotides; - at least one primer pair 1 comprising an antisense primer of sequence RT1, which is complementary to a fragment of target sequence A, said primer having a size of 6 to 70 nucleotides, and at least one primer pair 2 capable of amplifying target sequence B, a sense primer of sequence FW1 or RT1 and sequence FW2, where sequence FW2 is complementary to a fragment of target sequence B, and where the sequences FW1 or RT1 and FW2 are adjacent to form a single primer of 12 to 140 nucleotides in size; - at least one primer pair 2 comprising an antisense primer of sequence FW1 or RT1 and sequence RT2, wherein sequence RT2 is complementary to a fragment of target sequence B, and wherein the sequences FW1 or RT1 and RT2 are adjacent to form a single primer having a size of 12 to 140 nucleotides.

[0043] Thus, the present invention also provides a hybrid or chimeric primer pair comprising: a sense primer (or first oligonucleotide) of sequence FW1 or RT1 and sequence FW2, where sequence FW2 is complementary to a fragment of target sequence B and sequence FW1 or RT1 corresponds to the sequence of the sense or antisense primer, respectively, of primer pair 1 present at a higher concentration in the mixture, and where sequences FW1 or RT1 and FW2 are adjacent to form a single sense primer of 12 to 140 nucleotides in length; and an antisense primer (or second oligonucleotide) of sequence FW1 or RT1 and sequence RT2, where sequence RT2 is complementary to a fragment of target sequence B, and sequence RT1 or FW1 corresponds to the sequence of the antisense or sense primer, respectively, of primer pair 1 present in the mixture at an equal or higher concentration, and where the sequences FW1 or RT1 and FW2 are adjacent to form a single sense primer of 12 to 140 nucleotides in length.

[0044] According to a variant, the present invention also provides a hybrid or chimeric primer pair, a sense or antisense primer (or first oligonucleotide) of sequence FW1 or RT1, wherein sequence FW1 or RT1 is complementary to a fragment of target sequence A that is present at a higher concentration in the mixture, and said sequence is between 6 and 70 nucleotides in length; a hybrid or chimeric sense or antisense primer (or second oligonucleotide) of sequences FW1 and RT1, or of sequences RT1 and FW1, which primer is present in the mixture at a lower concentration compared to the first oligonucleotide of sequence FW1 or RT1, and wherein sequences FW1 and RT1, or RT1 and FW1, are adjacent to form a single primer of 12 to 140 nucleotides in length.

[0045] During duplex relay PCR, according to the present invention, the PCR reaction occurs in a mixture containing primer pair 1 capable of hybridizing and amplifying a first nucleic acid sequence (A) and primer pair 2 capable of hybridizing and amplifying a second nucleic acid sequence (B), as in standard duplex PCR. However, duplex relay PCR differs from standard duplex PCR in the use of a hybrid or chimeric primer in the second primer pair and, optionally, a sense or antisense primer in the first primer pair.

[0046] Thus, by virtue of the second PCR cycle, primer pair 1 is able to amplify the amplicon produced by primer pair 2, and therefore target sequence B as well.

[0047] Duplex relay PCR specifically involves contacting a sample with a reaction mixture comprising primer pair 1 capable of amplifying a nucleic acid sequence (A) and primer pair 2 capable of amplifying a second nucleic acid sequence (B).

[0048] Primer pair 1 consists of a first sense primer (e.g., sequence FW1) and a second antisense primer (e.g., sequence RT1), each capable of hybridizing to a complementary sequence of one of the strands of nucleic acid (A) and amplifying a sequence corresponding to the complementary sequence of the strand between the two primers. The complementary sequence is target sequence (A).

[0049] Primer pair 2 consists of a first sense primer (e.g., sequence FW1-FW2) and a second antisense primer (e.g., sequence RT1-RT2), each of which hybridizes to the complementary sequence of one of the strands of nucleic acid (B) and can amplify the sequence corresponding to the complementary sequence of the strand between the two primers. The complementary sequence is target sequence (B).

[0050] Furthermore, the reaction mixture contains all the reagents and conditions necessary to carry out PCR amplification. The reagents can be present in the reaction mixture initially or added after contacting the sample. The necessary reagents include, in particular, dNTPs and polymerase, which can be selected by those skilled in the art depending on the PCR and reaction conditions. The polymerase is preferably Thermophilus aquaticus polymerase or the "Klenow" fragment of the polymerase.

[0051] Once all components are present in the reaction mixture, amplification can begin.

[0052] The various steps of the PCR reaction are known to those skilled in the art. A PCR cycle includes a denaturation step, the purpose of which is to separate the double strands to obtain two single strands, followed by a hybridization step. During this step, a primer recognizes and then hybridizes with a complementary sequence present in one of the previously denatured single strands. An extension step then takes place, allowing the polymerase to synthesize a strand complementary to the single strand. This strand is synthesized from the free dNTPs present in the reaction mixture. The duration of this step depends on the length of the sequence to be amplified.

[0053] The PCR reaction continues with a second cycle, a third cycle, etc., each containing the same steps. Thus, at the end of the PCR, amplification of the sequence contained between the primers is achieved. This sequence corresponds to the amplicon.

[0054] Thus, the amplification of the first and second nucleic acid sequences is based on the amplification of the target nucleic acid sequence (A) and the target nucleic acid sequence (B) present in the sample, preferably the simultaneous amplification of the target nucleic acid sequence (A) and the target nucleic acid sequence (B) present in the sample.

[0055] The method according to the invention is characterized in that the first primer of primer pair 2 further comprises, at its 5', the sequence of the first primer of primer pair 1, and the second primer of primer pair 2 further comprises, at its 5', the sequence of the second primer of primer pair 1. The sequences of the first primer of pair 2 and the first primer of pair 1 are adjacent, and the sequences of the second primer of pair 2 and the second primer of pair 1 are also adjacent.

[0056] According to a variant, the second primer of primer pair 2 further comprises at its 5' position the sequence of the first or second primer of primer pair 1. The sequences of the first primer of pair 2 and the first primer of pair 1 are adjacent, and the sequences of the first or second primer of pair 2 and the second primer of pair 1 are also adjacent.

[0057] Preferably, the first primer of primer pair 2 comprises a nucleic acid sequence capable of hybridizing to a complementary sequence of a sequence to be amplified, such as target sequence (B), and further comprises, at its 5', the sequence of the first or second primer of primer pair 1, and the second primer of primer pair 2 comprises a nucleic acid sequence capable of hybridizing to a complementary sequence of a sequence to be amplified, such as target sequence (B), and further comprises, at its 5', the sequence of the first or second primer of primer pair 1. The nucleic acid sequence capable of hybridizing to a complementary sequence to be amplified, such as target sequence (B), differs between primer 1 and primer 2. In particular, primer 1 may be a sense primer, and primer 2 may be an antisense primer.

[0058] Preferably, the first primer pair can amplify a first sequence (A) and a second sequence (B) by virtue of the effect of the second PCR cycle. Indeed, during the first PCR cycle, the first primer pair can amplify the first sequence (A), and the second primer pair can amplify the second sequence (B). At the end of the first PCR cycle, a nucleic acid sequence comprising the sequence of the first primer of the first pair and the complementary sequence of sequence (A), a nucleic acid sequence comprising the sequence of the second primer of the first pair and the complementary sequence of sequence (A), a nucleic acid sequence comprising the sequence of the first primer of the second pair and the complementary sequence of sequence (B), and a nucleic acid sequence comprising the sequence of the second primer of the second pair and the complementary sequence of sequence (B) are generated.

[0059] Indeed, the first primer of the second pair comprises the sequence of the first primer of the first pair and a sequence capable of hybridizing to sequence (B), and the second primer of the second pair comprises the sequence of the second primer of the first pair and a sequence capable of hybridizing to sequence (B). Thus, during the second cycle, primer pair 1 can amplify the nucleic acid sequence generated during the first cycle (Figure 1).

[0060] According to one embodiment, the sequence of the first primer of the first pair is selected from SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, the sequence of the second primer of the first pair is selected from SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, the sequence of the first primer of the second pair is selected from SEQ ID NO: 7 or SEQ ID NO: 9, and the sequence of the second primer of the second pair is selected from SEQ ID NO: 8 or SEQ ID NO: 10. The sequences are listed in Table 1 below. [Table 1]

[0061] For diagnostic purposes, the aim is to target the variable region of the gene 16S, which is present in all bacteria.

[0062] The 16S gene contains conserved and variable regions, specifically nine variable regions and ten conserved regions (see Figure 5). The variable regions are numbered V1 to V9 and are all surrounded by conserved regions. Those skilled in the art can fully determine the relevant regions on the 16S gene based on general knowledge. Tremblay et al., 2015 (Frontiers in Microbiology) describes one of these many methods for amplifying and sequencing the relevant regions, for example, to identify bacteria present in a sample.

[0063] Therefore, according to a preferred embodiment, the sequence of interest corresponds to the variable sequence of a single gene or at least two distinct genes, more preferably the sequence of interest corresponds to the variable sequence of the 16S gene, and the primers are capable of hybridizing with at least 12 nucleotides of the conserved sequence of the 16S gene. Preferably, the sense primer is capable of hybridizing with at least 12 nucleotides of the conserved sequence of the 16S gene at the 5' position of the variable sequence of the 16S gene, and the antisense primer is capable of hybridizing with at least 12 nucleotides of the conserved sequence of the 16S gene at the 3' position of the variable sequence of the 16S gene.

[0064] In the context of the present invention, the inventors designed primers that specifically hybridize on conserved regions adjacent to variable regions of interest, such as V1-V2, V3, V4-V6, and V7-V9. Thus, the generated amplicons correspond to the sequences of variable regions V1-V2, V3, V4-V6, or V7-V9, and the sequences are specific to one pathogen.

[0065] According to a preferred embodiment, the sequence of the first primer (sense primer) of the first pair is selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 24, and the sequence of the second primer (antisense primer) of the first pair is selected from SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.

[0066] Preferably, the sequence of the first primer (sense primer) of the first pair is selected from SEQ ID NO: 14 or SEQ ID NO: 15, and the sequence of the second primer (antisense primer) of the first pair is selected from SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 26, and SEQ ID NO: 27.

[0067] The sequence of the first primer (sense primer) of the second pair is selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: S4, or SEQ ID NO: 35, and the sequence of the second primer of the second pair is selected from SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38. The sequences are listed in Table 1B below. [Table 2]

[0068] The first primer pair targeting the variable regions V1 to V2 of the 16S gene preferably consists of: a sense primer having at least 70% sequence homology with SEQ ID NO: 14 or SEQ ID NO: 15, and - an antisense primer having at least 70% sequence homology with SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 26 or SEQ ID NO: 27.

[0069] The second pair of hybrid or chimeric primers targeting the variable regions V4 to V6 of the gene 16S preferably consists of: a sense primer having at least 70% sequence identity with SEQ ID NO: 28 or SEQ ID NO: 29 or SEQ ID NO: 30, and - an antisense primer having at least 70% sequence homology with SEQ ID NO: 31 or SEQ ID NO: 32 or SEQ ID NO: 33.

[0070] The second or third primer pair targeting the variable regions V7 to V9 of the 16S gene preferably consists of: a sense primer having at least 70% sequence identity with SEQ ID NO: 34 or SEQ ID NO: 35, and - an antisense primer having at least 70% sequence homology with SEQ ID NO: 36 or SEQ ID NO: 37 or SEQ ID NO: 38.

[0071] Preferably, the 70% sequence homology relates specifically to the first 10 nucleotides at the 3' position of the sequence.

[0072] More preferably, the first primer pair targeting the variable regions V1 to V2 of the 16S gene consists of: - a sense primer of SEQ ID NO: 14 or SEQ ID NO: 15, and -Antisense primer of SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 26 or SEQ ID NO: 27.

[0073] The second pair of hybrid or chimeric primers targeting the variable regions V4 to V6 of the gene 16S preferably consists of: - a sense primer of SEQ ID NO: 28 or SEQ ID NO: 29 or SEQ ID NO: 30, and - antisense primer SEQ ID NO: 31 or SEQ ID NO: 32 or SEQ ID NO: 33.

[0074] The second or third primer pair targeting the variable regions V7 to V9 of the 16S gene preferably consists of: - a sense primer of SEQ ID NO: 34 or SEQ ID NO: 35, and - antisense primer SEQ ID NO: 36 or SEQ ID NO: 37 or SEQ ID NO: 38.

[0075] During the method according to the invention, the concentration of primer pair 2 may be less than, greater than, or equal to the concentration of primer pair 1. It is therefore possible to enhance or not enhance one primer pair compared to another primer pair, thus enhancing the amplification of one sequence of interest.

[0076] According to a particularly preferred embodiment of the present invention, the concentration of primer pair 2 is equal to or less than the concentration of primer pair 1, more preferably less.If the concentration of primer pair 2 is lower than the concentration of primer pair 1, primer pair 2 is disadvantageous to the first pair.Therefore, the amplicon generated by primer pair 1 exists in a larger amount.Then, when sequencing is performed, the amplicon of target A is represented in excess, so it is preferentially sequenced.Therefore, its sequence can be obtained more quickly.

[0077] The method according to the invention allows for the production of amplicons by primer pair 1 at least four times as much as those produced by primer pair 2. Thus, the amplicons produced by the second pair are present in lower amounts and are not removed as quickly during sequencing.

[0078] The concentration ratio of primer pair 1 to primer pair 2 is preferably 1:1 to 1000:1, even more preferably 1:1 to 100:1. According to a particularly advantageous embodiment, the concentration ratio of primer pair 1 to primer pair 2 is 10:1, more preferably 25:1. Such a ratio may correspond, for example, to a concentration of primer pair 1 of 1 mM and a concentration of primer pair 2 of 0.1 mM or 0.04 mM.

[0079] According to another embodiment, the method according to the invention may further comprise contacting the sample with a third primer pair capable of amplifying a third nucleic acid sequence (C). The first primer of primer pair 3 comprises a nucleic acid sequence capable of hybridizing to complementary sequence (C) and further comprises a sequence 5' corresponding to the sequence of the first or second primer of primer pair 1. The second primer of primer pair 3 comprises a nucleic acid sequence capable of hybridizing to complementary sequence (C) and further comprises a sequence 5' corresponding to the sequence of the first or second primer of primer pair 1.

[0080] The method according to the invention therefore comprises three different primer pairs that make it possible to amplify three different target nucleic acid sequences, in particular sequence A, sequence B and sequence C. The method is a PCR called triplex relay.

[0081] Sequences B and C are preferably amplified by hybrid or chimeric sense and antisense primers, each comprising the sequence of the sense primer of primer pair 1 present in a higher concentration, and the sequence of either the sense or antisense primer of primer pair 1 present in a higher concentration.

[0082] Thus, the method according to the invention uses: at least one primer pair 1 capable of amplifying target sequence A, a sense primer of sequence FW1 that is complementary to a fragment of target sequence A, the primer having a size of 6 to 70 nucleotides; ■ at least one primer pair 1 comprising an antisense primer of sequence RT1 that is complementary to a fragment of target sequence A, the primer having a size of 6 to 70 nucleotides; and at least one hybrid or chimeric primer pair 2 capable of amplifying target sequence B, (ii) a sense primer of sequence FW1 or RT1 and sequence FW2, wherein sequence FW2 is complementary to a fragment of target sequence B, and the sequences FW1 or RT1 and FW2 are adjacent to form a single primer having a size of 12 to 140 nucleotides; ■ at least one hybrid or chimeric primer pair 2 comprising an antisense primer of sequence FW1 or RT1 and sequence RT2, wherein sequence RT2 is complementary to a fragment of target sequence B, and the sequences FW1 or RT1 and RT2 are adjacent to form a single primer having a size of 12 to 140 nucleotides; and at least one primer pair 3 capable of amplifying target sequence C, (ii) a sense primer of sequence FW1 and sequence FW3, wherein sequence FW3 is complementary to a fragment of target sequence C, and the sequences FW1 and FW3 are adjacent to form a single primer having a size of 12 to 140 nucleotides; ■ At least one primer pair 3 comprising an antisense primer of sequence FW1 or RT1 and sequence RT3, wherein sequence RT3 is complementary to a fragment of target sequence C, and the sequences FW1 or RT1 and RT3 are adjacent to form a single primer of 12 to 140 nucleotides in size.

[0083] During the method, the concentration of primer pair 3 is less than, greater than, or equal to the concentration of primer pair 1, and / or the concentration of primer pair 2 is less than, greater than, or equal to the concentration of primer pair 1. Thus, it is possible to promote or not promote one primer pair compared to another primer pair. Preferably, the concentration of primer pair 3 is less than the concentration of primer pair 1, and / or the concentration of primer pair 2 is less than the concentration of primer pair 1.

[0084] Preferably, the concentration of primer pair 1 is higher than the concentration of the second and / or third primer pair, and the concentration of primer pair 2 is equal to the concentration of primer pair 3.

[0085] According to one embodiment, the concentration of primer pair 1 is greater than the concentrations of the second and third primer pairs, and the concentration of primer pair 2 is greater than the concentration of primer pair 3. Thus, the first pair is promoted relative to the second pair, which is also promoted relative to the third pair.

[0086] According to another embodiment, the method according to the invention may further comprise contacting the sample with a fourth primer pair, termed a hybrid or chimeric, capable of amplifying a fourth nucleic acid sequence (D). The first primer of primer pair 4 comprises a nucleic acid sequence capable of hybridizing to complementary sequence (D) and further comprises a sequence 5' corresponding to the sequence of the first or second primer of primer pair 1. The second primer of primer pair 4 comprises a nucleic acid sequence capable of hybridizing to complementary sequence (D) and further comprises a sequence 5' corresponding to the sequence of the first or second primer of primer pair 1.

[0087] Preferably, the concentration of the fourth primer pair is equal to or less than the concentration of primer pair 1, and the concentrations of the second, third, and fourth pairs are the same.

[0088] According to another embodiment, the method according to the invention may further comprise contacting the sample with an nth primer pair capable of amplifying the nth nucleic acid sequence. The first primer of primer pair n comprises a nucleic acid sequence capable of hybridizing to the target-complementary sequence and further comprises a sequence 5' corresponding to the sequence of the first or second primer of primer pair 1. The second primer of primer pair n comprises a nucleic acid sequence capable of hybridizing to the target-complementary sequence and further comprises a sequence 5' corresponding to the sequence of the first or second primer of primer pair 1.

[0089] Preferably, the concentration of the nth primer pair is less than or equal to the concentration of primer pair 1, and the concentrations of the second, third, fourth and nth pairs are the same.

[0090] Within the meaning of the present invention, "n" means a positive natural number, which allows enumerating objects by counting each one as 1. Thus, the list of natural numbers is infinite, as are the integers as their single successors, i.e., the integers immediately greater than it.

[0091] Furthermore, the reaction medium making it possible to carry out the method according to the invention may contain one or more specific probes bound to fluorophores, the emission spectrum of which is different from the others, so that each product of the PCR reaction is measured in real time by said specific probe.

[0092] Thus, according to one embodiment, the method according to the invention further comprises a step of quantification of the amplicons by using probes coupled to fluorophores, preferably "TaqMan" probes.

[0093] Such probes according to the invention are listed in Table 2 below. [Table 3]

[0094] According to another embodiment, the method according to the invention comprises a step of detection of the amplicons, and thus of the microorganisms, by fluorescent probes, fluorescent marking, melting curves, nested PCR, quantitative PCR, reverse transcription PCR or DNA sequencing.

[0095] If the detection of the amplicon is performed by sequencing, the method further comprises the step of sequencing the amplified nucleic acid or amplicon.

[0096] According to a particularly preferred embodiment, the method of the invention allows the detection and identification of microorganisms (bacteria, yeasts, viruses, etc.) involved in at least one infectious disease, preferably bacteria.

[0097] The method according to the invention can also be used in oncology for the diagnosis of genetic diseases and in general methods in molecular biology.

[0098] In another aspect, the present invention relates to a kit comprising primer pair 1 comprising SEQ ID NO:1 and SEQ ID NO:2, or SEQ ID NO:3 and SEQ ID NO:4, or SEQ ID NO:5 and SEQ ID NO:6, and primer pair 2 comprising SEQ ID NO:7 and SEQ ID NO:8, or SEQ ID NO:9 and SEQ ID NO:10.

[0099] According to a preferred embodiment, the present invention relates to a kit comprising: Primer pair 1 targeting the variable region V1-V2 of the 16S gene, consisting of: ■ a sense primer having at least 70% sequence homology with SEQ ID NO: 14 or SEQ ID NO: 15, and ■ an antisense primer having at least 70% sequence homology with SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 26 or SEQ ID NO: 27; Primer pair 2 targeting the variable regions V4 to V6 of the 16S gene, consisting of: a sense primer having at least 70% sequence identity with SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30; and ■ An antisense primer having at least 70% sequence homology with SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0100] According to another preferred embodiment, the present invention relates to a kit comprising: Primer pair 1 targeting the variable region V1-V2 of the 16S gene, consisting of: ■ a sense primer having at least 70% sequence homology with SEQ ID NO: 14 or SEQ ID NO: 15, and ■ an antisense primer having at least 70% sequence homology with SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 26 or SEQ ID NO: 27; Primer pair 2 targeting the variable regions V7 to V9 of the 16S gene, consisting of: ■ a sense primer having at least 70% sequence identity with SEQ ID NO: 34 or SEQ ID NO: 35, and ■ An antisense primer having at least 70% sequence homology with SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

[0101] According to another embodiment, the present invention relates to a kit comprising: Primer pair 1 targeting the variable region V1-V2 of the 16S gene, consisting of: ■ a sense primer having at least 70% sequence homology with SEQ ID NO: 14 or SEQ ID NO: 15, and ■ an antisense primer having at least 70% sequence homology with SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 26 or SEQ ID NO: 27; Primer pair 2 targeting the variable regions V4 to V6 of the 16S gene, consisting of: a sense primer having at least 70% sequence identity with SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30; and ■ an antisense primer having at least 70% sequence homology with SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33; Primer pair 3 targeting the variable regions V7 to V9 of the 16S gene, consisting of: ■ a sense primer having at least 70% sequence identity with SEQ ID NO: 34 or SEQ ID NO: 35, and ■ An antisense primer having at least 70% sequence homology with SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

[0102] The kit according to any of the above embodiments preferably comprises a primer with 70% sequence homology, particularly with respect to the first 10 nucleotides at the 3' position of the sequence.

[0103] According to a particularly preferred embodiment, the present invention relates to a kit comprising: Primer pair 1 targeting the variable region V1-V2 of the 16S gene, consisting of: ■ A sense primer of SEQ ID NO: 14 or SEQ ID NO: 15, and ■ Antisense primer SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 26 or SEQ ID NO: 27, Primer pair 2 targeting the variable regions V4 to V6 of the 16S gene, consisting of: ■ A sense primer of SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, and (iii) Antisense primer: SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0104] According to a particularly preferred embodiment, the present invention relates to a kit comprising: Primer pair 1 targeting the variable region V1-V2 of the 16S gene, consisting of: ■ A sense primer of SEQ ID NO: 14 or SEQ ID NO: 15, and ■ Antisense primer SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 26 or SEQ ID NO: 27, Primer pair 2 targeting the variable regions V7 to V9 of the 16S gene, consisting of: ■ A sense primer of SEQ ID NO: 34 or SEQ ID NO: 35, and ■ Antisense primer: SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

[0105] According to another particularly preferred embodiment, the present invention relates to a kit comprising: Primer pair 1 targeting the variable region V1-V2 of the 16S gene, consisting of: ■ A sense primer of SEQ ID NO: 14 or SEQ ID NO: 15, and ■ Antisense primer SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 26 or SEQ ID NO: 27, Primer pair 2 targeting the variable regions V4 to V6 of the 16S gene, consisting of: ■ A sense primer of SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, and ■ Antisense primer SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33; Primer pair 3 targeting the variable regions V7 to V9 of the 16S gene, consisting of: ■ A sense primer of SEQ ID NO: 34 or SEQ ID NO: 35, and ■ Antisense primer: SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

[0106] The kit according to the invention is preferably capable of carrying out the method of the invention.

[0107] The present invention also relates to the use of the kit according to the invention for detecting microorganisms present in the case of an infection, such as bacteria, viruses or yeasts. The present invention preferably relates to the use of the kit for diagnosing an infection in a subject or plant.

[0108] The present invention also relates to the use of a kit according to the invention for detecting at least one genetic disease or at least one cancer. [Example]

[0109] Example 1 of the method according to the invention for amplifying two sequences of specific genes of Staphylococcus aureus and Escherichia coli (Fig. 2A / 2B / 2C) material and method The relay PCR reaction was performed as follows: 0.2 mM dNTPs, 4 mM MgCl, 0.05 units of FastStart polymerase (Roche), and buffer according to the supplier's conditions. The TaqMan probe for primer pair 1 was 0.25 mM, and the probe for primer pair 2 was 0.1 mM. Primers were set at different concentrations: 1 mM for standard duplex, 1 mM for primer pair 1 in duplex-relay, and 0.1 or 1 mM for primer pair 2 in duplex-relay. The cycle was as follows: denaturation at 95°C for 10 minutes, followed by 55 cycles of 95°C for 15 seconds, 55°C for 15 seconds, and 68°C for 15 seconds. Approximately 100 equivalent genome copies of each tested bacterium were added to the mix to perform the amplification.

[0110] result Amplification of the two sequences of interest was performed by standard duplex PCR, relay PCR according to the invention with a 10:1 ratio of primer pair 1:primer pair 2, and relay PCR according to the invention with a 1:1 ratio of primer pair 1:primer pair 2.

[0111] Within the context of standard duplex PCR, two primer pairs were used at high concentrations. The primer pair allowing the amplification of a specific sequence of the gene Staphylococcus aureus was formed from primers with sequences SEQ ID NO: 3 and SEQ ID NO: 4, while the pair allowing the amplification of Escherichia coli was composed of primers with sequences SEQ ID NO: 1 and SEQ ID NO: 2. The results are shown in Figure 2A and Table 3. Accordingly, the inventors observed an overlap in Cq (the number of cycles at which the fluorescent signal begins to be detected), which means that identical amounts of bacterial genome were indeed added to the PCR mix. Furthermore, identical amounts of Escherichia coli and Staphylococcus aureus amplicons were generated.

[0112] During duplex relay PCR with a 10:1 ratio, primer pair 1 was formed from primers with sequences SEQ ID NO:2 and SEQ ID NO:3, and primer pair 2 was composed of primers with sequences SEQ ID NO:7 and SEQ ID NO:8. The first primer pair allowed for the amplification of a specific sequence for the Staphylococcus aureus gene, while the second hybrid or chimeric primer pair allowed for the amplification of a specific sequence for the Escherichia coli gene. The results are shown in Figure 2B and Table 3. Accordingly, we observed a Cq offset in the curves between the Staphylococcus aureus and Escherichia coli amplicons. This delay introduced by duplex relay PCR was 2.09 cycles (35.27 - 33.18) for a 10:1 primer ratio, or approximately a factor of 4 (2^1.99). This resulted in an approximately four-fold decrease in the amount of amplicon differential for the Escherichia coli gene compared to the Staphylococcus aureus amplicon.

[0113] During relay PCR with a 1:1 ratio, the primer pair used was identical to that used in relay PCR (ratio 10:1). The results are shown in Figure 2C and Table 3. Accordingly, we also observed a Cq offset in the curves between the Staphylococcus aureus and Escherichia coli amplicons. This delay introduced by duplex relay PCR was 1.79 cycles (35.68 - 33.89), i.e., approximately a factor of 3.5 (2^1.79). A similar factorial difference in the amount of amplicon was also observed here. [Table 4]

[0114] Example 2 of the method according to the invention for amplifying two sequences of specific genes of Klebsiella pneumoniae and Staphylococcus aureus (Fig. 3A / 3B / 3C) Materials and methods were the same as those described in Example 1.

[0115] result Amplification of the two sequences of interest was carried out by standard duplex PCR, duplex relay PCR according to the invention with a 10:1 ratio of primer pair 1:primer pair 2, and duplex relay PCR according to the invention with a 1:1 ratio of primer pair 1:primer pair 2.

[0116] Within the context of standard duplex PCR, two primer pairs were used at high concentrations. The primer pair allowing the amplification of a specific sequence of the gene Klebsiella pneumoniae was formed from the primers with the sequences SEQ ID NO: 5 and SEQ ID NO: 6, while the pair allowing the amplification of Staphylococcus aureus was composed of the primers with the sequences SEQ ID NO: 3 and SEQ ID NO: 4. The results are shown in Figure 3A and Table 4. Thus, the inventors observed an overlap in Cq (the number of cycles at which the fluorescent signal begins to be detected), which means that the same amount of bacterial genome was actually added to the PCR mix. Furthermore, the same amount of Klebsiella pneumoniae and Staphylococcus aureus amplicons was generated.

[0117] During relay PCR with a 10:1 ratio, primer pair 1 was formed from primers with sequences SEQ ID NO:5 and SEQ ID NO:6, and primer pair 2 was composed of primers with sequences SEQ ID NO:9 and SEQ ID NO:10. The first primer pair allowed for the amplification of a specific sequence for the Klebsiella pneumoniae gene, while the second chimeric primer pair allowed for the amplification of a specific sequence for the Staphylococcus aureus gene. The results are shown in Figure 3B and Table 4. Accordingly, the inventors observed a Cq offset in the curves between the Klebsiella pneumoniae and Staphylococcus aureus amplicons. This delay introduced by duplex relay PCR was 1.99 cycles (35.35 - 33.36) for a 10:1 primer ratio, i.e., approximately a factor of 4 (2^1.99). This resulted in an approximately four-fold lower differential abundance for the Staphylococcus aureus amplicon compared to the Klebsiella pneumoniae amplicon.

[0118] During relay PCR with a 1:1 ratio, the primer pair used was identical to that used in relay PCR (ratio 10:1). The results are shown in Figure 3C and Table 4. We also observed a Cq offset in the curves between the Klebsiella pneumoniae and Staphylococcus aureus amplicons. This delay introduced by duplex relay PCR was 2.59 cycles (36.43 to 33.84), i.e., approximately a factor of 6 (2^2.59). This leads to approximately six-fold less differential amplicon abundance for the Staphylococcus aureus gene compared to the Klebsiella pneumoniae amplicon. [Table 5]

[0119] Example 3 of the method according to the invention compared to standard multiplex PCR for the amplification of two sequences of the gene 16S of Klebsiella pneumoniae (Figure 6) material and method The relay PCR reaction was performed as follows: 0.2 mM dNTPs, 4 mM MgCl, 0.05 units of FastStart polymerase (Roche), and buffer according to the supplier's conditions. Primer pairs were set at different concentrations: 1 mM for standard simplex, 1 mM for standard duplex, 1 mM for primer pair 1 in duplex-relay, and 0.04 mM for primer pair 2 in duplex-relay. The cycle was as follows: denaturation at 95°C for 10 minutes, followed by 65 cycles of 95°C for 15 seconds, 55°C for 15 seconds, and 68°C for 30 seconds. Approximately 100 equivalent genome copies of Klebsiella pneumoniae were added to the mix to perform the amplification.

[0120] result Amplification of the two sequences of interest was performed by standard duplex PCR (Figure 6, line 3) and duplex relay PCR (Figure 6, line 4) according to the invention with a primer pair 1:primer pair 2 ratio of 25:1.

[0121] Line 1 represents the amplicon of the 5' region of the 16S gene (covering variable regions V1 to V2) (control PCR).

[0122] Line 2 represents the amplicon of the central region of the gene 16S (covering variable regions V4 to V6) (control PCR).

[0123] Line 3 represents the amplicons in standard multiplex PCR of the V1-V2 and V4-V6 regions of the 16S gene.

[0124] Line 4 represents the amplicon of regions V1-V2 and V4-V4 of gene 16S using the method according to the present invention (multiplex relay PCR).

[0125] In the context of standard duplex PCR, two primer pairs were used at high concentrations (line 3). The primer pair enabling the amplification of sequences V1 and V2 of the 16S gene of Klebsiella pneumoniae was formed by primers with sequences SEQ ID NO: 14 and SEQ ID NO: 16, while the pair enabling the amplification of sequences V4 to V6 of the 16S gene of Klebsiella pneumoniae was composed of primers with sequences SEQ ID NO: 20 and SEQ ID NO: 23. The results are shown in Figure 6. The inventors observed the absence of an amplicon for regions V1 and V2 of the 16S gene (line 3 and arrows on the left), indicating that the standard conditions for multiplex PCR were not compatible with the simultaneous amplification of two regions of the 16S gene.

[0126] During relay PCR with a ratio of 25:1, primer pair 1 was formed from primers with sequences SEQ ID NO:14 and SEQ ID NO:16, and primer pair 2 was composed of primers with sequences SEQ ID NO:28 and SEQ ID NO:33. The first primer pair was capable of amplifying regions V1-V2 of the 16S gene of Klebsiella pneumoniae, and the second chimeric primer pair was capable of amplifying regions V4-V6 of the 16S gene of Klebsiella pneumoniae. The results are shown in Figure 6. Thus, the inventors observed that the relay PCR conditions allowed simultaneous coamplification of two regions of the 16S gene (line 4), and that the amplicons of regions V1-V2 were present in greater amounts than the central region (despite their smaller size, the signals of amplicons V1-V2 were stronger).

[0127] Therefore, the method according to the invention makes it possible to co-amplify two target regions, contrary to standard multiplex PCR. The specific structure of the chimeric primer according to the invention therefore makes it possible to overcome the problems of the prior art, in particular the problems of multiplex PCR.

[0128] Example 4 of the method according to the invention compared with standard multiplex PCR for the amplification of three sequences of the gene 16S of Klebsiella pneumoniae (Figure 7) material and method The relay PCR reaction was performed as follows: 0.2 mM dNTPs, 4 mM MgCl, 0.05 units of FastStart polymerase (Roche), and buffer according to the supplier's conditions. Primer pairs were set at different concentrations: 1 mM for standard triplex, 1 mM for triplex relay primer pair 1, and 0.04 mM for triplex relay primer pairs 2 and 3. The cycle was as follows: denaturation at 95°C for 10 minutes, followed by 65 cycles of 95°C for 15 seconds, 55°C for 15 seconds, and 68°C for 18 seconds. Approximately 100 equivalent genome copies of Klebsiella pneumoniae were added to the mix to perform the amplification.

[0129] result In the context of a standard triplex PCR, three primer pairs were used at high concentrations (line 1): the primer pair allowing the amplification of specific sequences (V1 to V2) of the 16S gene of Klebsiella pneumoniae was formed by the primers with the sequences SEQ ID NO: 14 and SEQ ID NO: 16, the pair allowing the amplification of specific sequences (V4 to V6) of the 16S gene of Klebsiella pneumoniae was composed of the primers with the sequences SEQ ID NO: 20 and SEQ ID NO: 23, and the pair allowing the amplification of specific sequences (V7 to V9) of the 16S gene of Klebsiella pneumoniae was composed of the primers with the sequences SEQ ID NO: 24 and SEQ ID NO: 25.

[0130] In the context of triplex relay PCR (line 2), the primer pair enabling the amplification of specific sequences (V1-V2) of the 16S gene of Klebsiella pneumoniae was formed from primers with sequences SEQ ID NO: 14 and SEQ ID NO: 16. The pair enabling the amplification of specific sequences (V4-V6) of the 16S gene of Klebsiella pneumoniae was composed of primers with sequences SEQ ID NO: 28 and SEQ ID NO: 33. The pair enabling the amplification of specific sequences (V7-V9) of the 16S gene of Klebsiella pneumoniae was composed of primers with sequences SEQ ID NO: 34 and SEQ ID NO: 36. The primer pair targeting regions V1-V2 was set at a high concentration, while the other hybrid or chimeric primer pairs were set at a low concentration. The results are shown in Figure 7.

[0131] We observed that the targeted regions, particularly the variable regions V1 and V2 of the 16S gene of Klebsiella pneumoniae, were not sufficiently amplified by standard triplex PCR. In contrast, triplex relay PCR was able to amplify three regions of the 16S gene of Klebsiella pneumoniae: regions V1-V2 (343 bp), regions V7-V9 (413 bp by standard duplex PCR and 447 bp by duplex relay PCR), and regions V4-V6 (509 bp by standard duplex PCR and 543 bp by duplex relay PCR).

[0132] Thus, the method according to the invention makes it possible to co-amplify three targeted regions.

[0133] Example 5 of the method according to the invention using primer pair 1 comprising one primer at a high concentration and one hybrid or chimeric primer at a low concentration (Figure 8). material and method Two relay PCR reactions were performed in the following manner: 0.2 mM dNTPs, 4 mM MgCl, 0.05 units of FastStart polymerase (Roche), and buffer according to the supplier's conditions. Under the first duplex relay conditions, primers were set at different concentrations: 1 mM for the two primers in pair 1 and 0.04 mM for primer pair 2. Under the second duplex relay conditions, primers were set at different concentrations: 1 mM for the first standard primer in pair 1, 0.04 mM for the second chimeric primer in pair 1, and 0.04 mM for the chimeric primer in pair 2. The cycles were as follows: denaturation at 95°C for 10 minutes, followed by 55 cycles of 95°C for 15 seconds, 55°C for 15 seconds, and 68°C for 45 seconds. Approximately 100 equivalent genome copies of Klebsiella pneumoniae were added to the mix to perform the amplification.

[0134] result Within the context of the first duplex relay PCR (line 1), the primer pair allowing the amplification of sequences V1 to V2 of the 16S gene of Klebsiella pneumoniae is formed from primers of sequences SEQ ID NO: 14 and SEQ ID NO: 16 at a high concentration, and the pair allowing the amplification of regions V7 to V9 of the 16S gene of Klebsiella pneumoniae is composed of chimeric primers of sequences SEQ ID NO: 34 and SEQ ID NO: 36 at a low concentration.

[0135] In the context of the second duplex relay PCR (line 2), one single primer of pair 1 (SEQ ID NO: 14) was set at a high concentration, while the other primer (SEQ ID NO: 26), a hybrid primer, was set at a low concentration. The primer pair enabling the amplification of sequences V1V2 of the 16S gene of Klebsiella pneumoniae was formed from the primers of sequences SEQ ID NO: 14 and SEQ ID NO: 26, while the pair enabling the amplification of regions V7 to V9 of the 16S gene of Klebsiella pneumoniae was composed of the primers of sequences SEQ ID NO: 34 and SEQ ID NO: 37. The results are shown in Figure 8.

[0136] The inventors observed that under two conditions, amplicons corresponding to regions V1-V2 (343 bp (line 1) for the first duplex relay PCR, and 349 bp (line 2) for the second duplex relay PCR) and V7-V9 (447 bp (line 1) for the first duplex relay PCR, and 453 bp (line 2) for the second duplex relay PCR) were successfully amplified. However, we note that the use of a high concentration of paired single primers seems to further facilitate relay PCR according to the present invention. Furthermore, the brightness of the amplicon V1-V2 pieces, which are identical to the V7-V9 pieces, suggests that the amount of amplicon V1-V2 is much larger than that of amplicon V7-V9, and that amplicon V1-V2 is smaller. Thus, this variant of the inventive method retains the preferred amplification of regions where a high concentration of primers is used.

Claims

1. 1. A method for amplifying at least two different nucleic acid sequences of interest present in a sample using a primer pair 1 capable of amplifying a first nucleic acid sequence of interest (A) and at least one primer pair 2 capable of amplifying a second nucleic acid sequence of interest (B), comprising: a. contacting the sample with the primer pair 1 capable of amplifying the nucleic acid sequence (A) and the primer pair 2 capable of amplifying the second nucleic acid sequence (B), i. the primer pair 1 is formed from a first primer and a second primer capable of amplifying a nucleic acid corresponding to the first sequence (A); ii. A contacting step, wherein the primer pair 2 is formed from a first primer and a second primer capable of amplifying a nucleic acid corresponding to the second sequence (B); b. adding selected reagents and conditions that allow PCR amplification; c. amplifying the first and second nucleic acid sequences (A and B); Including, the sequence of the first primer of the primer pair 2 further comprises the sequence of the first or second primer of the primer pair 1 at its 5' end, and the sequence of the second primer of the primer pair 2 further comprises the sequence of the first or second primer of the primer pair 1 at its 5' end. method.

2. 2. The method of claim 1, wherein the sequence of the first primer of primer pair 2 and the sequence of the first or second primer of primer pair 1 are adjacent, and the sequence of the second primer of primer pair 2 and the sequence of the first or second primer of primer pair 1 are adjacent.

3. 3. The method of claim 1, wherein the second primer of primer pair 1 is a hybrid primer that contains the sequence of the first primer of primer pair 1 at its 5' end, and the sequences are adjacent.

4. The method according to any one of claims 1 to 3, wherein the concentration of the primer pair 2 is equal to or lower than the concentration of the primer pair 1.

5. The method according to any one of claims 1 to 4, wherein the concentration ratio of the primer pair 1 to the primer pair 2 is 1:1 to 1000:

1.

6. The method according to any one of claims 1 to 5, wherein the concentration ratio of the primer pair 1 to the primer pair 2 is 1:1 to 100:

1.

7. 7. The method of claim 1, further comprising contacting the sample with a primer pair 3 capable of amplifying a nucleic acid sequence corresponding to a third nucleic acid sequence (C), wherein the sequence of a first primer of primer pair 3 further comprises, at its 5' end, the sequence of the first or second primer of primer pair 1, and the sequence of a second primer of primer pair 3 further comprises, at its 5' end, the sequence of the first or second primer of primer pair 1.

8. The method of claim 7 , wherein the concentration of primer pair 1 is greater than the concentration of primer pair 2 and / or primer pair 3.

9. 9. The method of claim 7 or 8, wherein the concentration of primer pair 2 is equal to the concentration of primer pair 3.

10. 10. The method of any one of claims 7 to 9, further comprising contacting the sample with a primer pair 4 capable of amplifying a fourth nucleic acid sequence (D), wherein the sequence of a first primer of primer pair 4 further comprises, at its 5' end, the sequence of the first or the second primer of primer pair 1, and the sequence of a second primer of primer pair 4 further comprises, at its 5' end, the sequence of the first or the second primer of primer pair 1.

11. 11. The method of claim 10, wherein the concentration of primer pair 4 is less than the concentration of primer pair 1.

12. The method of claim 10 or 11, wherein the concentrations of primer pair 2, primer pair 3, and primer pair 4 are the same.

13. The method according to any one of claims 1 to 12, further comprising the step of sequencing the nucleic acid amplified by said amplifying step.

14. The method according to any one of claims 1 to 13, wherein the amplification of said nucleic acid sequence of interest makes it possible to detect and identify at least one microorganism involved in at least one infectious disease.

15. 15. The method of claim 14, wherein the microorganisms are identified by fluorescent probes, fluorescent marking, melting curves, nested PCR, quantitative PCR, reverse transcription PCR, or DNA sequencing.

16. A kit comprising: Primer pair 1 targeting the variable region V1-V2 of the gene 16S, composed of: ■ A sense primer of SEQ ID NO: 14 or SEQ ID NO: 15, and ■ Antisense primer SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 26 or SEQ ID NO: 27, Primer pair 2 targeting the variable regions V4 to V6 of the gene 16S, consisting of: ■ A sense primer of SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, and ■ Antisense primer SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, Includes a kit.

17. A kit comprising: Primer pair 1 targeting the variable region V1-V2 of the gene 16S, composed of: ■ A sense primer of SEQ ID NO: 14 or SEQ ID NO: 15, and ■ Antisense primer SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 26 or SEQ ID NO: 27, Primer pair 2 targeting the variable region V7 to V9 of the gene 16S, consisting of: ■ A sense primer of SEQ ID NO: 34 or SEQ ID NO: 35, and ■ Antisense primer SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, Includes a kit.

18. A kit comprising: Primer pair 1 targeting the variable region V1-V2 of the gene 16S, composed of: ■ A sense primer of SEQ ID NO: 14 or SEQ ID NO: 15, and ■ Antisense primer SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 26 or SEQ ID NO: 27, Primer pair 2 targeting the variable regions V4 to V6 of the gene 16S, consisting of: ■ A sense primer of SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, and ■ Antisense primer SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, Primer pair 3 targeting the variable regions V7 to V9 of said gene 16S, consisting of: ■ A sense primer of SEQ ID NO: 34 or SEQ ID NO: 35, and ■ Antisense primer SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, Includes a kit.

19. Use of a kit according to any one of claims 16 to 18 for carrying out the method according to any one of claims 1 to 15 and for detecting at least one microorganism present in the case of at least one infectious disease.

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