Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/686—Polymerase chain reaction [PCR]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/16—Primer sets for multiplex assays

Definitions

• the invention relates to the field of the detection and amplification of at least one nucleic acid of interest, in particular in infectious diagnosis.
• the invention thus relates to a method for amplifying at least two distinct nucleic acid sequences present in a sample from a pair of primers 1 amplifying a first nucleic acid (A) and at least one pair of primers. hybrids or chimeras 2 amplifying a second nucleic acid (B).
• the first primer such as a forward primer of primer pair 2 further comprises in 5 'the sequence of the first primer of primer pair 1
• the second primer of primer pair 2 further comprises in 5 ′ the sequence of the first or of the second primer of the pair of primers 1.
• the invention also relates to the primers, a kit capable of implementing said method and its uses.
• PCR Polymerase Chain Reaction
• Coli is suspected, a pair of primer allowing the amplification of a nucleic acid specific for E. Coli can be used.
• the origin of the infection is not known, it is necessary to use pairs of primers specific to each of the pathogens to be detected.
• a so-called simplex PCR approach a single pair of primers is used and a PCR reaction must be reproduced as many times as necessary with respect to the number of viruses, bacteria or yeasts that one wishes to identify as responsible for the infection. .
• PCR approach multiplex, all the pairs of primers are added in a single reaction and all the pathogens sought will be identified at one time.
• Multiplex PCR is a PCR allowing the simultaneous amplification of several different nucleic acid sequences from the same biological sample. It allows several targeted amplifications to be carried out in a single reaction. Multiplex PCR is therefore particularly suitable in infectious diagnosis. During infectious diagnosis, pairs of different primers are used to target several bacteria, viruses or yeasts during the same PCR reaction in the presence of the target biological sample.
• the pairs of primers used in multiplex PCR need to be highly optimized so that all the primers can work at the same temperature during PCR, making it possible in particular to avoid the phenomenon of cross reactions.
• multiplex PCR is able to reveal at least one pathogen in community-acquired pneumonia in 77% of hospitalized patients, compared to 39% by culture (NJ Gadsby CD Russell MP McHugh Comprehensive molecular testing for respiratory pathogens in community -acquired pneumonia. Clin Infect Dis 2016).
• it can detect up to three times more pathogens in the stool than traditional 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).
• a single test can demonstrate the presence in the sample of several infectious agents in less than two hours.
• Today, panels are already offered to clinicians for meningitis, sexually transmitted infections, respiratory infections and infectious enterocolitis.
• multiplex PCR is limited to the detection of a predefined quantity of pathogens, of which the pairs of primers targeting at least one fragment of a specific gene for said pathogens are included in the panel.
• the detection kit can comprise a very large number of primers therefore making it possible to detect a large quantity of microorganisms.
• a pathogen is detected by a pair of primers whose amplicon is specific to said pathogen.
• a high concentration of different primers in a single test generates a contradictory deleterious effect.
• a phenomenon of competition between the primers appears, resulting in an inhibition of the primers.
• nonspecific amplifications are much more likely due to the multitude of primers used. Consequently, multiplexing that is too high induces very poor detection sensitivity.
• the invention aims to solve the problems and drawbacks of the prior art and in particular to avoid or limit the degradation of the sensitivity of the multiplex PCR which can result in the non-amplification or poor amplification of targeted nucleic acids because of the phenomenon of competition between the primers when they are present in one and the same test in too large quantities as well as to suppress or limit the degradation of the sensitivity of the multiplex PCR linked to the nonspecific amplifications which consume undesirably the reagents of 'amplification.
• the invention relates to a method for amplifying at least two distinct nucleic acid sequences of interest present in a sample from a pair of primers 1 capable of amplifying a first sequence of acids. nucleic acids of interest (A) and at least one pair of hybrid primers 2 capable of amplifying a second sequence of nucleic acids of interest (B), said method comprising the following steps: a. bringing the sample into contact with the pair of primers 1 capable of amplifying the first sequence of nucleic acids (A) and the pair of primers 2 capable of amplifying the second sequence of nucleic acids (B),
• the one pair of primers consisting of a first primer and a second primer capable of amplifying a nucleic acid corresponding to the first nucleic acid sequence (A)
• the pair of primers consisting of a 2 first primer and a second primer capable of amplifying a nucleic acid corresponding to the second nucleic acid sequence (B)
• B the pair of primers consisting of a 2 first primer and a second primer capable of amplifying a nucleic acid corresponding to the second nucleic acid sequence
• the sequence of the first primer of the pair of primers 2 further comprises in 5 'the sequence of the first primer of the pair of primer 1 and the sequence of the second primer of the pair of primers 2 furthermore comprises, in 5 ′, the sequence of the second primer of the pair of primers 1.
• the sequence of the first primer of the pair of primers 2 (called chimeric or hybrid) further comprises, at 5 ′, the sequence of the first or of the second primer of the pair of primer 1 and the sequence of the second primer of primer pair 2 further comprises in 5 'the sequence of the first or second primer of primer pair 1.
• the concentration of the pair of primers 2 is less than or equal to that of the pair of primers 1, more preferably less.
• the method according to the invention can also comprise a third pair of primers B, a fourth pair of primers 4 etc., each comprising a first primer, the sequence of which further comprises, at 5 ′, the sequence of the first or of the second. primer of the pair of primers 1 and a second primer, the sequence of which also comprises, in 5 ′, the sequence of the first or of the second primer of the pair of primers 1.
• 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).
• the first oligonucleotide of the pair of primers 1, 2, 3 or 4 can be a forward or reverse primer and the second oligonucleotide of the pair of primers 1, 2, 3 or 4 can be an reverse or forward primer.
• the method according to the invention is a multiplex PCR and makes it possible to carry out differential quantitative amplifications, that is to say that the targets are not amplified in the same way leading to differences in quantities produced for the same number. cycle between targets.
• Said PCR according to the invention is also called Relay PCR or PCR according to the invention in the remainder of the description.
• the PCR according to the invention makes it possible in particular to detect and identify microorganisms such as bacteria, viruses, yeasts involved in infectious diseases. Diseases can be detected in a subject such as an animal or human, or in a plant.
• the invention also relates to the primers and kits comprising specific primers, a detection kit capable of implementing the method of the invention and its use for detecting the microorganisms present during infectious diseases.
• FIG. 1 represents the principle of the PCR Relay according to the invention.
• FIG. 2A represents a standard Duplex (multiplex with 2 targets) PCR assay with a pair of primers 1 targeting Staphylococcus aureus and a pair of primers 2 targeting Escherichia coli.
• the ratio of primer torque 1: primer pair 2 is equal to 1: 1.
• black amplification curves targeting a specific gene for Escherichia coli.
• gray amplification curves targeting a specific gene of Staphylococcus aureus.
• FIG. 2B represents a relay PCR assay according to the invention comprising a pair of primers 1 targeting Staphylococcus aureus and a pair of primers 2 targeting Escherichia coli.
• the ratio of primer torque 1: primer torque 2 is equal to 10: 1.
• black amplification curves targeting a specific gene for Escherichia coli.
• gray amplification curves targeting a specific gene of Staphylococcus aureus.
• FIG. 2C represents a relay PCR assay according to the invention comprising a pair of primers 1 targeting Staphylococcus aureus and a pair of primers 2 targeting Escherichia coli.
• the ratio of primer torque 1: primer pair 2 is equal to 1: 1.
• black amplification curves targeting a specific gene for Escherichia coli.
• gray amplification curves targeting a gene specific for Staphylococcus aureus.
• FIG. 3A represents a standard Duplex PCR assay with a pair of primers 1 targeting Klebsiella pneumoniae and a pair of primers 2 targeting Staphylococcus aureus.
• the ratio of primer torque 1: primer pair 2 is equal to 1: 1.
• black amplification curves targeting a specific gene for Klebsiella pneumoniae.
• gray amplification curves targeting a specific gene of Staphylococcus aureus.
• FIG. 3B represents a relay PCR assay according to the invention comprising a pair of primers 1 targeting Klebsiella pneumoniae and a pair of primers 2 targeting Staphylococcus aureus.
• the ratio of primer torque 1: primer torque 2 is equal to 10: 1.
• black amplification curves targeting a specific gene for Klebsiella pneumoniae.
• gray amplification curves targeting a specific gene of Staphylococcus aureus.
• FIG. 3C represents a relay PCR assay according to the invention comprising a pair of primers 1 targeting Klebsiella pneumoniae and a pair of primers 2 targeting Staphylococcus aureus.
• the ratio of primer torque 1: primer pair 2 is equal to 1: 1.
• black amplification curves targeting a specific gene for Klebsiella pneumoniae.
• gray amplification curves targeting a specific gene of Staphylococcus aureus.
• FIG. 4 represents the structure of a pair of primers used in standard PCR capable of amplifying a target sequence, for example A comprising a sense primer of sense sequence FW1 and an antisense primer of sequence RT1; and the structure of a pair of hybrid or chimeric primers according to the invention used in Relay PCR capable of amplifying a target sequence, for example B.
• the pair of hybrid or chimeric primers comprises a sense primer of sequence FW1 + FW2 and an antisense primer of sequence RT1 + RT2.
• the FW2 sequence is capable of hybridizing with a region of the target sequence B, the FW1 sequence corresponds to the sequence of the sense primer of the primer pair 1 present in greater concentration in the mixture, and said sequences FW1 and FW2 are contiguous to form a single forward primer between 12 and 140 nucleotides in length.
• the pair of hybrid or chimeric primers also comprises an antisense primer of sequence RT1 + RT2, the sequence RT2 is complementary to a region of the target sequence B, the sequence RT1 corresponds to the sequence of the antisense primer of the pair of primer 1 present in greater concentration in the mixture, said sequences FW1 and FW2 are contiguous to form a single primer with a length of between 12 and 140 nucleotides.
• the pair of primers 1 present in greater concentration in the mixture is capable of amplifying the amplicon generated by the pair of primer 2, during the first cycle, and therefore of also amplifying the target sequence B.
• the pair of primers 1 is capable of amplifying the target sequence A but also the target sequence B from the second cycle of PCR.
• Figure 5 shows the sequence of the bacterial 16S gene.
• Said sequence comprises: 9 variable regions specific to each species, and 10 regions conserved between species, contiguous to said variable regions. Variable regions are numbered VI through V9.
• the conserved regions make it possible to define, according to the knowledge of those skilled in the art, the nucleotide sequence of the sense and antisense primers capable of hybridizing to these conserved regions surrounding at least one variable region of interest (for example VI to V2) .
• Figure 6 is an agarose gel representing PCR results according to the standard Duplex (2-target multiplex) method or according to the method of the present invention Duplex Relais. Under all conditions, 100 copies of the Klebsiella pneumoniae genome were added. Lane 1: amplicon of the 5 ′ region of the 16S gene (amplicon covering the variable areas VI to V2) obtained with primers targeting areas conserved contiguous to said variable areas in high concentration (PCR control). 2: amplicon of the central region of the 16S gene (amplicon covering the variable areas V4 to V6) obtained with primers targeting areas conserved contiguous to said variable areas in high concentration (PCR control).
• 3 standard multiplex PCR amplicon of the 5 ′ (V1-V2) and central (V4-V6) regions of the 16S gene with two pairs of primers targeting each variable zone respectively, the primers being in high concentrations.
• 4 2 amplicon regions of 16S gene according to the method of the present invention Duplex Relay where the first pair of primers target the 5 'region (V1-V2) and the 2nd pair of primers or chimeric hybrid comprises a sequence capable of targeting the central region (V4-V6) but also the sequence of the first pair of primers, said primers are used at high (first pair of primers) and low concentrations (second pair of primers) respectively.
• the arrows indicate the amplification or non-amplification of the amplicon of interest (here the V1-V2 region of the 16S gene).
• FIG. 7 is an agarose gel representing results of PCR according to the standard Triplex (3-target multiplex) method or according to the method of the present invention (Triplex Relais). Under all conditions 100 copies of the Klebsiella pneumoniae genome were added. 1: amplicons obtained with a standard Triplex PCR, the primers being at ImM each. 2: amplicons obtained with a Triplex Relay PCR where the chimeric or hybrid primers were used at a low concentration of 0.04 mM.
• the Triplex Relais amplicons V4-V6 and V7-V9 are slightly larger than their standard Triplex counterpart because of the length of the chimeric primers which increase their size after polymerization.
• FIG. 8 is an agarose gel representing results of PCR according to the relay multiplex PCR method comprising a single standard primer in high concentration. Under all conditions 100 copies of the Klebsiella pneumoniae genome were added. 1: amplicons obtained with a Duplex Relay PCR comprising a pair of primer 1 in high concentration targeting the V1-V2 region of the 16S gene and a pair of primer 2 hybrids or chimeras in low concentration targeting the V7-V9 region of the 16S gene .
• amplicons obtained with a Duplex Relay PCR comprising a pair of primer 1 targeting the V1-V2 region of the 16S gene consisting of a sense primer in high concentration and a hybrid or chimeric antisense primer in low concentration comprising 5 ' the sequence of the sense primer and a pair of primer 2 hybrids or chimeras in low concentration targeting the V7-V9 region of the 16S gene.
• the reverse primer of the first pair as well as the sense and reverse primers of the second pair are hybrid or chimeric primers. Only the directional primer of the primer pair 1 is present in high concentration.
• the term “patient” is understood to mean a subject who may be a human being or an animal, preferably a human being or a mammal.
• the subject is a human patient, regardless of age or sex. Newborns, infants and children are also included.
• diagnosis means a test which aims to detect and identify one or more pathology (s), in particular the detection and identification of infectious diseases in a subject or a plant in order to be able to administer to the subject or to the plant a treatment corresponding to the pathology.
• the term “pair of primers” or “pair of oligonucleotides” means two oligonucleotides making it possible to amplify a target sequence in the context of a PCR.
• primer or “oligonucleotide” or “primer” is meant within the meaning of the invention a short segment of nucleic acids of one to several tens of nucleotides. Said primers are capable of hybridizing with a complementary sequence. In the context of the invention, they are capable of hybridizing with a fragment of the target sequence.
• the target sequence can be a sequence of nucleic acids (DNA or RNA).
• FW1 sequence means the sequence of an oligonucleotide of one to a few tens of nucleotides, in particular the sequence of a sense primer capable of hybridizing with the sequence complementary to the sequence d 'interest.
• RT1 sequence 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 sequence complementary to the sequence of interest.
• the term “pair of primers capable of amplifying a nucleic acid sequence of interest” means a pair of primers comprising a sense primer and an antisense primer capable of amplifying a determined sequence of interest by PCR.
• the nucleic acid sequence of interest can be a sequence present in the variable part of the 16S gene of a bacterium. Said primers being designed to target said sequence of interest according to the general knowledge of those skilled in the art.
• the term “amplification” is understood to mean the amplification of the target sequence during a PCR reaction and therefore the increase in the number of copies of the target sequence.
• the primers hybridize with the complementary sequence denatured beforehand, followed by the elongation of the strands by the action of the polymerase. The reaction continues with several successive cycles making it possible to increase the number of copies of the amplicon.
• amplicon is intended to mean a nucleic acid fragment corresponding to the sequence amplified during PCR and delimited by the two primers used. The amplicon is therefore a copy of the target sequence.
• hybrid primer or “chimeric primer” means a primer comprising a nucleic acid sequence capable of hybridizing with a strand complementary to the target sequence and at 5 ′ an acid sequence. nucleic acid corresponding to the sequence of a primer belonging to another pair of primer, for example the sequence of a forward or reverse primer of another pair of primer.
• the pair of primers 2, B and 4 consists of hybrid or chimeric primers.
• the primer pair 1 comprises a “standard” primer and a hybrid or chimeric primer.
• the first oligonucleotide of the pair of primers 2 comprises a sequence capable of hybridizing with a specific nucleic acid sequence (target B) and at 5 ′ a nucleic acid sequence corresponding to the sequence of the first oligonucleotide of the pair of primers 1 capable of hybridizing with another specific nucleic acid sequence (target A).
• concentration ratio is understood to mean the concentration ratio of the pair of primers 1 relative to a second pair of primers such as the pair of primers 2.
• a ratio of 1: 1 corresponds to an identical concentration of the first couple and the second couple and a ratio of 10: 1 corresponds to a concentration 10 times greater of the first couple compared to the second.
• the subject of the invention is therefore a novel so-called multiplex PCR method.
• So-called standard multiplex PCRs are known which make it possible to identify a very large number of target sequences.
• Such standard multiplex PCRs require the presence in high concentration of each pair of primers targeting a particular nucleic acid.
• each pair of primers is therefore present at the same high concentration during the multiplex PCR reaction. From at least 4 or 5 pairs of different primers, the reaction conditions begin to deteriorate.
• the appearance of a phenomenon of competition between the primers results in an inhibition of the specific hybridizations of the primers.
• nonspecific amplifications can occur by the multiplicity of primers used.
• standard multiplex PCRs have very poor detection sensitivity.
• the invention relates to a novel multiplex PCR method known as the Multiplex Relay PCR (or Relay PCR).
• a novel multiplex PCR method known as the Multiplex Relay PCR (or Relay PCR).
• Relay PCR either the method according to the invention, a single pair of primers or a single primer is used in high concentration, all the other primers are in very low concentration. Thanks to the invention, it is possible to maintain an extreme sensitivity for all the targeted nucleic acids.
• nucleic acids it is also possible to promote the amplification of certain nucleic acids over others (quantitative amplification asymmetry or differential quantitative amplification), in particular the nucleic acids of target A, over others. These nucleic acids preferentially amplified will thus be present in greater quantity. In diagnosis, favoring certain nucleic acids has a medical advantage because some nucleic acids are more informative than others.
• the invention relates to a method for amplifying at least two distinct nucleic acid sequences of interest present in a sample from a pair of primers 1 capable of amplifying a first sequence of nucleic acids of interest ( A) and at least one pair of primers 2 capable of amplifying a second nucleic acid sequence of interest (B), said method comprising the following steps: a. bringing the sample into contact with the pair of primers 1 capable of amplifying the first sequence of nucleic acids (A) and the pair of primers 2 capable of amplifying the second sequence of nucleic acids (B), i .
• the pair of primers 1 consisting of a first primer and a second primer capable of amplifying a nucleic acid corresponding to the first nucleic acid sequence (A)
• the pair of primers 2 consisting of a first primer and a second primer capable of amplifying a nucleic acid corresponding to the second nucleic acid sequence (B)
• B second nucleic acid sequence
• the sequence of the first primer of the pair of primers 2 further comprises in 5 'the sequence of the first primer of the pair of primer 1 and the sequence of the second primer of the pair of primers 2 further comprises in 5 ′ the sequence of the second primer of the pair of primers 1.
• step c. The amplification of the first and second nucleic acid sequence (A and B) is carried out by an amplification reaction based on a polymerization (elongation) of the nucleic acid sequence ( A) and the nucleic acid sequence (B) present in the sample
• the sequence of the first primer of the pair of primers 2 further comprises, in 5 ′ the sequence of the first primer or of the second primer of the pair of primers 1 and the sequence of the second primer of the pair of primers 2 further comprises, in 5 ′, the sequence of the first or of the second primer of the pair of primers 1.
• the sequence of the first primer of the primer pair 2 and the sequence of the first or second primer of the primer pair 1 are contiguous and the sequence of the second primer of the primer pair 2 and the sequence of the first or the second primer of the pair of primers 1 are contiguous.
• said sequences are not contiguous but are spaced apart by a few nucleotides with a spacer (or “linker” or “spacer”) of a size between 1 and 12 nucleotides.
• the method according to the invention is characterized in that: the sequence of the second primer of the pair of primers 1 further comprises in 5 ′ the sequence of the first primer of said pair of primer 1, said sequences are contiguous and the sequence of the first primer of primer pair 2 further comprises in 5 'the sequence of the first primer of primer pair 1 and the sequence of the second primer of primer pair 2 further comprises further 5 'the sequence of the first primer of the pair of primers 1, said sequences are contiguous.
• sequence of the second primer of the pair of primers 1 further comprises, at 5 ′, the sequence of the first primer of said pair of primer 1 and / or the sequence of the first primer of the pair of primers 2 further comprises in 5 'the sequence of the first primer of primer pair 1 and the sequence of the second primer of primer pair 2 further comprises in 5 ′ the sequence of the first primer of primer pair 1, said sequences are spaced a few nucleotides apart with a spacer (or “linker” or “spacer”) of a size of between 3 and 12 nucleotides.
• the method uses: at least one pair of primers 1 capable of amplifying a target sequence A comprising: a sense primer of sequence FW1 complementary to a fragment of target sequence A, said primer being of a size between 6 and 70 nucletoids, and an antisense primer of sequence RT1 complementary to a fragment of the target sequence A, said primer being of a size between 6 and 70 nucletoids, and at least one pair of primers 2 capable of amplifying a target sequence B comprising: a forward primer of sequence FW1 or RT1 and of sequence FW2, the sequence FW2 being complementary to a fragment of the target sequence B, said sequences FW1 or RT1 and FW2 being contiguous to form a single primer with a size of between 12 and 140 nucletoids, and an antisense primer of sequence FW1 or RT1 and of RT2 sequence, the RT2 sequence being complementary to a fragment of the target sequence B, said FW1 or RT1 and RT2 sequence being complementary to a fragment of the
• the invention also relates to a pair of hybrid or chimeric primers comprising: a sense primer (or first oligonucleotide) of sequence FW1 or RT1 and of sequence FW2, the sequence FW2 is complementary to a fragment of the target sequence B, the FW1 or RT1 sequence corresponds respectively to the sequence of the sense or antisense primer of primer pair 1 present in greater concentration in the mixture, said sequences FW1 or RT1 and FW2 are contiguous to form a single sense primer d 'a length of between 12 and 140 nucleotides, and an antisense primer (or second oligonucleotide) of sequence FW1 or RT1 and sequence RT2, the sequence RT2 is complementary to a fragment of the target sequence B, the sequence RT1 or FW1 corresponds respectively to the sequence of the antisense or sense primer of the primer pair 1 present in an equal or greater concentration in the mixture, said sequences FW1 or RT1 and FW2 are contiguous to form
• the invention also relates to a pair of hybrid or chimeric primers comprising: a sense or antisense primer (or first oligonucleotide) of sequence FW1 or RT1, the FW1 or RT1 sequence is complementary to a fragment of the target sequence A, present in greater concentration in the mixture, said sequences are between 6 and 70 nucleotides in length, and a hybrid sense or antisense primer or chimera (or second oligonucleotide) of sequence FW1 and of sequence RT1 or of sequence RT1 and of sequence FW1, said primers being present in a lower concentration compared to the first oligonucleotide of sequence FW1 or RT1 in the mixture, said sequences FW1 and RT1 or RT1 and FW1 are contiguous to form a single primer between 12 and 140 nucleotides in length.
• a sense or antisense primer or first oligonucleotide of sequence FW1 or RT1 sequence is complementary to a
• the PCR reaction takes place in a mixture comprising a pair of primers 1 capable of hybridizing and amplifying a first sequence of nucleic acids (A) and a pair of primers 2 capable of hybridizing and amplifying a second sequence of nucleic acids (B) as is the case for a standard Duplex PCR.
• the Duplex Relay PCR differs from the standard Duplex PCR by the fact of using hybrid or chimeric primers for the second pair of primers, and possibly for a forward or reverse primer of the first pair of primers.
• the primer pair 1 is able to also amplify the amplicon generated by the primer pair 2 and therefore the target sequence B.
• the Duplex Relay PCR comprises in particular bringing the sample into contact with the reaction mixture comprising the pair of primers 1 capable of amplifying the nucleic acid sequence (A) and the pair of primers 2 capable of amplifying the second sequence of nucleic acids (B).
• the pair of primers 1 consists of a first sense primer (for example of sequence FW1) and of a second antisense primer (for example of sequence RT1) capable of each hybridizing to the sequence complementary to one of the strands. of nucleic acid (A) and amplifying the sequence corresponding to the sequence of the complementary strand between the two primers. Said complementary sequence being the target sequence (A).
• the pair of primers 2 consists of a first sense primer (for example of sequence FW1-FW2) and of a second antisense primer (for example of sequence RT1-RT2) capable of each hybridizing to the complementary sequence d '' one of the strands of the nucleic acid (B) and amplifying the sequence corresponding to the sequence of the complementary strand between the two primers.
• Said complementary sequence being the target sequence (B).
• the reaction mixture includes all the reagents and conditions necessary to perform PCR amplification. Said reagents possibly being initially present in the reaction mixture or added after bringing the sample into contact.
• the reagents required include in particular dNTPs and the polymerase that a person skilled in the art will be able to choose depending on the PCR and the reaction conditions.
• the polymerase is the polymerase from Thermophilus aquaticus or the so-called Klenow fragment of this polymerase.
• a PCR cycle includes a denaturation step which aims to separate the double strand in order to obtain two single strands followed by the hybridization step.
• the primers will recognize the complementary sequence present on one of the single strands previously denatured and will then hybridize.
• An elongation step follows which allows the polymerases to synthesize the complementary strand of the single strand. This strand is synthesized from the free dNTPs present in the reaction medium. The duration of this step depends on the length of the sequence to be amplified.
• the PCR reaction continues with a second cycle, third cycle etc. which each include the same steps. At the end of the PCR, an amplification of the sequence between the primers is thus obtained. Said sequence corresponding to the amplicon.
• the amplification of the first and second nucleic acid sequence is based on an amplification of the target nucleic acid sequence (A) and of the target nucleic acid sequence (B) present in the sample, preferably simultaneous amplification of the target nucleic acid sequence (A) and the target nucleic acid sequence (B) present in the sample.
• the method according to the invention is characterized in that the first primer of the pair of primers 2 further comprises in 5 ′ the sequence of the first primer of the pair of primers 1, and the second primer of the pair of primers 2 comprises furthermore in 5 'the sequence of the second primer of pair of primers 1.
• the sequences of the first primer of pair 2 and of the first primer of pair 1 are contiguous and the sequences of the second primer of pair 2 and of the second primer of pair 1 are also contiguous.
• the second primer of the pair of primers 2 further comprises, in 5 ′ the sequence of the first or of the second primer of the pair of primers 1.
• the sequences of the first primer of the pair 2 and of the first primer of couple 1 are contiguous and the sequences of the first or second primer of couple 2 and of the second primer of couple 1 are also contiguous.
• the first primer of the pair of primers 2 comprises a sequence of nucleic acids capable of hybridizing with the sequence complementary to the sequence to be amplified, such as the target sequence (B) and, in addition, in 5 ′ the sequence of the first or second primer of the pair of primers 1, and the second primer of the pair of primers 2 comprises a nucleic acid sequence capable of hybridizing with the sequence complementary to the sequence to be amplified, such as the target sequence ( B) and in addition, at 5 ′, the sequence of the first or second primer of the pair of primers 1.
• the nucleic acid sequence capable of hybridizing with the complementary sequence to be amplified, such as the target sequence (B) being separate for primer 1 and primer 2.
• primer 1 can be a forward primer and primer 2 a reverse primer.
• the first pair of primers is able to amplify the first sequence (A) and the second sequence (B) from the second cycle of PCR.
• the first pair of primers is able to amplify the first sequence (A) and the second pair of primers is able to amplify the second sequence (B).
• nucleic acid sequences are generated comprising the sequence of the first primer of the first pair and the sequence complementary to sequence (A), nucleic acid sequences comprising the sequence of second primer of the first pair and the sequence complementary to sequence (A), nucleic acid sequences comprising the sequence of the first primer of the second pair and the sequence complementary to sequence (B), and nucleic acid sequences comprising the sequence of the second primer of the second pair and the sequence complementary to sequence (B).
• the first primer of the second pair comprises the sequence of the first primer of the first pair and of 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 of a sequence capable of hybridizing to sequence (B ).
• the pair of primers 1 is able to amplify the nucleic acid sequences generated during the first cycle (FIG. 1).
• the sequence of the first primer of the first pair is chosen 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 chosen from among SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6
• the sequence of the first primer of the second pair is chosen from SEQ ID NO: 7 or SEQ ID NO: 9
• the sequence of the second primer of the second pair is chosen from SEQID NO: 8 or SEQID NO: 10.
• the 16S gene comprises conserved regions and variable regions, in particular 9 variable regions and 10 conserved regions (cf. FIG. 5). Variable regions are numbered VI through V9 and are all surrounded by conserved regions. Those skilled in the art, by virtue of their general knowledge, are perfectly able to determine said regions on the 16S gene. Tremblay et al. 2015 (Frontiers in Microbiology) describes, for example, one of these many methods for amplifying and sequencing these regions in order to identify the bacteria present in a sample.
• the sequences of interest correspond to variable sequences of a single gene or of at least two distinct genes, more preferably the sequences of interest correspond to the variable sequences of the 16S gene and said primers. are capable of hybridizing with at least ten nucleotides of a conserved sequence of the 16S gene.
• the sense primer can hybridize with at least ten nucleotides of a sequence conserved from the 16S gene in 5 'of a variable sequence of said 16S gene and the antisense primer can hybridize with at least ten nucleotides of a conserved sequence of the 16S gene at B 'of a variable sequence of said 16S gene.
• the inventors have designed primers making it possible to hybridize specifically on the conserved regions contiguous with the variable regions of interest such as V1-V2, V3, V4-V6 and V7-V9.
• the amplicon generated corresponds to the sequences of the variable regions V1-V2 or V3 or V4-V6 or V7-V9, the sequence of which is specific to a pathogen.
• the sequence of the first primer (sense primer) of the first pair is chosen 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;
• the sequence of the second primer (reverse primer) of the first pair is chosen 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.
• the sequence of the first primer (sense primer) of the first pair is SEQ ID NO: 14 or SEQ ID NO: 15 and the sequence of the second primer (antisense primer) of the first pair is chosen from SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 26 and SEQ ID NO: 27.
• the sequence of the first primer (sense primer) of the second pair is chosen from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: S4 or SEQ ID NO: 35; the sequence of the second primer of the second pair is chosen 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. Said sequences being listed in Table IB below.
• the first pair of primers targeting the V1-V2 variable regions of the 16S gene consists of: a forward 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.
• the second pair of hybrid or chimeric primers targeting the variable regions V4 to V6 of the 16S gene consists of: - a forward primer having at least 70% sequence homology with SEQ
• the second pair or the third pair of primers targeting the variable regions V7 to V9 of the 16S gene consists of: a sense primer having at least 70% sequence homology 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.
• the sequence homology of 70% concerns, in particular the first ten nucleotides in 3 'of said sequence.
• the first pair of primers targeting the V1-V2 variable regions of the 16S gene consists of: a sense primer of SEQ ID NO: 14 or SEQ ID NO: 15, and an antisense primer SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 26 or
• the second pair of hybrid or chimeric primers targeting the variable regions V4 to V6 of the 16S gene consists of: a sense primer of SEQ ID NO: 28 or SEQ ID NO: 29 or SEQ ID NO: 30 and of an antisense primer SEQ ID NO: 31 or SEQ ID NO: 32 or SEQ ID NO: 33.
• the second pair or the third pair of primers targeting the variable regions V7 to V9 of the 16S gene consists of: a sense primer of SEQ ID NO: 34 or SEQ ID NO: 35 and an antisense primer SEQ ID NO: 36 or SEQ ID NO: 37 or SEQ ID NO: 38.
• the concentration of the pair of primers 2 may be less than, greater than or equal to that of the pair of primers 1. It is thus possible to favor or not a pair of primers over another. and therefore the amplification of a sequence of interest.
• the concentration of the pair of primers 2 is less than or equal to that of the pair of primers 1, more preferably less.
• the concentration of the pair of primers 2 is lower than that of the pair of primers 1, the pair of primers 2 is disadvantaged with respect to the first pair.
• the amplicons generated by the pair of primers 1 will thus be present in greater quantity.
• sequencing is then carried out, the amplicons of target A will be over-represented and will therefore be sequenced preferentially. Obtaining their sequence will then be faster.
• the method according to the invention makes it possible to generate a quantity of amplicons by the pair of primers 1 at least 4 times greater than the amplicons generated by the pair of primers 2.
• the amplicons generated by the second pair will therefore be present in lower quantity and will come out slower during sequencing.
• the concentration ratio between the pair of primers 1 and the pair of primers 2 is between 1: 1 and 1000: 1, even more preferably between 1: 1 and 100: 1.
• the concentration ratio between the pair of primers 1 and the pair of primers 2 is equal to 10: 1, more preferably equal to 25: 1.
• Such a ratio can, for example, correspond to a concentration of the pair of primers 1 equal to ImM and a concentration of the pair of primers 2 equal to 0.1 mM or 0.04 mM.
• the method according to the invention may further comprise the sample contacting with a 3 rd pair of primers adapted to amplify a 3 rd nucleic acid sequence (C).
• the first primer of the pair of primers 3 comprises a nucleic acid sequence capable of hybridizing with the complementary sequence (C) and, in addition, at 5 ′ the sequence corresponding to the sequence of the first primer or of the second primer of the pair of primers 1.
• the second primer of the pair of primers 3 comprises a nucleic acid sequence capable of hybridizing with the complementary sequence (C) and, in addition, in 5 'the sequence corresponding to the sequence of the first or of the second primer of primer pair 1.
• the method according to the invention thus comprises 3 pairs of different primers making it possible to amplify 3 different target nucleic acid sequences, in particular an A sequence, a B sequence and a C sequence.
• Said method is a so-called Triplex Relay PCR.
• 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 greater concentration and the sequence of the sense or antisense primer of pair d primer 1 present in highest concentration.
• the method according to the invention uses: at least one pair of primers 1 capable of amplifying a target sequence A comprising:
• a reverse primer complementary sequence RT1 to a fragment of the target sequence A said primer being of a size between 6 and 70 nuclétoides, and at least one pair of primers hybrids or chimeras 2 capable of amplifying a target sequence B comprising:
• sequence FW1 or RT1 and of sequence FW2 being complementary to a fragment of the target sequence B, said sequence FW1 or RT1 and FW2 being contiguous to form a single primer with a size of between 12 and 140 nucletoids, and
• a reverse primer of FW1 sequence or sequence RT1 and RT2 RT2 sequence being complementary to a fragment of the target sequence B, said FW1 sequence RT1 or RT2 and being contiguous to form a single primer of a size between 12 and 140 nucletoids, and at least one pair of primers B capable of amplifying a target sequence C comprising:
• sequence FW1 and FW3 being complementary to a fragment of the target sequence C, said sequence FW1 and FW3 being contiguous to form a single primer with a size of between 12 and 140 nucletoids
• an antisense primer of sequence FW1 or RT1 and sequence RT3 the RT3 sequence being complementary to a fragment of the target sequence C, said sequence FW1 or RT1 and RT3 being contiguous to form a single primer with a size of between 12 and 140 nucletoides.
• the concentration of the primer pair 3 is less than or greater than or equal to that of the primer pair 1 and / or the concentration of the primer pair 2 is less than or greater than or equal to that of the primer pair 1. It is thus possible to favor or not a pair of primers over another.
• the concentration of the primer pair 3 is less than that of the primer pair 1 and / or the concentration of the primer pair 2 is less than that of the primer pair 1.
• the concentration of the pair of primers 1 is greater than that of the second and / or of the third pair of primers and the concentration of the pair of primers 2 is equal to that of the pair of primers 3.
• the concentration of the pair of primers 1 is greater than that of the second and third pair of primers and the concentration of the pair of primers 2 is greater than that of the pair of primers 3.
• the first couple is favored over the second and the second is also favored over the third.
• the method according to the invention can further comprise the bringing the sample into contact with a fourth pair of so-called hybrid or chimeric primers capable of amplifying a fourth sequence of nucleic acids (D).
• the first primer of the pair of primers 4 comprises a nucleic acid sequence capable of hybridizing with the complementary sequence (D) and, in addition, in 5 'the sequence corresponding to the sequence of the first or of the second primer of the pair of primers 1.
• the second primer of the pair of primers 4 comprises a nucleic acid sequence capable of hybridizing with the complementary sequence (D) and, in addition, in 5 'the sequence corresponding to the sequence of the first or of the second primer of primer pair 1.
• the concentration of the fourth pair of primers is less than or equal to the concentration of the pair of primers 1 and the concentration of the second, third and fourth pair is identical.
• the method according to the invention can further comprise bringing the sample into contact with an n th pair of primers capable of amplifying an n th sequence of nucleic acids.
• the first primer of the pair of primers n comprises a sequence of nucleic acids capable of hybridizing with the target complementary sequence and, in addition, in 5 'the sequence corresponding to the sequence of the first or of the second primer of the pair of primers 1.
• the second primer of the pair of primers n comprises a nucleic acid sequence capable of hybridizing with the target complementary sequence and, in addition, in 5 'the sequence corresponding to the sequence of the first or of the second primer of the pair of primers 1.
• the concentration of the n th pair of primers is less than or equal to the concentration of the pair of primers 1 and the concentration of the second, of the third, of the fourth pair and of the n th pair is identical.
• n is understood to mean a natural integer which is a positive number making it possible to count objects each counting as one.
• an integer has a unique successor, that is, an integer immediately above it, and the list of natural integers is infinite.
• reaction medium making it possible to implement the method according to the invention can comprise one or more specific probe (s) coupled to a fluorophore whose emission spectrum is different from the others.
• each product of the PCR reaction is measured in real time by said specific probe (s).
• the method according to the invention further comprises a step of quantifying the amplicons by using probes coupled to a fluorophore, preferably so-called TaqMan probes.
• the method according to the invention comprises a step of detecting amplicons and therefore microorganisms by fluorescent probes, fluorescent labeling, melting curve, nested PCR, quantitative PCR, reverse-transcription PCR or by sequencing of DNA.
• the method further comprises a step of sequencing the amplified nucleic acids or amplicons.
• the method according to the invention makes it possible to detect and identify microorganisms (bacteria, yeasts, viruses, etc.) involved in at least one infectious disease, preferably bacteria.
• microorganisms bacteria, yeasts, viruses, etc.
• the method according to the invention can also be used for the diagnosis of genetic diseases, in oncology and in general in molecular biology.
• the invention relates to a kit comprising a pair of primers 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 a pair of primers 2 comprising SEQ ID No. 7 and SEQ ID No. 8 or SEQ ID No. 9 and SEQ ID No. 10.
• the invention relates to a kit comprising: a pair of primers 1 targeting the V1-V2 variable regions 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, a pair of primers 2 targeting the V4-V6 variable regions of the 16S gene consisting of:
• ⁇ a sense primer having at least 70% sequence homology 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.
• the invention relates to a kit comprising: a pair of primers 1 targeting the V1-V2 variable regions 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, a pair of primers 2 targeting the V7-V9 variable regions of the 16S gene consisting of:
• ⁇ a sense primer having at least 70% sequence homology 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,
• the invention relates to a kit comprising: a pair of primers 1 targeting the V1-V2 variable regions 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, a pair of primers 2 targeting the variable regions V4-V6 of the 16S gene consisting of:
• ⁇ a sense primer having at least 70% sequence homology 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, a pair of primers 3 targeting the variable regions V7-V9 of the 16S gene consisting of:
• ⁇ a sense primer having at least 70% sequence homology 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.
• the kit according to any one of the preceding embodiments comprises primers whose sequence homology of 70% relates, in particular, to the first ten nucleotides in 3 ′ of said sequence.
• the invention relates to a kit comprising: a pair of primers 1 targeting the V1-V2 variable regions of the 16S gene consisting of:
• the invention relates to a kit comprising: a pair of primers 1 targeting the V1-V2 variable regions of the 16S gene consisting of:
• the invention relates to a kit comprising: a pair of primers 1 targeting the V1-V2 variable regions of the 16S gene consisting of:
• a pair of primers 2 targeting the V4-V6 variable regions of the 16S gene consisting of:
• kit according to the invention is suitable for implementing the method according to the invention.
• the invention also relates to the use of the kit according to the invention for detecting the microorganisms present during infectious diseases, such as bacteria, viruses or yeasts.
• the invention relates to the use of the kit for diagnosing an infectious disease in a subject or a plant.
• the invention also relates to the use of the kit according to the invention for detecting at least one genetic disease or at least one cancer.
• Example Example 1 of the method according to the invention for the amplification of two sequences of a specific gene for Staphylococcus aureus and Escherichia coli Figures 2A / 2B / 2C
• the Relay PCR reactions were carried out as follows: 0.2 mM of dNTP, 4 mM of MgCl2, 0.05 units of FastStart polymerase (Roche) and the buffer according to the conditions of the supplier.
• the TaqMan probes for primer pair 1 were 0.25 mM while those for primer pair 2 were 0.1 mM.
• the pairs of primers were set at different concentrations: ImM for the standard duplexes, ImM for the pair of primers 1 in the Duplex-Relays and 0, lmM or ImM for the pair of primers 2 in the Duplex-Relays.
• the cycles were as follows: denaturation at 95 ° C 10min then 55 cycles at 95 ° C 15sec / 55 ° C 15sec / 68 ° C 15sec. About 100 equivalent genome copies of each of the bacteria tested were added to the mixes to carry out the amplifications.
• the amplification of the two sequences of interest was carried out by a standard duplex PCR, by PCR Relay according to the invention with a ratio of primer pair 1: primer pair 2 equal to 10: 1 and by Relay PCR according to the invention. the invention with a ratio of primer torque 1: primer pair 2 equal to 1: 1.
• the pair of primers making it possible to amplify the specific sequence of the Staphylococcus aureus gene consists of the primers of sequence SEQ ID NO: 3 and SEQ ID NO: 4 and the pair making it possible to amplify Escherichia coli consists of the primers of sequence SEQ ID NO: 1 and SEQ ID NO: 2.
• the results are shown in Figure 2A and Table 3. The inventors thus observed a superposition of Cqs (number of cycles initiating detection of the fluorescence signal) meaning that an identical quantity of bacterial genome has been added to the PCR mix. . In addition, as many amount of Escherichia coli and Staphylococcus aureus amplicons were generated.
• the pair of primers 1 consists of the primers of sequences SEQ ID NO: 2 and SEQ ID NO: 3 and the pair of primers 2 consists of the primers of sequences SEQ ID NO: 7 and SEQ ID NO: 8.
• the first pair of primers makes it possible to amplify the specific sequence of the Staphylococcus aureus gene and the second pair of hybrid or chimeric primers makes it possible to amplify the specific sequence of the Escherichia coli gene.
• the results are shown in FIG. 2B and Table 3. The inventors thus observed a Cq shift of the curves between the amplicons Staphylococcus aureus and Escherichia coli.
• SA values for Staphylococcus aureus
• EC values for Esche ri chia coli.
• Example 2 of the method according to the invention for the amplification of two sequences of a specific gene for Klebsiella pneumoniae and for Staphylococcus aureus Figures 3A / 3B / 3C
• the amplification of the two sequences of interest was carried out by a standard Duplex PCR, and by Duplex-Relay PCR according to the invention with a ratio of primer pair 1: primer pair 2 equal to 10: 1 and by PCR Duplex-Relay according to the invention with a ratio of primer torque 1: primer pair 2 equal to 1: 1.
• the two pairs of primers were put in strong concentration.
• the pair of primers making it possible to amplify the specific sequence of the Klebsiella pneumoniae gene consists of the primers of sequence SEQ ID NO: 5 and SEQ ID NO: 6 and the pair making it possible to amplify Staphylococcus aureus consists of the primers of sequences SEQ ID NO: 3 and SEQ ID NO: 4.
• the results are represented in FIG. 3A and Table 4.
• the inventors thus observed a superposition of the Cqs (number of cycles initiating detection of the fluorescence signal) meaning that an identical quantity of bacterial genome has indeed been added to the PCR mix. .
• as many amount of Klebsiella pneumoniae and Staphylococcus aureus amplicons were generated.
• the pair of primers 1 consists of the primers of sequences SEQ ID NO: 5 and SEQ ID NO: 6 and the pair of primers 2 consists of the primers of sequences SEQ ID NO : 9 and SEQ ID NO: 10.
• the first pair of primers makes it possible to amplify the specific sequence of the Klebsiella pneumoniae gene and the second pair of chimeric primers makes it possible to amplify the specific sequence of the Staphylococcus aureus gene.
• the results are shown in FIG. 3B and Table 4. The inventors thus observed a Cq shift of the curves between the amplicons Klebsiella pneumoniae and Staphylococcus aureus.
• the delay caused by the Duplex-Relay PCR was 1.99 cycles (35.35-33.36) for the primer ratio 10: 1, ie approximately a factor of 4 (2 L 1.99). It leads to a difference in quantity of approximately 4 times less amplicons relating to the Staphylococcus aureus gene compared to amplicons of Klebsiella pneumoniae.
• the primer pairs used are identical to those used in Relay PCR (10: 1 ratio).
• the results are presented in FIG. 3C and Table 4. The inventors also observed a Cq shift of the curves between the amplicons Klebsiella pneumoniae and Staphylococcus aureus.
• the delay caused by the Duplex-Relay PCR was 2.59 cycles (36.43-33.84), or approximately a factor of 6 (2 L 2.59). It leads to a difference in the quantity of approximately 6 times less amplicons relating to the Staphylococcus aureus gene compared to the amplicons of Klebsiella pneumoniae. [Table 4]
• KP values for Klebsiella pneumoniae SA: values for Staphylococcus aureus
• Example 3 of the method according to the invention compared with a standard multiplex PCR for the amplification of two sequences of a 16S gene of Klebsiella pneumoniae (FIG. 6)
• the Relay PCR reactions were carried out as follows: 0.2mM of dNTP, 4mM of MgCl2, 0.05 units of FastStart polymerase (Roche) and the buffer according to the conditions of the supplier.
• the pairs of primers were set at different concentrations: ImM for the standard simplex, ImM for the standard Duplex, ImM for the pair of primers 1 in the Duplex Relays and 0.04 mM for the pair of primers 2 in the Duplex Relays .
• the cycles were as follows: denaturation at 95 ° C 10min then 65 cycles at 95 ° C 15sec / 55 ° C 15sec / 68 ° C 30sec. About 100 equivalent genome copies of Klebsiella pneumoniae were added to the mixes to achieve amplifications.
• Line 1 represents the amplicon of the 5 'region of the 16S gene (covering the variable areas VI to V2) (PCR control).
• Line 2 represents the amplicon of the central region of the 16S gene (covering the variable areas V4 to V6) (PCR control).
• Lane 3 represents the standard multiplex PCR amplicons of the V1-V2 and V4-V6 regions of the 16S gene.
• Line 4 represents the amplicons of the V1-V2 and V4-V6 regions of the 16S gene with the method according to the present invention (PCR multiplex relay),
• the two pairs of primers were placed in high concentration (Row 3).
• the pair of primers making it possible to amplify the V1V2 sequence of the 16S gene of Klebsiella pneumoniae consists of the primers of sequence SEQ ID NO: 14 and SEQ ID NO: 16 and the couple making it possible to amplify the V4-V6 sequence of the 16S gene of Klebsiella pneumoniae consists of the primers of sequences SEQ ID NO: 20 and SEQ ID NO: 23.
• the results are shown in Figure 6.
• the inventors observed (line 3 and left arrow) that there is no amplicon of the V1-V2 region of the 16S gene demonstrating that the standard conditions of multiplexed PCR do not. are not compatible with the simultaneous amplification of the 2 regions of the 16S gene.
• the pair of primers 1 consists of the primers of sequences SEQ ID NO: 14 and SEQ ID NO: 16 and the pair of primers 2 consists of the primers of sequences SEQ ID NO : 28 and SEQ ID NO: 33.
• the first pair of primers makes it possible to amplify the V1-V2 region of the 16S gene of Klebsiella pneumoniae and the second pair of chimeric primers makes it possible to amplify the V4-V6 region of the 16S gene of Klebsiella pneumoniae.
• the results are represented in FIG. 6.
• the inventors have thus observed that the conditions of the relay PCR make it possible to simultaneously co-amplify the 2 regions of the 16S gene (Line 4) and that the amplicons of the V1-V2 region are in addition. large quantity than those in the region central (stronger signal for the V1-V2 amplicon while its size is smaller).
• the method according to the present invention makes it possible to co-amplify the two targeted regions, unlike a standard multiplex PCR.
• the particular structure of the chimeric primers according to the present invention thus makes it possible to overcome the problems of the prior art, in particular of multiplex PCR.
• Example 4 of the method according to the invention compared with a standard multiplex PCR for the amplification of three sequences of the 16S gene of Klebsiella pneumoniae (FIG. 7)
• the Relay PCR reactions were carried out as follows: 0.2 mM of dNTP, 4 mM of MgCl2, 0.05 units of FastStart polymerase (Roche) and the buffer according to the conditions of the supplier.
• the primer pairs were set at different concentrations: ImM for standard Triplexes, ImM for primer pair 1 in Triplex Relays and 0.04 mM for primer pair 2 and primer pair 3 in Triplexes Relay.
• the cycles were as follows: denaturation at 95 ° C 10min then 65 cycles at 95 ° C 15sec / 55 ° C 15sec / 68 ° C 18sec. About 100 equivalent genome copies of Klebsiella pneumoniae were added to the mixes to carry out the amplifications.
• the pair of primers making it possible to amplify the specific sequence (V1-V2) of the 16S gene of Klebsiella pneumoniae consists of the primers of sequence SEQ ID NO: 14 and SEQ ID NO: 16
• the couple making it possible to amplify the specific sequence (V4-V6) of the 16S gene of Klebsiella pneumoniae consists of the primers of sequences SEQ ID NO: 20 and SEQ ID NO: 23
• the pair making it possible to amplify the specific sequence (V7-V9) of the 16S gene of Klebsiella pneumoniae is consisting of the primers of sequences SEQ ID NO: 24 and SEQ ID NO: 25.
• the pair of primers making it possible to amplify the specific sequence (V1V2) of the 16S gene of Klebsiella pneumoniae consists of the primers of sequence SEQ ID NO: 14 and SEQ ID NO: 16
• the pair making it possible to amplify the specific sequence (V4-V6) of the 16S gene of Klebsiella pneumoniae consists of the primers of sequences SEQ ID NO: 28 and SEQ ID NO: 33
• the pair making it possible to amplify the specific sequence (V7-V9) of the 16S gene of Klebsiella pneumoniae consists of the primers of sequences SEQ ID NO: 34 and SEQ ID NO: 36 .
• the primers of the pair targeting the V1-V2 region are placed in high concentration while the hybrid or chimeric primers of the other pairs are placed in low concentration. The results are shown in Figure 7.
• the Triplex Relais is capable of amplifying the 3 regions of the 16S gene of Klebsiella pneumo niae, namely region VI to V2 (343pb), region V7 to V9 (413 bp in standard Duplex PCR and 447 bp in Duplex Relay PCR) and region V4 to V6 (509 bp in standard Duplex PCR and 543 bp in Relay Duplex PCR).
• the method according to the present invention makes it possible to co-amplify the three targeted regions.
• Example 5 of the process according to the invention with a pair of primer 1 comprising a primer in high concentration and a hybrid or chimeric primer in low concentration (FIG. 8).
• the two Relay PCR reactions were carried out as follows: 0.2 mM of dNTP, 4 mM of MgCl2, 0.05 units of FastStart polymerase (Roche) and the buffer according to the conditions of the supplier.
• the primers were put at different concentrations: ImM for the two primers of pair 1 and 0.04 mM for the pair of primers 2.
• the primers were put at different concentrations: ImM for the first standard primer of couple 1, 0.04 m M for the second chimeric primer of couple 1, and 0.04 mM for the chimeric primers of couple 2.
• the cycling was as follows: denaturation at 95 ° C 10min then 55 cycles at 95 ° C 15sec / 55 ° C 15sec / 68 ° C 45sec. About 100 equivalent genome copies of Klebsiella pneumoniae were added to the mixes to carry out the amplifications.
• the pair of primers making it possible to amplify the V1-V2 region of the 16S gene of Klebsiella pneumoniae consists of the primers of sequence SEQ ID NO: 14 and SEQ ID NO: 16 in high concentration
• the torque allowing to amplify the V7-V9 region of the 16S gene of Klebsiella pneumoniae consists of chimeric primers of sequences SEQ ID NO: 34 and SEQ ID NO: 36 in low concentration.
• a single primer of pair 1 was put in high concentration, SEQ ID NO: 14, the other primer being a hybrid primers put in low concentration SEQ ID NO: 26 .
• the pair of primers making it possible to amplify the V1V2 region of the 16S gene of Klebsiella pneumoniae consists of the primers of sequence SEQ ID NO: 14 and SEQ ID NO: 26, the couple making it possible to amplify the V7-V9 region of the 16S gene of Klebsiella pneumoniae consists of the primers of the sequences SEQID NO: 34 and SEQID NO: 37.
• the results are represented in FIG. 8.
• the inventors observed that under the 2 conditions, the amplicons corresponding to the V1-V2 regions (343 bp for the first Duplex Relay PCR (line 1) and t349 bp for the second Duplex Relay PCR (line 2) and V7-V9 (447 bp for the first Duplex Relay PCR (line 1) and 453 bp for the second Duplex Relay PCR (line 2) are well amplified.
• V7-V9 447 bp for the first Duplex Relay PCR (line 1) and 453 bp for the second Duplex Relay PCR (line 2) are well amplified.
• the fact of using a single primer of couple 1 in high concentration seems to facilitate PCR Relay according to the present invention even more.
• the brightness of the V1-V2 amplicon band identical to that of the V7-V9 band suggests that the amount of VI-V2 amplicon is much greater. to that of the V7-V9 amplicons, the V1-V2 amplicons being smaller

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