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

Abstract

The invention relates to a method for the in vitro determination of 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 exhibiting fluorescence when inserted into an amplicon, the method comprising the implementation of a plurality of steps, including the processing of the Gaussian curve model so as to determine the presence or absence of the type of amplicon in the biological sample.

Description

[0001] TITLE: In vitro method for determining the presence or absence of at least one amplicon

[0002] FIELD OF INVENTION

[0003] 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).

[0004] STATE OF THE ART

[0005] To detect a specific type of amplicon in a biological sample, a polymerase chain reaction (PCR) is performed, followed by heating the sample so that the amplicons of the target type dehybridize, meaning their strands separate. This heating step is also called melting. During this step, successive fluorescence measurements are taken, and a melt curve is constructed, representing the change in fluorescence as a function of temperature. The negative primary derivative of this curve, which represents the change in fluorescence with temperature, is then calculated, and the temperature corresponding to the maximum change in fluorescence is determined.This temperature is considered the melting temperature, which is specific to a particular 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.

[0006] However, this technique has drawbacks. Indeed, the determined melting temperature varies depending on the heating rate of the sample (and therefore the temperature gradient within the sample). 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, depending on the sample heating rate, the detection systems, and other factors, different melting temperatures are determined for the same sample. This can lead to detection errors of a type of amplicon, i.e., false positives or false negatives. DESCRIPTION OF THE INVENTION

[0007] One aim 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.

[0008] Another objective 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.

[0009] 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:

[0010] - a) 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;

[0011] - b) measurement and acquisition during heating, for different temperatures, of the fluorescence of the intercalating agent;

[0012] - c) calculation of an evolution, as a function of temperature, of a negative variation of the fluorescence measured with respect to temperature;

[0013] - 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);

[0014] - e) processing of the Gaussian curve model in order to determine the presence or absence of the amplicon type in the biological sample.

[0015] Depending on advantageous and non-limiting characteristics, taken alone or in any combination:

[0016] - step e) includes 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 type of amplicon in the biological sample is determined as a function of the melting temperature;

[0017] - Step e) comprises a step e2) for determining the standard deviation of the Gaussian curve model and a step e3) for 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. - said Gaussian curve models 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 Gaussian curve model;

[0018] - we seek to determine the presence or absence of at least two types of amplicons in the biological sample and in which, in step d), we determine 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) and in which, in step e), we determine the presence or absence of the types of amplicons in the biological sample according to the models;

[0019] 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:

[0020] - a suitable heating device to heat 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;

[0021] - a fluorometer suitable for measuring the fluorescence of the intercalating agent;

[0022] - a processing unit configured for:

[0023] - acquire, during heating, for different temperatures, the fluorescence of the intercalating agent;

[0024] - calculate an evolution, as a function of temperature, a negative variation of the fluorescence measured with respect to temperature;

[0025] - 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;

[0026] - to process the Gaussian curve model in such a way as to determine the presence or absence of the amplicon type in the biological sample.

[0027] According to one feature of the invention, the heating device is a Peltier module.

[0028] According to one feature of the invention, the biological sample is placed in a chamber of an analysis card.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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 Gaussian curve model.

[0033] 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 readability and simplifies demodulation and the determination of the peak of interest for amplicons.

[0034] DESCRIPTION OF THE FIGURES

[0035] 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:

[0036] Fig. 1 schematically illustrates an in vitro system for determining the presence or absence of at least one type of amplicon in a biological sample that has undergone polymerase chain amplification;

[0037] Fig. 2 represents an in vitro method for determining the presence or absence of at least one type of amplicon in a biological sample that has undergone polymerase chain amplification;

[0038] Fig. 3 illustrates a curve of the evolution of the fluorescence of an intercalating agent as a function of temperature in the sample;

[0039] 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;

[0040] Fig. 5 illustrates a Gaussian curve obtained by Gaussian fitting applied to the curve in figure 3;

[0041] Fig. 6 illustrates, for the same 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;

[0042] Fig. 7 illustrates a standard curve representing, for an amplicon, the correspondence between the determined melting temperature and the standard deviation;

[0043] 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.

[0044] Fig. 9 is a graphical representation of the comparison of Gaussian curves when the thickness of the reaction chamber varies.

[0045] Fig. 10 is a graphical representation of the comparison of Gaussian curves when the thickness of the reaction chamber varies in a multiplexing case.

[0046] Fig. 11 is a graphical representation following figure 10.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] With reference to Figure 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 that has undergone polymerase chain reaction (PCA or, in English, PCR for "polymerase chain reaction").

[0049] As illustrated in Figure 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.

[0050] Since the biological sample has undergone polymerase chain reaction, if the biological sample contained amplicons of a certain type of amplicon prior to polymerase chain reaction, the biological sample is assumed to have a high concentration of that type of amplicon.

[0051] An amplicon is a segment of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). An amplicon of the type we are trying to detect in the sample is, for example, a segment of DNA from a virus such as the hepatitis B, C, and D viruses. An amplicon consists of two strands.

[0052] 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. Therefore, 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.

[0053] 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.

[0054] The biological sample includes an intercalating agent capable of fluorescence when intercalated into an amplicon. The intercalating agent is able to intercalate into an amplicon, that is, to insert 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.

[0055] Analysis card 1 is advantageously suited to be the site of polymerase chain amplification.

[0056] Analysis card 1 therefore advantageously includes a chamber adapted to receive a biological sample (not yet undergoing 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.

[0057] The chambers and tanks are shaped like shells or "blisters" in English.

[0058] Alternatively, the reaction chamber(s) can be formed in a plate or array, generally defined as a flat surface element of 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 faces.

[0059] 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.

[0060] 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 following description for the sake of simplicity.

[0061] Advantageously, the 101 heating device is a Peltier module. A Peltier module exploits the Peltier effect, by which an electric current is converted into a temperature difference.

[0062] System 100 includes a fluorometer 102 adapted to measure the fluorescence of the intercalating agent in the biological sample.

[0063] The system 100 further includes a processing unit 103, such as a processor. The processing unit 103 is configured to:

[0064] - acquire, during the heating of the biological sample by the heating device 101, for different temperatures, the fluorescence of the intercalating agent;

[0065] - calculate an evolution, as a function of temperature, of a negative variation of the fluorescence measured with respect to temperature;

[0066] - 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;

[0067] - to process the Gaussian curve model in such a way as to determine the presence or absence of the amplicon type in the biological sample. With reference to Figure 2, an in vitro method for determining the presence or absence of at least one amplicon type in a biological sample that has undergone polymerase chain reaction is proposed.

[0068] As explained previously, the biological sample may contain one or more amplicon types. In other words, the biological sample is likely to contain one or more amplicons of the type being detected. If the biological sample contains one or more amplicons, the sample, and in particular the intercalating agent sandwiched between the amplicon strands, will fluoresce.

[0069] 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.

[0070] 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 allows the type of amplicon present in the biological sample to be determined.

[0071] When the two strands of the amplicon separate, the intercalating agent that was inserted between them is released and ceases to fluoresce. Therefore, the melting temperature corresponds to the temperature at which a decrease in fluorescence is observed. It is thus necessary to monitor the fluorescence during step a) heating.

[0072] The maximum heating temperature depends on the type of amplicon being detected. The biological sample is advantageously heated to a temperature higher than the temperature specific to the amplicon type. 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 being detected.

[0073] 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 within the sample, which facilitates the analysis of fluorescence data and makes it easier to detect the amplicon type. However, we will see that the method presented allows us to determine the presence of a specific amplicon type even when the heating rate is high, for example, greater than 5°C per second or even greater than 10°C per second, and therefore when the temperature gradient within the sample is significant.

[0074] 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.

[0075] The fluorometer 102 measures the fluorescence of the intercalating agent during heating. Preferably, at least one temperature value per second is measured. The processing unit 103 acquires the fluorescence during heating. In other words, the processing unit 103 acquires the measurements taken by the fluorometer 102.

[0076] Fluorescence measurements can be plotted against temperature. Figure 3 illustrates an example of how measured fluorescence changes with 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 in the biological sample (which may be of the same or different types) separate, and the intercalating agent is released, thus ceasing to emit fluorescence.

[0077] The process includes a step (c), implemented by the processing unit 103, which calculates the temperature-dependent evolution of a negative variation in the measured fluorescence with respect to temperature. In other words, the negative derivative of the fluorescence temperature-dependent evolution (i.e., the negative derivative of the curve illustrated in Figure 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)).

[0078] Advantageously, to calculate the negative derivative, the derivative of the evolution of fluorescence as a function of temperature is first calculated.

[0079] 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.

[0080] Another way to calculate the derivative is to approximate (such as by regression or interpolation) the evolution of fluorescence as a function of temperature into a function and then calculate the derivative of that function.

[0081] In any case, a person skilled in the art knows how to calculate the derivative of a set of points.

[0082] Then, to obtain the negative derivative, simply reverse the sign of each point of the derivative.

[0083] 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 fluorescence as a function of temperature.

[0084] Advantageously, as illustrated in Figure 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.

[0085] 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).

[0086] In other words, a Gaussian "fitting" is performed, meaning that a Gaussian curve model is sought that most closely resembles the temperature-dependent evolution of the measured negative fluorescence value. Put another way, the Gaussian curve model is sought that best matches a curve representing the temperature-dependent evolution of the measured negative fluorescence value.

[0087] As illustrated in Figure 5, the Gaussian curve pattern includes a peak (like a Gaussian curve well known to a person skilled in the art).

[0088] Step d) has the advantage of removing noise from the temperature-dependent values ​​of the measured negative fluorescence variation. In other words, step d) cleans the data corresponding to the temperature-dependent negative fluorescence variation. This results in a more reliable and easier-to-analyze Gaussian curve model.

[0089] 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).

[0090] For this, 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 temperature-dependent evolution of the negative variation of the measured fluorescence with respect to temperature.

[0091] Advantageously, this first Gaussian curve model is then subtracted from the temperature-dependent negative fluorescence variation of the measured value. A second Gaussian curve model can then be determined by performing a Gaussian fitting on the temperature-dependent negative fluorescence variation of the measured value, subtracted from the first Gaussian curve model. This second Gaussian curve model includes the second-largest peak in the temperature-dependent negative fluorescence variation of the measured value.

[0092] 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.

[0093] This method allows, as we will see later, to determine the presence or absence of several types of amplicons in a biological sample in a simple and reliable way.

[0094] The process includes a step e) of processing, by the processing unit 103, the Gaussian curve model to determine the presence or absence of the amplicon type in the biological sample. The amplicon type whose presence or absence is to be determined in the biological sample is known in advance. In other words, the process does not seek to detect the presence of an amplicon whose type is unknown. Put another way, the target is known. For example, the process might seek to determine the presence of the amplicon type characteristic of the hepatitis B virus. 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 amplicon type 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 negative change in fluorescence measured with respect to temperature). The mean p is shown schematically in Figure 5.

[0095] In one embodiment, the presence or absence of the amplicon type in the biological sample is determined based on its melting point. To do this, the melting point obtained is compared to the theoretical melting point of the amplicon type being sought. If the melting points are substantially identical, the amplicon type in the biological sample is considered to be the one being sought. Otherwise, the amplicon type in the biological sample is considered not to be the one being sought.

[0096] An advantageous embodiment will be described, which is particularly beneficial when the Gaussian curve model is unreliable. For example, the peak within the Gaussian curve model may be imprecise and spread out, as illustrated in Figure 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 being sought.

[0097] 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 σ. The standard deviation is proportional to the full width at half maximum (FWHM) of the Gaussian curve. Indeed, we have:

[0098] [Math. 1] width at mid — height = 2 V21n (2)o ~ 2.3548 a.

[0099] The width at half height L is shown schematically in figure 5.

[0100] The standard deviation characterizes the temperature gradient, i.e., the temperature distribution, within the biological sample. The larger the standard deviation, the greater the temperature gradient within the biological sample, which may indicate a malfunction of the heating device 101 (which heats too slowly) or incorrect positioning of the biological sample relative to the heating device 101. We will see that the standard deviation can therefore be used to verify the proper functioning of the system 100 for determining the presence or absence of a particular type of amplicon in the biological sample.

[0101] Advantageously, the process includes a step e3) for determining the presence of the amplicon type in the biological sample if a point on a pre-established standard curve specific to that amplicon type has an abscissa approximately equal to the standard deviation and an ordinate approximately equal to the melting point. In other words, both the standard deviation and the mean, i.e., the melting point, of the Gaussian curve model are used to determine the presence or absence of the amplicon type. A standard curve is used for this purpose. This standard curve is used in the same way as a nomogram.

[0102] The standard curve represents, for the type of amplicon whose presence is being 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 Figure 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 being determined). The standard curve is specific to the amplicon type.

[0103] The Gaussian curve models from which the standard curve is established are obtained by implementing steps a) to d) of this procedure using standard biological samples, each containing the type of amplicon whose presence or absence is to be determined. Gaussian curve models for amplicon type A are illustrated in Figure 8.

[0104] 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 this procedure 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.

[0105] The heating rate of the biological sample in step a) differs for each Gaussian curve model, resulting in a different temperature gradient for each standard biological sample. This allows for the generation of Gaussian curve models with different standard deviations, enabling the creation of a standard curve representing the melting temperature as a function of the standard deviation for the amplicon type whose presence or absence is being investigated. The standard curve for amplicon type A is illustrated in Figure 7.

[0106] By determining whether the standard curve includes a point whose x-coordinate corresponds to the standard deviation obtained for the biological sample in question and whose y-coordinate corresponds to the melting point obtained for the biological sample in question, 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.

[0107] By "corresponds", it is understood that we are looking for a point whose abscissa corresponds approximately to the standard deviation obtained for the biological sample considered and whose ordinate corresponds approximately to the melting temperature obtained for the biological sample considered.

[0108] According to an embodiment where the aim is to determine the presence or absence of several types of amplicons, 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 a second type of amplicon, and so on.

[0109] Thus, the present method allows for the simple and reliable determination of the presence or absence of a specific amplicon type in a biological sample. This method, particularly through the use of a calibration curve, offers the advantage of enabling reliable amplicon type detection even at high heating rates. Therefore, the heating step can be accelerated without compromising the reliability of amplicon type detection.

[0110] The steps of the presented process can be implemented successively, partially simultaneously, or simultaneously.

[0111] A method is proposed for testing System 100, which determines in vitro the presence or absence of at least one type of amplicon in a biological sample. This testing method aims to characterize the operation of System 100. It allows for the simple and reliable detection of malfunctions in System 100.

[0112] The control process includes the implementation of the steps of the determination process presented previously to determine in vitro the presence or absence of at least one type of control amplicon in a biological control sample.

[0113] By "control biological sample," we mean a biological sample known to contain a certain type of amplicon, referred to as the control amplicon type. In other words, the biological sample is known to contain the control amplicon type, 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 control amplicon type in the control biological sample using System 100 will result in the determination of the presence of the control amplicon type in the control biological sample. Otherwise, a malfunction of System 100 can be concluded.

[0114] The control procedure therefore includes the implementation of step f) to characterize the operation of system 100 based on the result of the step determining the presence or absence of the control amplicon type in the control biological sample. Step f) can be implemented by 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.

[0115] The malfunction could, for example, originate from a problem with the fluorometer 102. For instance, the fluorometer 102 might be acquiring erroneous fluorescence data. In this case, a corrective measure could be to adjust / configure the fluorometer 102 or to replace it with a non-malfunctioning one.

[0116] The malfunction could, for example, originate from a malfunction of the heating device 101. For instance, the heating device 101 might not heat sufficiently and / or quickly or slowly enough. Alternatively, or in addition, the biological sample might not be positioned correctly on the heating device 101 (for example, not close enough and / or not centered).

[0117] Advantageously, the control process includes a step g) of characterizing the operation of the heating device 101. Step g) can be implemented by the processing unit 103.

[0118] 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 us to characterize the temperature gradient in the control biological sample. The larger the standard deviation, the greater the temperature gradient in the control biological sample. An excessively large temperature gradient in the biological sample is undesirable, as 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, which is, for example, that communicated by a thermocouple to the processing unit 103 at that time t. The acquired fluorescence data may therefore not allow the presence of a type of amplicon to be determined in the sample.

[0119] At the end of step g), a malfunction of the heating device 101 may be detected. For example, a malfunction may be detected if the temperature gradient exceeds a threshold determined according to the instrument and its technical specifications. In this case, a corrective measure may 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 may be correctly repositioned on the heating device 101 (e.g., closer and / or more centered).

[0120] 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 y-axis and the temperature in degrees Celsius on the x-axis. Thus, in Figure 9, a dashed line represents a melting peak when the reaction chamber has a specific thickness e1, and a solid line represents a melting peak when the reaction chamber has a specific thickness e2, where e2 is less than e1. It can be seen that the temperature gradient is higher when the reaction chamber is thicker, and the melting peak width is greater for the thicker reaction chamber due to the higher temperature gradient.

[0121] Figures 10 and 11 show a first dashed curve representing a melting peak when the reaction chamber has a specific thickness el, a second dashed curve with one dot representing a melting peak when the reaction chamber has a specific thickness eZ, and a third dashed curve with two dots representing a melting peak when the reaction chamber has a specific thickness e2, where el is greater than e2. In Figure 11, a solid line represents the double peak with the reaction chamber of thickness e2. Reducing the thickness of the reaction chamber results in melting peaks with a narrower width, which allows for better identification and separation of multiple melting peaks.

Claims

DEMANDS 1. A method for in vitro determining 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: - a) 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; - b) measurement and acquisition during heating, for different temperatures, of the fluorescence of the intercalating agent; - c) calculation of an evolution, as a function of temperature, of a negative variation of the fluorescence measured 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 in order 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 model of the Gaussian curve and a step e3) of determining the presence of the type of amplicon in the biological sample if a point of 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 type of amplicon melting temperatures 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 temperature-dependent evolution of the negative variation of the measured fluorescence relative to the temperature calculated in step c) and in which, in step e), the presence or absence of the types of amplicons in the biological sample is determined according to the models.

6. 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 amplicon type separate if one or more amplicons of the amplicon type are present in the biological sample; - a fluorometer (102) adapted to measure the fluorescence of the intercalating agent; - a processing unit (103) configured for: - acquire, during heating, for different temperatures, the fluorescence of the intercalating agent; - calculate an evolution, as a function of temperature, a negative variation of the fluorescence measured 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. System (100) according to claim 6, wherein the heating device (101) is a Peltier module.

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 the processing of a fusion signal.

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