EP-4736166-A1 - MANUFACTURE OF POLYMERASE CHAIN AMPLIFICATION KITS OPTIMIZING AN AMPLIFICATION HYBRIDIZATION PHASE

Abstract

Disclosed is a method for digitally simulating a polymerase chain amplification of at least one target, comprising the simulation of a plurality of successive cycles of the amplification, each cycle comprising: a hybridization phase; an elongation phase; then a denaturation phase; wherein the hybridization phase comprises, for each cycle: obtaining, for the cycle, an initial value of a vector of concentrations of a plurality of nucleic acid molecules in the form of single strands; initializing a matrix of concentrations of duplexes formed by the hybridization of the plurality of nucleic acid molecules in the form of single strands; calculating the change in the matrix of concentrations during the hybridization phase by applying, to the matrix of concentrations, a matrix differential equation representing the kinetics of the duplex formation according to the concentrations of the plurality of nucleic acid molecules.

Inventors

• DRAZEK, LAURENT
• PAILLIER, François
• VIDAL, Céline

Assignees

• BIOMERIEUX

Dates

Publication Date

20260506

Application Date

20240624

Claims (1)

1. [Claim 1] A method (P4) for numerical simulation of a polymerase chain reaction of at least one target, said method comprising the simulation of a plurality of successive cycles of the amplification, each cycle comprising, sequentially: - a hybridization phase (S41) between the at least one target and a plurality of primers; - an elongation phase (S42); - then a denaturation phase (S43); wherein the hybridization phase comprises for each cycle: - obtaining (S411) an initial value ( ^̅^ 0 ) for the cycle of a vector ( ^̅^) of concentrations of a plurality of nucleic acid molecules in the form of single strands comprising the at least one target and the plurality of primers; - initializing (S412) a concentration matrix ( ^^ ) of duplexes formed by the hybridization of the plurality of nucleic acid molecules in the form of single strands; - calculating (S413), by successive time steps, the evolution of said concentration matrix during the hybridization phase, by applying to the concentration matrix a matrix differential equation representing the kinetics of formation of said duplexes as a function of the concentrations of the plurality of nucleic acid molecules, said matrix differential equation being parameterized by at least one association matrix comprising the association constants associated with each duplex and a dissociation matrix comprising the dissociation constants associated with each duplex. [Claim 2] Numerical simulation method according to claim 1, in which the differential equation integrates mass conservation equations of the form: in which: - ^^ ∗ represents the time elapsed since the start of the hybridization phase; - the notations ^^ ^^ and ^^ ^^ respectively designate the concentrations of two nucleic acid molecules in the form of single strands of indices i and j in the vector ^̅^ of concentrations of a plurality of nucleic acid molecules; - the notation ^^ ^^ ^^ denotes the concentration of a duplex formed by the hybridization of the pair of nucleic acid molecules in the form of single strands of indices x and y in the vector ^̅^ of concentrations of a plurality of nucleic acid molecules; - the notations ^^ ^^0 and ^^ ^^0 denote the values of ^^ ^^ and ^^ ^^ at ^^ ∗ = 0; or any other mathematically equivalent formulation. [Claim 3] A numerical simulation method according to claim 2, in which the matrix differential equation is of the following form: in which: - H is the concentration matrix of the duplexes; - T0 is the initial value for the cycle of the concentration vector of the plurality of molecules; - Kon is the association matrix; - K off is the dissociation matrix; - the operator ^^ ^^, ^^ represents a two-dimensional matrix comprising i rows and j columns all of whose elements are equal to 1; - the operator "diag()" designates an operator taking as a parameter a square matrix and extracting from this square matrix a vector having as dimension the number of rows or columns of the square matrix and containing all the elements of the diagonal of the square matrix. [Claim 4] Method according to any one of the preceding claims, each association or dissociation matrix comprises elements, and each element of the association matrix, or each element of the dissociation matrix associated with a pair of molecules belonging to said plurality of molecules is: - zero, if the variation in free enthalpy (ΔG) associated with the hybridization reaction of the molecules forming the pair is greater than a threshold; - respectively equal to an association constant, or to a dissociation constant of the hybridization reaction of the pair of molecules otherwise. [Claim 5] Method according to the preceding claim, in which the value of said threshold is chosen from at least two predefined threshold values, the lowest threshold value being reserved for pairs of molecules comprising a matched amplicon and primer. [Claim 6] A method according to any preceding claim, comprising a prior step of defining the plurality of molecules comprising: - an initialization of the plurality of nucleic acid molecules in single-strand form as the molecules initially present in the polymerase chain reaction; - an initial simulation of a plurality of cycles of the polymerase chain reaction, each cycle of the initial simulation comprising: - obtaining the hybridization reactions of the molecules forming each pair of molecules among said plurality of molecules having an affinity below a threshold; - a simulation of a hybridization phase implementing said hybridization reactions; - a simulation of an elongation phase; - a simulation of a denaturation phase; - an addition to said plurality of molecules of the additional molecules obtained at the end of the hybridization, elongation and denaturation phases. [Claim 7] Method according to claim 5, in which the prior step of defining the plurality of molecules comprises the execution of a number of cycles of the initial simulation between 3 and 7. [Claim 8] Method according to any one of the preceding claims, comprising, at the end of the simulation of said plurality of cycles, a subsequent step of displaying (S81) the temporal evolution of the concentration of at least one of said molecules. [Claim 9] Method (P8) according to any one of the preceding claims, comprising, a subsequent step of validating (S83) or modifying (S84) the plurality of primers and the initial value of the concentration vector for said plurality of primers in the first cycle of the reaction by comparing (S82) a value representative of the kinetics of a reaction to a threshold. [Claim 10] Method according to the preceding claim, in which the value representative of the kinetics is a value chosen from: - a threshold cycle (Ct); - a crossover point (Cp); - a final concentration of a nucleic acid molecule amplified by the reaction; - a concentration of amplicons at the end of the reaction. [Claim 11] Method according to one of claims 9 or 10, in which the modification of the plurality of primers comprises: - the identification of a target to be favored according to the results of the simulation of the polymerase chain reaction; - the identification, from the association matrix or the dissociation matrix, of a nucleic acid molecule in the form of a single strand forming a pair with a primer associated with said target; - the carrying out at least one modification of the polymerase chain reaction chosen from: - a reduction, in the initial concentration vector, of the initial concentration of said nucleic acid molecule in the form of a single strand forming a pair with the primer associated with said target; - a modification of the concentration of salts; - a modification of the hybridization temperature for at least one cycle; - a modification of the hybridization duration of at least one cycle. [Claim 12] Method (P9) of manufacturing (S91) a kit for the characterization of microorganisms included in a sample by using a polymerase chain reaction, said kit comprising a plurality of validated primers, method in which the plurality of primers and the concentration vector for said plurality of primers are obtained by a simulation method according to one of claims 9 to 11. [Claim 13] Kit (K1) for polymerase chain reaction manufactured by the method according to the preceding claim. [Claim 14] Computer program comprising instructions for implementing the method according to one of claims 1 to 11 when this program is executed by a processor. [Claim 15] A non-transitory recording medium readable by a computer on which is recorded a program for implementing the method according to one of claims 1 to 11 when this program is executed by a processor. [Claim 16] A method (P10) for characterizing microorganisms included in a sample, comprising: − preparing (S101) the sample so as to carry out a PCR on the prepared sample, said preparation comprising a step of adding a kit (K1) manufactured in accordance with a method of claim 12; − carrying out (S102) the PCR on the prepared sample; − characterizing (S103) the microorganisms according to the results of the PCR. [Claim 17] The method of claim 16, wherein the sample is taken from an animal or a human, said method comprising selecting an antimicrobial based on the characterization of the microorganisms present in said sample, and administering said antimicrobial to said animal or human. [Claim 18] The method of claim 16, wherein the sample is taken from an inanimate object, said method comprising selecting an antimicrobial based on the characterization of the microorganisms present in said sample, and applying said antimicrobial to said inanimate object.

Description

Description Title: MANUFACTURE OF POLYMERASE CHAIN AMPLIFICATION KITS OPTIMIZING A HYBRIDIZATION PHASE OF AMPLIFICATION Technical field [0001] The present disclosure relates to the field of polymerase chain reaction. More specifically, the present disclosure relates to the field of simulation and manufacturing of polymerase chain reaction kits. Prior art [0002] A polymerase chain reaction (also called a polymerase chain reaction, and abbreviated "PCR") is a reaction allowing the multiplication of nucleic acid molecules (for example DNA or RNA). At each reaction cycle, each nucleic acid molecule is duplicated. PCR amplification therefore allows the nucleic acid molecules to be multiplied exponentially, the concentration of nucleic acid being able to double at each reaction cycle. [0003] This allows PCR amplification to achieve, from even very small quantities of nucleic acids, relatively high concentrations, allowing their detection. Thus, PCR amplification is used in many biomedical applications, in particular the detection and characterization of pathogenic agents, because it allows the transformation of tiny quantities of nucleic acids representative of a pathogen in detectable quantities. [0004] A PCR amplification is called “singleplex” when it aims to amplify a single nucleic acid, or “multiplex” when it aims to amplify a plurality of different nucleic acids simultaneously. The molecules to be amplified can be called “targets” and the amplified molecules “amplicons”, it being understood that an amplicon can itself be amplified at one or more subsequent cycles. [0005] PCR amplification is generally carried out in a PCR kit applying different temperatures in order to trigger the successive steps of the amplification cycles. The PCR kit initially contains primers making it possible to initiate the amplification. [0006] The success and speed of PCR amplification depend on many parameters of the PCR kit, including the durations and temperatures of the different stages of the reaction, the sequences of the primers initially present in the kit, their concentration, the concentrations of monovalent and divalent salts, oligonucleotides in solution, the length of the expected amplicons (shorter amplicons also allowing shorter cycle times), etc. [0007] Generally speaking, when designing a PCR kit, the designer performs the following steps: a) identifies for each microorganism a list of specific potential targets (i.e. not shared with other microorganisms); b) chooses, based on his experience, potential primers from a list of possible primers; c) performs tests in real conditions; d) repeats the process if the PCR performance is not satisfactory. [0008] This methodology is all the more difficult when several targets are targeted. The design of a kit is therefore long and complex and relies on professional expertise specific to each designer. [0009] Anticipation of reactions on paper is also impossible, because the designer is very quickly overwhelmed by the number of reactions, so that it is impossible for the human mind to simulate on paper what will actually take place during the PCR. [0010] For example, it is impossible to solve a priori a system of differential equations representing the hybridization between all the molecules present in a PCR kit, in particular the amplicons, primers and intermediate products. State-of-the-art methods also do not allow numerical simulations of such a system to be performed in a reasonable time, due to the presence of numerous differential equations linked to each other. This considerably reduces the possibilities for designing and therefore improving PCR kits. [0011] There is therefore a need for a method for designing PCR kits that makes it possible to predict the course of hybridization phases in order to facilitate the selection of parameters facilitating the amplification of one or more intended targets. Abstract [0012] The present disclosure improves the situation. [0013] A method for numerical simulation of a polymerase chain reaction of at least one target is proposed, said method comprising the simulation of a plurality of successive cycles of the amplification, each cycle comprising, sequentially: a hybridization phase between the at least one target and a plurality of primers; an elongation phase; then a denaturation phase; in which the hybridization phase comprises for each cycle: obtaining an initial value for the cycle of a vector of concentrations of a plurality of nucleic acid molecules in the form of single strands comprising the at least one target and the plurality of primers; initializing a matrix of concentrations of duplexes formed by the hybridization of the plurality of nucleic acid molecules in the form of single strands; calculating, by successive time steps, the evolution of said concentration matrix during the hybridization phase, by applying to the concentration matrix a matrix differential equation representing the kinetics of formation of said du