Method for in vitro determination of the presence or absence of at least one amplicon

Abstract

The invention relates to a method for determining in vitro the presence or absence of at least one type of amplicon in a biological sample that has undergone polymerase chain reaction, an amplicon comprising two strands, the sample comprising at least one intercalating agent capable of being fluorescent when intercalated in an amplicon, the method comprising the implementation of several steps including the processing of the Gaussian curve model so as to determine the presence or absence of the amplicon type in the biological sample. Figure 1

Description

Title of the invention: Method for in vitro determination of the presence or absence of at least one amplicon. FIELD OF THE INVENTION

[0001] The present invention relates to the field of in vitro determination of the presence or absence of at least one type of amplicon in a biological sample, more specifically once the sample has undergone polymerase chain reaction (PCR). STATE OF THE ART

[0002] To detect a specific type of amplicon in a biological sample, it is known to perform polymerase chain reaction (PCR) followed by heating the biological sample so that the amplicons of the type to be detected dehybridize, i.e., the strands of the amplicons separate. This heating step is also called melting. During this heating step, successive measurements of the sample's fluorescence are acquired, and a melt curve is constructed, representing the change in fluorescence as a function of temperature in the sample. The negative primary derivative of this curve, which represents the change in fluorescence as a function of temperature, is then calculated, and the temperature corresponding to the maximum change in fluorescence is determined.This temperature is considered to be the melting temperature, which is specific to a type of amplicon. Based on the determined melting temperature, one can determine whether the type of amplicon being sought is present in the biological sample by comparing the determined melting temperature to the melting temperature specific to that amplicon type. If the determined melting temperature is close to the theoretical melting temperature, the amplicon type is considered to be present in the sample.

[0003] However, this technique has drawbacks. Indeed, depending on the heating rate of the sample (and therefore on the temperature gradient within the sample), the determined melting temperature varies. The determined melting temperature can also vary depending on other factors such as the geometry of the well in which the biological sample is placed, the type of liquid containing the biological sample, or the configuration of the detection system. Consequently, melting temperatures are determined based on the heating rate of the sample, the detection systems, and other factors. different for the same sample. This can lead to errors in detecting a type of amplicon, i.e. false positives or false negatives. Description of the invention

[0004] An object of the invention is to provide a solution for determining in vitro the presence or absence of at least one type of amplicon in a biological sample that is more reliable and easier to implement.

[0005] Another object of the invention is to provide a solution for rapidly, reliably and efficiently controlling systems to determine in vitro the presence or absence of at least one type of amplicon in a biological sample.

[0006] To this end, the invention relates to a method for determining in vitro the presence or absence of at least one type of amplicon in a biological sample that has undergone polymerase chain reaction, an amplicon comprising two strands, the sample comprising at least one intercalating agent capable of being fluorescent when intercalated in an amplicon, the method comprising the implementation of the following steps:

[0007] - a) heating the biological sample so that the two strands of each amplicons of the amplicon type separate if one or more amplicons of the amplicon type are present in the biological sample;

[0008] - b) measurement and acquisition during heating, for different temperatures, of the fluorescence of the intercalating agent;

[0009] - c) calculation of an evolution, as a function of temperature, of a negative variation of fluorescence measured in relation to temperature;

[0010] - d) determination of at least one model of a Gaussian curve corresponding to the evolution, as a function of temperature, of the negative variation of the fluorescence measured with respect to the temperature calculated in step c);

[0011] - e) processing the Gaussian curve model so as to determine the presence or not of the type of amplicon in the biological sample.

[0012] According to advantageous and non-limiting features, taken alone or in any combination:

[0013] - step e) includes a step el) of determining a melting temperature corresponding to the average of the Gaussian curve model and the presence or absence of the amplicon type in the biological sample is determined as a function of the melting temperature;

[0014] - step e) includes a step e2) of determining the standard deviation of the model of the Gaussian curve and a step e3) of determining the presence of the amplicon type in the biological sample if a point on a pre-established standard curve specific to the amplicon has an abscissa substantially equal to the standard deviation and an ordinate substantially equal to the melting temperature, said standard curve representing, for the amplicon type, melting temperatures obtained for different Gaussian curve models as a function of the standard deviation of the different Gaussian curve models. Advantageously, the thermal gradient varies with increasing thermal transition rate.

[0015] - said models of Gaussian curves from which the standard curve is established are obtained by implementing steps a) to d) of the present process, the heating rate of the biological sample in step a) being different for obtaining each model of Gaussian curve;

[0016] - we seek to determine the presence or absence of at least two types of amplicons in the biological sample and in which, at step d), at least two models of Gaussian curves corresponding to the evolution, as a function of temperature, of the negative variation of the fluorescence measured with respect to the temperature calculated in step c) are determined and in which, at step e), the presence or absence of the types of amplicons in the biological sample is determined according to the models;

[0017] The invention also relates to an in vitro system for determining the presence or absence of a type of amplicon in a biological sample that has undergone polymerase chain reaction, an amplicon comprising two strands, the sample comprising at least one intercalating agent capable of being fluorescent when intercalated in an amplicon, the system comprising:

[0018] - a heating device suitable for heating the biological sample so that the two strands of each amplicon of the amplicon type separate if one or more amplicons of the amplicon type are present in the biological sample;

[0019] - a fluorometer adapted to measure the fluorescence of the intercalating agent;

[0020] - a processing unit configured for:

[0021] - acquire, during heating, for different temperatures, the fluorescence of the intercalating agent;

[0022] - calculate an evolution, as a function of temperature, a negative variation of the fluorescence measured in relation to temperature;

[0023] - determine at least one model of a Gaussian curve corresponding to the evolution, as a function of temperature, the negative variation of the measured fluorescence relative to the calculated temperature;

[0024] - process the Gaussian curve model in such a way as to determine the presence or not of the type of amplicon in the biological sample.

[0025] According to a feature of the invention, the heating device is a Peltier module.

[0026] According to one feature of the invention, the biological sample is arranged in a chamber of an analysis card.

[0027] According to one feature of the invention, the reaction chamber(s) have a variable thickness. More specifically, the thickness varies by plastic or elastic deformation, the deformation being able to be caused by an actuator or any other means.

[0028] According to one feature of the invention, by varying the thickness of the reaction chamber, the thermal gradient is directly related to the standard deviation, i.e., the full width at half maximum (FWHM) of the Gaussian curve. This results in a narrower melting peak, and the temperature is closer to the actual temperature of the amplicons. Furthermore, this configuration allows, if the process according to the invention is used with thermal multiplexing (several probes with different melting temperatures in the same reservoir), for less spread-out, narrower melting peaks, which are therefore more easily demodulated and separable.

[0029] The invention also relates to a method for controlling a system (100) comprising implementing the steps of the method according to the invention to determine in vitro the presence or absence of at least one type of control amplicon in a biological control sample and implementing a step f) of characterizing the operation of the system (100) according to the result of the step of determining the presence or absence of the type of control amplicon in the biological control sample.

[0030] According to one feature of the invention, the control method includes a step g) of characterizing the operation of the heating device (101) as a function of a temperature gradient in the biological control sample determined from the standard deviation of the model of the Gaussian curve.

[0031] Finally, the invention also relates to using the determination method for processing a fusion signal. Indeed, thanks to the determination method according to the invention, it is possible to optimize the fusion signal so that it is less noisy or even noise-free, which improves signal readout and simplifies demodulation and the determination of the peak of interest for amplicons. DESCRIPTION OF FIGURES

[0032] Other features and advantages of the present invention will become apparent from the following description of a preferred embodiment. This description will be given with reference to the accompanying figures, including:

[0033] [Fig.l] schematically illustrates an in vitro determination system for the presence or absence of at least one type of amplicon in a biological sample that has undergone polymerase chain amplification;

[0034] [Fig.2] represents an in vitro method for determining the presence or absence of au minus one type of amplicon in a biological sample that has undergone polymerase chain reaction;

[0035] [Fig.3] illustrates a curve of the evolution of the fluorescence of an intercalating agent in function of the temperature in the sample;

[0036] [Fig.4] illustrates a curve of the evolution of the negative variation of the fluorescence of an intercalating agent as a function of temperature in the sample;

[0037] [Fig.5] illustrates a Gaussian curve obtained by Gaussian fitting applied to the curve of [Fig.3];

[0038] [Fig.6] illustrates, for the same amplicon, curves of the evolution of the variation negative fluorescence of an intercalating agent as a function of temperature in the sample, each curve being obtained by heating the sample at a different rate;

[0039] [Fig.7] illustrates a standard curve representing, for an amplicon, the correspondence between the determined melting temperature and the standard deviation;

[0040] [Fig.8] illustrates, for the same type of amplicon, curves of the evolution of the negative variation of the fluorescence of an intercalating agent as a function of temperature in the sample, each curve being obtained by heating the sample at a different rate, said curves being used to construct a standard curve of the type of amplicon.

[0041] [Fig.9] is a graphical representation of the comparison of Gaussian curves when the thickness of the reaction chamber varies.

[0042] [Fig. 10] is a graphical representation of the comparison of Gaussian curves when the thickness of the reaction chamber varies in a multiplexing case.

[0043] [Fig. 11] is a graphical representation following [Fig. 10]. DETAILED DESCRIPTION OF THE INVENTION

[0044] With reference to [Fig.1], a system is proposed for the in vitro determination of the presence or absence of at least one amplicon type in a biological sample E which has undergone polymerase chain reaction (PCA or, in English, PCR for "polymerase chain reaction").

[0045] As illustrated in [Fig.1], the biological sample E which has undergone polymerase chain amplification is advantageously contained in a reaction chamber 11 of an analysis card 1. The biological sample may include tissues and cells from a human or animal body and their derivatives, organs, blood, its components or its derivatives.

[0046] Since the biological sample underwent polymerase chain reaction, if the biological sample included amplicons of a type of amplicon before In polymerase chain reaction, the biological sample is assumed to have a high concentration of this type of amplicon.

[0047] An amplicon is a piece of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). An amplicon of the type whose presence in the sample is to be detected is, for example, a piece of DNA from a virus such as the hepatitis B, C, and D viruses. An amplicon comprises two strands.

[0048] By "in vitro determination of the presence or absence of at least one type of amplicon," it is understood that the aim is to determine the presence or absence of at least one species of amplicon. Consequently, if the presence of two types of amplicons is determined, it is understood that the presence of two different species of amplicons is determined. Two types of amplicons are different if they originate from different DNA or RNA. In other words, two different types of amplicons characterize, for example, two different viruses. It will be seen later that the system and method presented allow for the reliable and simple determination of the presence or absence of several different types of amplicons in a biological sample.

[0049] Furthermore, if the biological sample includes a certain type of amplicon, it is understood that the biological sample includes a multitude of amplicons of that certain type of amplicon.

[0050] The biological sample comprises an intercalating agent capable of being fluorescent when intercalated into an amplicon. The intercalating agent is capable of intercalating into an amplicon, that is, of inserting itself between the strands of an amplicon. The intercalating agent emits fluorescence when intercalated into an amplicon. Conversely, the intercalating agent does not emit fluorescence when it is not intercalated into an amplicon. Therefore, if the biological sample that has undergone polymerase chain reaction does not contain an amplicon, this biological sample (more precisely, the intercalating agent) does not emit fluorescence.

[0051] Analysis card 1 is advantageously adapted to be the site of polymerase chain amplification.

[0052] The analysis card 1 therefore advantageously includes a chamber adapted to receive a biological sample (not yet having undergone polymerase chain amplification), reservoirs (not shown) adapted to contain nucleic acid and / or specific primers made up of synthetic oligonucleotides and at least one reaction chamber 11 adapted to receive the biological sample having undergone polymerase chain amplification.

[0053] The chambers and tanks are in the shape of shells or "blisters" in English.

[0054] Alternatively, the reaction chamber(s) may be formed in a plate or "array", generally defined as a surface element a flat plate exhibiting a certain thickness, which is nevertheless very small compared to the dimensions of its flat surface. For example, the thickness is at least 10 times less than the widths and lengths of the plate's faces.

[0055] Advantageously, the chamber is in fluidic communication with at least one reservoir via a conduit or fluidic path. One reservoir is in fluidic communication with at least one other reservoir via a conduit or fluidic path. The reaction chambers 11 are in fluidic communication with at least one reservoir via a conduit or fluidic path.

[0056] The system 100 includes a heating device 101 adapted to heat the biological sample that has undergone polymerase chain reaction, which will be referred to as the "biological sample" in the rest of the description for the sake of simplicity.

[0057] Advantageously, the heating device 101 is a Peltier module. A Peltier module exploits the Peltier effect, by which an electric current is converted into a temperature difference.

[0058] The system 100 includes a fluorometer 102 adapted to measure the fluorescence of the intercalating agent in the biological sample.

[0059] The system 100 further comprises a processing unit 103 such as a processor. The processing unit 103 is configured to:

[0060] - acquire, during the heating of the biological sample by the device heating 101, for different temperatures, the fluorescence of the intercalating agent;

[0061] - calculate the evolution, as a function of temperature, of a negative variation of the fluorescence measured in relation to temperature;

[0062] - determine at least one model of a Gaussian curve corresponding to the evolution, as a function of temperature, of the negative variation of the measured fluorescence relative to the calculated temperature;

[0063] - process the Gaussian curve model in such a way as to determine the presence or not of the type of amplicon in the biological sample.

[0064] With reference to [Fig.2], a method is proposed for in vitro determination of the presence or absence of at least one type of amplicon in a biological sample that has undergone polymerase chain amplification.

[0065] As explained previously, the biological sample may comprise one or more types of amplicon. In other words, the biological sample is likely to comprise one or more amplicons of the type of amplicon that one seeks to detect. If the biological sample comprises one or more amplicons, the biological sample, in particular the intercalating agent sandwiched between the strands of the amplicons, emits fluorescence.

[0066] The process includes a step a) implemented by means of the heating device 101 of heating the biological sample so that the two strands of each amplicon of the amplicon type separate (this phenomenon is called dehybridization) if one or more amplicons of the amplicon type that one seeks to detect are present in the biological sample.

[0067] Indeed, when an amplicon is heated, the two strands of the amplicon separate when the amplicon reaches a temperature specific to the type of amplicon, called the melting temperature. This melting temperature makes it possible to determine the type of amplicon present in the biological sample.

[0068] When the two strands of the amplicon separate, the intercalating agent that was inserted between the two strands is released and ceases to emit fluorescence. Consequently, the melting temperature corresponds to the temperature at which a decrease in fluorescence is observed. It is therefore necessary to monitor the fluorescence during step a) heating.

[0069] The maximum heating temperature depends on the type of amplicon to be detected. The biological sample is advantageously heated to a temperature higher than the temperature specific to the type of amplicon. Preferably, the biological sample is heated to a temperature 10°C, and even more preferably 15°C, higher than the temperature specific to the type of amplicon to be detected.

[0070] As a general rule, the heating rate is less than 5°C per second, or even less than 2°C per second, or even less than 1°C per second. Indeed, the lower the heating rate, the smaller the temperature gradient in the sample, which facilitates the analysis of fluorescence data and makes it easier to detect the amplicon type. However, it will be seen that the method presented makes it possible to determine the presence of an amplicon type even if the heating rate is high, for example, greater than 5°C per second or even greater than 10°C per second, and therefore even if the temperature gradient in the sample is significant.

[0071] The process includes a step b) of measuring and acquiring, during heating at different temperatures, the fluorescence of the intercalating agent. Step b) is therefore carried out at least partially simultaneously with step a) of heating.

[0072] The fluorimeter 102 measures the fluorescence of the intercalating agent during heating. Preferably, at least one temperature value per second is measured.

[0073] The processing unit 103 acquires fluorescence during heating. In other words, the processing unit 103 acquires the measurements taken by the fluorometer 102.

[0074] Fluorescence measurements can be represented as a function of temperature. Figure 3 illustrates an example of the evolution of the measured fluorescence as a function of temperature. temperature. This curve is known as the melt curve. As can be seen, the higher the temperature, the lower the fluorescence because the strands of the amplicons contained in the biological sample (which may be of the same or different types of amplicons) separate and the intercalating agent is released and therefore ceases to emit fluorescence.

[0075] The method includes a step c), implemented by the processing unit 103, which calculates the evolution, as a function of temperature, of a negative variation of the measured fluorescence with respect to temperature. In other words, the negative derivative of the fluorescence evolution as a function of temperature (i.e., the negative derivative of the curve illustrated in [Fig. 4]) is calculated. Step c) can be implemented after step b) or simultaneously or partially simultaneously with the implementation of step b) (therefore potentially partially simultaneously with step a)).

[0076] Advantageously, to calculate the negative derivative, the derivative of the evolution of fluorescence as a function of temperature is first calculated.

[0077] To calculate this derivative, it is possible to calculate the derivative at each point of the curve representing the evolution of fluorescence as a function of temperature. The derivative value at a point corresponds to the slope of the tangent to the curve at that point.

[0078] Another way to calculate the derivative is to perform an approximation (such as a regression or an interpolation) of the evolution of fluorescence as a function of temperature into a function and then to calculate the derivative of this function.

[0079] In any event, a person skilled in the art knows how to calculate the derivative of a set of points.

[0080] Then, to obtain the negative derivative, it suffices to reverse the sign of each point of the derivative.

[0081] Figure 4 illustrates a curve of the evolution, as a function of temperature, of the negative variation of the fluorescence measured with respect to temperature, i.e. of the negative derivative of the evolution of the fluorescence as a function of temperature.

[0082] Advantageously, as illustrated in [Fig.4], if the biological sample contains at least one type of amplicon, a peak corresponding to the separation of the strands of the amplicon(s) of that type of amplicon can be distinguished on the curve.

[0083] The process includes a step d), implemented by the processing unit 103, of determining at least one model of a Gaussian curve corresponding to the evolution, as a function of temperature, of the negative variation of the fluorescence measured with respect to the temperature calculated in step c).

[0084] In other words, a Gaussian "fitting" is performed, that is to say, a Gaussian curve model is sought that most closely resembles the evolution, as a function of temperature, of the negative variation of the fluorescence measured by in relation to temperature. In other words, we are looking for the Gaussian curve model that best matches a curve representing the evolution, as a function of temperature, of the negative variation of the fluorescence measured with respect to temperature.

[0085] As illustrated in [Fig.5], the Gaussian curve pattern includes a peak (such as a Gaussian curve well known to the person skilled in the art).

[0086] Step d) has the advantage of removing the noise present in the temperature-dependent values ​​of the negative variation of the measured fluorescence with respect to temperature. In other words, step d) cleans the data corresponding to the temperature-dependent evolution of the measured negative variation of the fluorescence with respect to temperature. Consequently, a Gaussian curve model is obtained that is more reliable and easier to analyze.

[0087] According to a particular embodiment, the presence or absence of at least two types of amplicons is to be determined in the biological sample. Therefore, in step d), at least two models of Gaussian curves are determined corresponding to the evolution, as a function of temperature, of the negative variation of the fluorescence measured with respect to the temperature calculated in step c).

[0088] For this purpose, for example, a first Gaussian curve model is determined by performing a Gaussian fitting. This first Gaussian curve model includes the largest peak in the evolution, as a function of temperature, of the negative variation of the fluorescence measured with respect to temperature.

[0089] Advantageously, this first Gaussian curve model is then subtracted from the temperature-dependent evolution of the negative variation of the measured fluorescence with respect to temperature. A second Gaussian curve model can then be determined by performing a Gaussian fitting on the temperature-dependent evolution of the negative variation of the measured fluorescence with respect to temperature, subtracted from the first Gaussian curve model. This second Gaussian curve model includes the second largest peak in the temperature-dependent evolution of the negative variation of the measured fluorescence with respect to temperature.

[0090] If we seek to determine the presence or absence of a third (or even more) type of amplicon, the subtraction and fitting steps can be repeated.

[0091] This method allows, as will be seen later, to determine the presence or absence of several types of amplicons in a biological sample in a simple and reliable way.

[0092] The process includes a step e) of processing, by the processing unit 103, the Gaussian curve model so as to determine the presence or absence of the amplicon type in the biological sample. The amplicon type that is sought to Determining the presence or absence of an amplicon in the biological sample is known in advance. In other words, we are not trying to detect the presence of an amplicon whose type is unknown. Put another way, we know what we are looking for. For example, we might try to determine the presence of the type of amplicon characteristic of the hepatitis B virus.

[0093] Advantageously, step e) includes a step el) of determining a melting temperature from the Gaussian curve model. More precisely, the mean of the Gaussian curve model is determined, and this mean is considered to correspond to the melting temperature specific to the type of amplicon contained in the biological sample. The mean of a Gaussian curve model is known to those skilled in the art and is denoted p. This mean is the temperature corresponding to the largest ordinate value (i.e., the largest value of negative variation of the measured fluorescence with respect to temperature). The mean p is shown schematically in [Fig. 5].

[0094] According to a certain embodiment, the presence or absence of the amplicon type in the biological sample is determined based on the melting temperature. To this end, the obtained melting temperature is compared to the theoretical melting temperature of the amplicon type to be detected. If the melting temperatures are substantially identical, the amplicon type in the biological sample is considered to be the one to be detected. Otherwise, the amplicon type in the biological sample is considered not to be the one to be detected.

[0095] An advantageous embodiment will be described, which is particularly advantageous in cases where the Gaussian curve model is unreliable. For example, the peak included in the Gaussian curve model may be imprecise and spread out, as illustrated in [Fig. 6]. This makes it difficult to determine a usable melting temperature value. Indeed, the melting temperature obtained from such a Gaussian curve model does not correspond to the melting temperature specific to the type of amplicon that one seeks to detect.

[0096] Advantageously, step e) includes a step e2) for determining the standard deviation of the Gaussian curve model. The standard deviation of a Gaussian curve model is known to those skilled in the art and is denoted π. The standard deviation is proportional to the full width at half maximum (FWHM) of the Gaussian curve. Indeed, we have:

[0097] ictrggw at mid-height = 2^21n (2) a « 2.3548 a

[0098] The width at half height L is shown schematically in [Fig.5].

[0099] The standard deviation characterizes the temperature gradient, i.e., the temperature distribution, in the biological sample. The larger the standard deviation, the more The temperature gradient in the biological sample is significant, which may indicate a malfunction of the heating device 101 (which heats too slowly) or a poor positioning of the biological sample relative to the heating device 101. We will see that the standard deviation can therefore be used to check the proper functioning of the system 100 for determining the presence or absence of a type of amplicon in the biological sample.

[0100] Advantageously, the method includes a step e3) of determining the presence of the amplicon type in the biological sample if a point on a pre-established standard curve specific to the amplicon type has an abscissa substantially equal to the standard deviation and an ordinate substantially equal to the melting temperature. In other words, both the standard deviation and the mean, i.e., the melting temperature, of the Gaussian curve model are used to determine the presence or absence of the amplicon type. For this purpose, a standard curve is used. This standard curve is used in the same way as a nomogram.

[0101] The standard curve represents, for the type of amplicon whose presence is to be determined, melting temperatures obtained for different Gaussian curve models as a function of the standard deviation of the different Gaussian curve models. An example of a standard curve is illustrated in [Fig. 7]. A standard curve is associated with a certain type of amplicon (i.e., the type of amplicon whose presence in the biological sample is to be determined). The standard curve is specific to the amplicon type.

[0102] The Gaussian curve models from which the standard curve is established are obtained by carrying out steps a) to d) of the present process using standard biological samples, each containing the type of amplicon whose presence or absence is to be determined. Gaussian curve models for an amplicon type A are illustrated in [Fig. 8].

[0103] The standard curve is advantageously specific to system 100. By system-specific, it is understood that the standard curve is preferably specific to a certain model of system 100 (the standard curve is therefore specific to all systems 100 of the same model), or even, preferably, specific to a single system 100. In other words, the standard curve was constructed from models of Gaussian curves obtained by implementing steps a) to d) of the present method using system 100. Indeed, each system 100 may have different parameters and / or configurations, which can lead to different results depending on the system. It is therefore advantageous for the standard curve to be specific to system 100.

[0104] The heating rate of the biological sample in step a) is different for obtaining each Gaussian curve model such that the gradient of The temperature differs for each standard biological sample. This allows for the creation of Gaussian curve models with different standard deviations, enabling the determination of a standard curve that represents the evolution of the melting temperature as a function of the standard deviation for the type of amplicon whose presence or absence is being investigated. The standard curve for amplicon type A is illustrated in [Fig. 7].

[0105] By determining whether the standard curve includes a point whose abscissa corresponds to the standard deviation obtained for the biological sample under consideration and whose ordinate corresponds to the melting temperature obtained for the biological sample under consideration, one can conclude whether or not the amplicon type is present in the biological sample. If such a point exists, it is determined that the amplicon type is present in the sample.

[0106] By "corresponds", it is understood that we are looking for a point whose abscissa corresponds substantially to the standard deviation obtained for the biological sample considered and whose ordinate corresponds substantially to the melting temperature obtained for the biological sample considered.

[0107] According to an embodiment in which the presence or absence of several types of amplicons is to be determined, step e) is carried out independently for each model of the Gaussian curve obtained. In other words, the presence or absence of a first type of amplicon is determined, then of a second type of amplicon, etc.

[0108] Thus, the present method allows for the simple and reliable determination of the presence or absence of a particular type of amplicon in a biological sample. This method, particularly through the use of a calibration curve, has the advantage of enabling reliable detection of the amplicon type even at high heating rates. Therefore, the heating step can be accelerated without compromising the reliability of amplicon type detection.

[0109] The steps of the presented process can be carried out successively, partially simultaneously or simultaneously.

[0110] A method is proposed for testing system 100 for the in vitro determination of the presence or absence of at least one type of amplicon in a biological sample. The testing method aims to characterize the operation of system 100. The testing method allows for the simple and reliable detection of malfunctions in system 100.

[0111] The control process includes implementing the steps of the determination process described above to determine in vitro the presence or absence of at least one type of control amplicon in a biological control sample.

[0112] By “control biological sample” is meant a biological sample known to contain a certain type of amplicon, referred to as type of a control amplicon. In other words, it is known that the biological sample contains the type of control amplicon, for example, a type of amplicon specific to the hepatitis B virus. Therefore, it is expected that the implementation of the determination procedure to determine in vitro the presence or absence of the type of control amplicon in the biological control sample using system 100 will result in the determination of the presence of the type of control amplicon in the biological control sample. Otherwise, a malfunction of system 100 can be concluded.

[0113] The control process therefore includes implementing a step f) of characterizing the operation of system 100 based on the result of the step of determining the presence or absence of the control amplicon type in the control biological sample. Step f) can be implemented by the processing unit 103. If the presence of the control amplicon type is determined, the operation of system 100 is considered good. If the presence of the control amplicon type is not determined (i.e., the control amplicon type is not detected), the operation of system 100 is considered poor. In other words, system 100 is malfunctioning. In this case, corrective measures can be implemented.

[0114] The malfunction may, for example, originate from a malfunction of the fluorometer 102. For example, the fluorometer 102 may acquire erroneous fluorescence data. In this case, a corrective measure may consist of adjusting / configuring the fluorometer 102 or replacing the fluorometer 102 with a fluorometer that does not malfunction.

[0115] The malfunction may, for example, originate from a malfunction of the heating device 101. For example, the heating device 101 may not heat sufficiently and / or quickly or slowly enough. Alternatively, or in addition, the biological sample may not be positioned correctly on the heating device 101 (for example, not close enough and / or not centered).

[0116] Advantageously, the control method includes a step g) of characterizing the operation of the heating device 101. Step g) can be implemented by the processing unit 103.

[0117] More specifically, in step g), the operation of the heating device 101 is characterized as a function of a temperature gradient in the control biological sample determined from the standard deviation of the Gaussian curve model. Indeed, the standard deviation of the Gaussian curve model allows the temperature gradient in the control biological sample to be characterized. The larger the standard deviation, the larger the temperature gradient in the control biological sample. A temperature gradient in the biological sample that is too This is not desirable because it indicates that the temperature of the biological sample is not homogeneous. Consequently, the amplicons in the biological sample will not dehybridize at the same time depending on their position within the sample. However, at a given time t, a fluorescence measurement will be acquired for a single temperature value, such as the one transmitted by a thermocouple to processing unit 103 at that time t. Therefore, the acquired fluorescence data may not be sufficient to determine the presence of a particular type of amplicon in the sample.

[0118] At the end of step g), a malfunction of the heating device 101 can be detected. For example, a malfunction can be detected if the temperature gradient exceeds a threshold determined according to the instrument and its technical specifications. In this case, a corrective measure can be implemented, such as adjusting / setting the heating device 101 or replacing it with a new heating device. Alternatively, or in addition, the biological sample can be correctly repositioned on the heating device 101 (for example, closer and / or more centered).

[0119] Figures 9, 10, and 11 illustrate an embodiment in which the reaction chamber varies in thickness, with the peak height in RFUs (Relative Fluorescence Units) on the ordinate and the temperature in degrees Celsius on the abscissa. Thus, in [Fig. 9], a first dashed curve represents a melting peak when the reaction chamber has a specific thickness e1, and a second solid curve represents a melting peak when the reaction chamber has a specific thickness e2, e2 being less than e1. It can be seen that the thermal gradient is higher when the reaction chamber is thicker and that the melting peak width is greater for the thicker reaction chamber due to the higher thermal gradient.

[0120] In figures 10 and 11, we observe a first dashed curve representing a melting peak when the reaction chamber has a determined thickness e1, a second discontinuous dashed curve with one dot representing a melting peak when the reaction chamber has a determined thickness e2, a third discontinuous dashed curve with two dots representing a melting peak when the reaction chamber has a determined thickness e2;

[0121] el being greater than e2. In [Fig. 11], a solid line represents the double peak with the reaction chamber of thickness e2. The reduction in the thickness of the reaction chamber allows for melting peaks with a narrower width, which allows for better determination in cases of multiple melting peaks and better separation of these peaks.

Claims

Demands

1. A method for determining in vitro the presence or absence of at least one type of amplicon in a biological sample that has undergone polymerase chain reaction, an amplicon comprising two strands, the sample comprising at least one intercalating agent capable of being fluorescent when intercalated in an amplicon, the method comprising carrying out the following steps: - a) heating the biological sample so that the two strands of each amplicon of the type of amplicon separate if one or more amplicons of the type of amplicon are present in the biological sample; - b) measuring and acquiring, during heating, at different temperatures, the fluorescence of the intercalating agent; - c) calculating a temperature-dependent trend of a negative variation in the measured fluorescence with respect to temperature;- d) determination of at least one model of a Gaussian curve corresponding to the evolution, as a function of temperature, of the negative variation of the fluorescence measured with respect to the temperature calculated in step c); - e) processing of the Gaussian curve model so as to determine the presence or absence of the amplicon type in the biological sample.

2. A method according to claim 1, wherein step e) comprises a step el) of determining a melting temperature corresponding to the average of the model of the Gaussian curve and the presence or absence of the amplicon type in the biological sample is determined as a function of the melting temperature.

3. A method according to claim 2, wherein step e) comprises a step e2) of determining the standard deviation of the Gaussian curve model and a step e3) of determining the presence of the amplicon type in the biological sample if a point on a pre-established amplicon-specific standard curve has an abscissa substantially equal to the standard deviation and an ordinate substantially equal to the melting temperature, said standard curve representing melting temperatures for the amplicon type obtained for different models of Gaussian curves as a function of the standard deviation of the different models of Gaussian curves.

4. A method according to claim 3, wherein said models of Gaussian curves from which the standard curve is established are obtained by implementing steps a) to d) of the present method, the heating rate of the biological sample in step a) being different for obtaining each model of Gaussian curve.

5. A method according to any one of claims 1 to 4, wherein the presence or absence of at least two types of amplicons in the biological sample is determined, and wherein, in step d), at least two models of Gaussian curves corresponding to the evolution, as a function of temperature, of the negative variation of the fluorescence measured with respect to the temperature calculated in step c), are determined, and wherein, in step e), the presence or absence of the types of amplicons in the biological sample is determined according to the models.

6. A system (100) for the in vitro determination of the presence or absence of a type of amplicon in a biological sample that has undergone polymerase chain reaction, an amplicon comprising two strands, the sample comprising at least one intercalating agent capable of being fluorescent when intercalated in an amplicon, the system (100) comprising: - a heating device (101) adapted to heat the biological sample so that the two strands of each amplicon of the type of amplicon separate if one or more amplicons of the type of amplicon are present in the biological sample; - a fluorometer (102) adapted to measure the fluorescence of the intercalating agent; - a processing unit (103) configured to: - acquire, during heating, at different temperatures, the fluorescence of the intercalating agent; - calculate a temperature-dependent, negative variation of the measured fluorescence with respect to temperature;- determine at least one model of a Gaussian curve corresponding to the evolution, as a function of temperature, of the negative variation of the measured fluorescence with respect to the calculated temperature; - to process the Gaussian curve model in order to determine the presence or absence of the amplicon type in the biological sample.

7. 7. System (100) according to claim 6, wherein the heating device (101) is a Peltier module.

8. 8. System (100) according to any one of claims 6 and 7, wherein the biological sample is disposed in a chamber of an analysis card (1).

9. A method for controlling a system (100) according to claims 6 to 8, the method comprising carrying out the steps of the method according to any one of claims 1 to 5 to determine in vitro the presence or absence of at least one type of control amplicon in a biological control sample and carrying out a step f) of characterizing the operation of the system (100) as a function of the result of the step of determining the presence or absence of the type of control amplicon in the biological control sample.

10. Method according to claim 9, comprising a step g) of characterizing the operation of the heating device (101) as a function of a temperature gradient in the control biological sample determined from the standard deviation of the model of the Gaussian curve.

11. Use of the method according to any one of claims 1 to 5 for processing a fusion signal.

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