EP-4736167-A1 - MANUFACTURE OF POLYMERASE CHAIN AMPLIFICATION KITS OPTIMIZING AN AMPLIFICATION DENATURATION 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, sequentially: a hybridization phase; an elongation phase; then a denaturation phase; wherein, for each cycle: the hybridization phase comprises: 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; calculating a matrix of concentrations of partial duplexes formed by the hybridization of the plurality of nucleic acid molecules in the form of single strands; the denaturation phase comprises: multiplying a vector of the concentration of elongated duplexes resulting from the application of the elongation phase to the concentrations of duplexes, by an elongated duplex denaturation tensor.

Inventors

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

Assignees

• bioMérieux

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, for each cycle: - the hybridization phase comprises: - 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; - calculating a matrix of concentrations of partial duplexes formed by the hybridization of the plurality of nucleic acid molecules in the form of single strands, each element of said matrix representing the concentration of the duplexes formed by the hybridization of a pair of said plurality of nucleic acid molecules in the form of single strands, independently of the hybridization position of said pair; - the denaturation phase comprises: - multiplying a vector of concentration of elongated duplexes, resulting from the application of the elongation phase to the concentrations of duplexes, by a tensor of denaturation of the elongated duplexes ( ^̿^ ^ ∗ ^) in order to obtain an initial value of vector of concentrations of a plurality of nucleic acid molecules in the form of single strands for the following cycle. [Claim 2] Method according to the preceding claim, in which: - at least one duplex results from the pairing between two primers; - the elongation phase simulates the concentration of each duplex resulting from the pairing between two primers into a single concentration of elongated duplex resulting from the pairing between two primers; - the coefficients of the denaturation tensor are defined so as to distribute each concentration of elongated duplex resulting from the pairing between two primers into nucleic acid molecules in the form of single strands corresponding to an elongation of the duplex resulting from the pairing between the two primers in both directions. [Claim 3] Method according to the preceding claim, in which, for each hybridization reaction between two primers: - the concentrations of elongated duplexes comprise a concentration of an elongated virtual duplex equal to the sum of the concentrations of the elongated duplexes formed by the elongation in each of the two directions of the duplexes formed by the hybridization of the two primers; - the coefficients of the denaturation tensor of the elongated duplexes corresponding to the distribution of the concentration of the elongated virtual duplex towards each of the two primers, and each of the two nucleic acids in the form of single strands resulting from the elongation in one of the two directions are all equal to 0.5. [Claim 4] A method according to any preceding claim, wherein, for each set of multiple hybridization reactions of a single-stranded nucleic acid by a primer at multiple locations respectively: - the duplex concentrations and the elongated duplex concentrations respectively comprise a concentration of a virtual duplex equal to the sum of the concentrations of the duplexes formed by the multiple hybridization reactions, and a concentration of an elongated virtual duplex equal to the sum of the concentrations of the elongated duplexes formed by the elongation of the duplexes formed by the multiple hybridization reactions; - the coefficients of the denaturation tensor of the elongated duplexes corresponding to the distribution of the concentration of the elongated virtual duplex towards each single-stranded nucleic acid associated with one of the multiple hybridization reactions are respectively equal to the ratio between the interaction energy of the multiple hybridization reaction divided by the sum of the interaction energies of all the multiple hybridization reactions with said single-stranded nucleic acid. [Claim 5] Method according to any one of the preceding claims, in which the elongation phase comprises: - obtaining the concentrations of the elongated duplexes by multiplying the concentrations of the duplexes by elongation coefficients. [Claim 6] Method according to the preceding claim, in which: - the elongation phase further comprises updating the concentration vector of the duplexes, representing at the end of the elongation phase the concentration of the non-elongated duplexes; - the denaturation phase further comprises multiplying said duplex concentration vector by a denaturation tensor of the non-elongated duplexes ( ^̿^ ^^ ) to update the initial value for the next cycle of the vector of concentrations of the plurality of nucleic acid molecules in the form of single strands. [Claim 7] The method of the preceding claim, wherein, during the denaturation phase, the same denaturation coefficient is applied to a non-elongated duplex and to the corresponding elongated duplex to update the vector of concentrations of the plurality of nucleic acid molecules in the form of single strands, and the duplex concentrations for the next cycle. [Claim 8] A method (P8) according to any preceding claim, 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 9] A method of manufacturing a kit for characterizing microorganisms included in a sample by using a polymerase chain reaction, said kit comprising a plurality of validated primers, wherein the plurality of primers and the concentration vector for said plurality of primers are obtained by a simulation method according to one of claims 1 to 7. [Claim 10] A kit for polymerase chain reaction manufactured by the method according to the preceding claim. [Claim 11] Computer program comprising instructions for implementing the method according to one of claims 1 to 8 when this program is executed by a processor. [Claim 12] 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 8 when this program is executed by a processor. [Claim 13] Method for characterizing microorganisms included in a sample, comprising: − Preparing the sample so as to perform a PCR on the prepared sample, said preparation comprising a step of adding a kit manufactured in accordance with a method of claim 1 to 8; − Carrying out the PCR on the prepared sample. − Characterizing the microorganisms according to the results of the PCR. [Claim 14] The method of claim 13, wherein the sample is taken from an animal or a human being, said method comprising choosing an antimicrobial according to the characterization of the microorganisms present in said sample, and administering said antimicrobial to said animal or human being. [Claim 15] The method of claim 13, wherein the sample is taken from an inanimate object, said method comprising choosing an antimicrobial according to 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 DENATURATION PHASE OF THE 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 phase, taking into account the fact that hybridization between the different molecules may have occurred in different places. 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 denaturation phases, including in the case of multiple hybridizations, 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 diff