WO2019110620A1

WO2019110620A1 - Method for detecting the presence of a biochemical element

Info

Publication number: WO2019110620A1
Application number: PCT/EP2018/083552
Authority: WO
Inventors: Luc Montagnier
Original assignee: Fondation Luc Montagnier
Priority date: 2017-12-04
Filing date: 2018-12-04
Publication date: 2019-06-13
Also published as: CH714402A1

Abstract

Method for detecting a biochemical element in a solution, characterised in that it comprises the following steps: - high dilution (E2) of the solution, according to a dilution ratio of less than or equal to 10-3; - amplification (E3) of a certain trace of a biochemical element in the solution highly diluted by polymerase chain reaction (PCR); - detection of the presence of a biochemical element in the amplified solution by means of an electrophoresis step (E4) and/or by sequencing (E5) a fragment obtained, in particular by the Sanger method, and subsequently analysing (E6) the result.

Description

Method for detecting the presence of a biochemical element

The present invention relates to a method for detecting the presence of a biochemical element in a solution. It is particularly applicable to the detection of infectious agents in the blood of a patient. It allows the diagnosis of a certain disease, the discovery of elements responsible for a disease, and the improvement of the development of a therapy associated with a disease. The search for the presence of biochemical elements of bacteria type, or of bacterial forms, of viruses, etc., is very useful in many situations, in particular for medical applications, to make a diagnosis as relevant as possible. In these medical applications, bacterial or viral research is for example often performed from a blood plasma from which DNA is extracted. Such research is complex because it is necessary to avoid any bacterial contamination of the blood samples, which would distort the results. In addition, the existing methods implement filtering domains and other traditional steps, which prove to be insufficient in certain situations. As a result, certain bacteria or viruses, and more generally certain biochemical elements, especially when they are present in small quantities, remain very difficult or impossible to detect, which leads to misdiagnosis and inappropriate therapeutic treatments.

The present invention aims to overcome these drawbacks of traditional procedures of the state of the art and seeks a solution for detecting the presence of a biochemical element more sensitive, more precise, and therefore more reliable. In addition, the invention also seeks a solution for detecting the presence of a biochemical element that is easy to implement and at a minimum cost. For this purpose, the invention relates to a method for detecting a biochemical or chemical element in a solution, in particular an element present in a very small quantity, characterized in that it comprises the following steps:

- high dilution of the solution, according to a dilution ratio of less than or equal to 10 ³ ;

amplification of a certain trace of a biochemical element in the highly diluted solution by PCR polymerase chain reaction;

- Search for the presence of a biochemical element in the amplified solution by an electrophoresis step and / or by sequencing a fragment obtained, in particular by the method of Sanger, and analysis of the result.

The high dilution of the solution may comprise a diluted at a lower dilution than or equal to 10 ^4, or 10 ^-8, or 10 ¹⁰ or 10 ^13, or even at a lower dilution than or equal to 10 ^15, and / or at a dilution rate of between 10 ³ and 10 ³ inclusive, or even between 10 ¹ and 10 ² inclusive.

The process is advantageously repeated, from the amplification step, with several samples of the solution corresponding to several different high dilutions.

The method may further comprise a high agitation sub-step of the highly diluted solution prior to the amplification step. The high dilution step may comprise a dilution sub-step which comprises a series of successive dilutions of the solution, in water uncontaminated, with stirring for at least 10 seconds, or even at least 15 seconds, between all or part of the dilutions of the series of successive dilutions.

The amplification step can carry out a polymerase chain reaction PCR comprising at least 45 thermocycles, and / or using as polymerase that known by its trade name PerFecTa.

The high dilution step may include a substep of sterile water filtration.

The method for detecting a biochemical element may comprise one or more filtration steps of the solution, in particular by 450 nm and / or 100 nm filters, before the amplification step.

The biochemical element may be bacterial or viral in nature.

The solution can be a blood plasma.

The method of detecting a biochemical element may include a step of preparing the solution by adding an anticoagulant to a blood sample, and then separating the plasma by centrifuging the blood at a rate of about 3000 trs / sec. min for about five minutes at a controlled temperature of about 20 ° C.

The biochemical element may be Borrelia burgdorferi or Sutterella.

The invention also relates to a method of analyzing a patient, in particular suffering from Lyme disease or autism, characterized in that it comprises a step of analyzing a patient's plasma by setting of a method for detecting a biochemical element in this plasma as described above.

The invention also relates to a method of analyzing a disease, characterized in that it comprises the following steps:

method of detecting at least one biochemical element as previously described on body fluids of several persons suffering from the disease to be analyzed;

comparison of the results obtained between the different persons, and detection of the common biochemical element (s) for these different persons, to conclude that the disease is of infectious type caused by this or these biochemical element (s) (s) common.

The invention also relates to a definition of an antibiotic-based medication applied to Lyme disease, autism, rheumatoid arthritis, or Alzheimer's disease, after it is detected by a method as described previously.

The invention also relates to an antibiotic treatment applied to a disease analyzed by an analysis method as described above.

Finally, the invention also relates to a device for detecting a biochemical element for implementing the method for detecting at least one biochemical element as described above, characterized in that it comprises:

a diluter robot, which implements the dilution of a solution and a stirring of a microtube comprising the diluted solution; and

a thermocycler implementing amplification by PCR polymerase chain reaction. These objects, features and advantages of the present invention will be set forth in detail in the following description of a particular embodiment made in a non-limiting manner in relation to the appended figures among which:

FIG. 1 represents a logic diagram of the steps of the method for detecting the presence of a biochemical element in a solution according to one embodiment of the invention. Figures 2a and 2b show respectively the results of the application of the method according to the embodiment of the invention obtained on a patient of 14 years and his mother, both affected by Lyme disease. According to one embodiment of the invention, the method of detecting the presence of a biochemical element will now be described in the context of an exemplary search from a blood plasma. A particularity of this detection comes from the fact that this biochemical or chemical element sought is generally present in trace form, that is to say at a very low level in the solution obtained. It comes from an element present in the initial blood. In particular, this element may be present in the blood at a rate of less than 1 ng / l, and even of the order of a few pg / l, that is to say less than or equal to 100 pg / l, or even less or equal to 50 μg / l, or even less than or equal to 10 μg / l.

This method comprises a first preliminary step E1 preparation of blood plasma.

For this, it is possible to take between 2 and 5 ml of blood of a patient in the presence of an anticoagulant such as EDTA (ethylene diamine tetra acetic acid). The blood is then centrifuged at a speed of 3000 rpm for five minutes at a controlled temperature of 20 ° C. This operation allows to separate and recover the blood plasma. Blood plasma is stored at -20 ° C if not used immediately. It is stored at a temperature of 4 ° C if used immediately.

Then, according to a first dilution sub-step E11 of this preparation step E1, a plasma sample is diluted in sterile water at a ratio of 1: 100. This dilution step in the preparation has the function of obtaining a larger amount of plasma, which can be useful if many analyzes are planned. This sub-step therefore remains optional.

A second filtration sub-step E12 comprises the successive passage of the diluted plasma through 450 nm and 100 nm filters, for example using Millex filters marketed by Millipore. This filtration can potentially eliminate all or almost all bacteria and much, if not all, of the plasma viruses. It remains the smallest bodies, including traces of DNA and RNA. Thus, as will be detailed later, the detection method is based first of all on the detection of a trace of a biochemical element, particularly nucleic acid or nucleotides, which we will also call DNA trace. , from this biochemical element or corresponding to this chemical element, before it can be concluded a posteriori to the initial presence of the biochemical element in the plasma analyzed. As a reminder, the method is adapted to an initial presence of an element in a very small quantity, and which is therefore in the form of a trace only in the solution coming from the blood plasma thus prepared. In particular, the method is suitable for detecting said element that is present at a level of less than or equal to 100 μg / l, or even less than or equal to 50 μg / l, or even less than or equal to 10 μg / 1. This preliminary preparation step E1 being finalized, the method according to the embodiment of the invention, which consists of a method of ultrasensitive detection of a biochemical element in a solution, by means of the detection of a trace of this element, such as a nucleotide, a nucleic acid or trace or DNA sequence of this element in the solution, will now be described.

Note that these steps are performed in a class II biosafety cabinet. The operator wears protective clothing, such as a lab coat, nitrile gloves and a mask.

The method first comprises a second dilution step E2. This step is carried out from sterile water.

The method then implements a first substep of filtration E21 of sterile water, with a filter at 220 nm, to remove any microbial bodies that would contaminate the water. This sub-step is recommended but optional, it depends on the quality of the sterile water available.

Finally, the method implements a second substep of dilution E22 as such. According to the embodiment, this sub-step comprises a series of successive dilutions in the uncontaminated water of the solution to be analyzed, resulting from the preceding steps, that is to say the blood plasma in this embodiment.

According to the embodiment of the invention, several polypropylene microtubes of 1.5 ml can be filled with 0.9 ml of water each. Then, 0.1 ml of a plasma sample is added to the first microtube, then 0.1 ml of this first microtube is taken and transferred to the second microtube, and so on. This allows to chain several decimal dilutions in series. Advantageously, after each filling of a microtube, or after each decimal dilution, the filled microtube is vortexed at maximum speed for at least 10 seconds, preferably at least 15 seconds, before proceeding with further dilution. A number of at least 10 dilutions, or even at least 20, is thus achieved.

According to the embodiment, this number of dilutions is advantageously between 10 and 20 inclusive, or even beyond 20 dilutions. In other words, the dilution ratio is less than or equal to 10 ^10, or between 10 ¹⁰ and 10 ²⁰ inclusive, or even less than or equal to 10 ^20th.

However, this dilution depends on the biochemical element sought and the process can begin to give results for high dilutions of four decimal dilutions, ie a dilution rate of 10 ⁴ . As a remark, this dilution rate corresponds to the total dilution ratio, that is to say taking into account any dilutions made before the implementation of this second step E2, for example during the preparation step E1 mentioned previously. .

This second step E2 thus makes it possible to obtain a very highly diluted solution, which we will call highly diluted plasma in this embodiment for the rest of the description.

Note, the method according to the embodiment provides a strong stirring of the solution before the amplification step which will be described below. This process according to the embodiment also advantageously provides a strong agitation of the solution during the realization of this substep dilution, by the succession of sub-steps of dilutions and agitations. The stirring is more generally carried out between all or part of the successive dilutions. Furthermore, stirring is vigorous agitation, vortexing at high speed, for example in a device at 50 Hz at a speed of 2700 rpm, for at least ten seconds, or even at least fifteen seconds.

Of course, the E2 dilution step may alternatively include other dilutions than decimal dilutions.

Advantageously, this dilution step E2 is carried out by a diluter robot, which comprises an arm for automatically carrying out the operations of opening a microtube, sampling a predefined dose with precision, closing the microtube, payment of a dose in a neighboring microtube, etc. In addition, this dilutor robot also uses a microtube stirring function. For this, it may comprise a rotary support for microtube, allowing its agitation by rotating it. Alternatively, another stirring means is possible. Advantageously, the speed and / or the stirring time is adjustable.

The method then implements a third step E3 for amplifying traces of DNA in the highly diluted solution.

According to the embodiment of the invention, this third step uses a PCR polymerase chain reaction method.

For this, several samples of highly diluted plasma can be used to increase the reliability of the process. These samples may correspond to several distinct and / or identical dilution ratios.

By way of example, according to the embodiment, a highly diluted plasma sample of 5 ml is poured into a 0.2 ml microtube, in which a polymerase adapted to the PCR method, such as that known, is added. by its commercial name of PerFecTa ThoughMix of the company Quantabio, and 0,2 mM of each selected primer, to obtain a total volume of 20 mI. For example, in the case where the bacterium sought is Borrelia burgdorferi, for example responsible for Lyme disease, then the PCR method can be implemented in a thermocycler, for example as known by its trade name Nexus GSX1 / GX2e marketed by the company Eppendorf, as follows: a first denaturation step is initiated at 95 ° C for 5 minutes, followed by 45 temperature cycles, each comprising a period of 30 seconds at 95 ° C, then 30 seconds at 61 ° C, then 40 seconds at 72 ° C, before a final incubation of 15 minutes at 72 ° C. The sample or samples thus treated are then cooled to a temperature of 4 ° C.

This approach is conducted simultaneously or in parallel, from the same sample of the highly diluted solution or several separate samples, for each biochemical element sought. Naturally, the primers used during the PCR process will be adapted to the desired biochemical element. Note, in the case where the biochemical element (s) are not known, the method uses a relatively generic primer, that is to say compatible with a multitude of DNA sequences. The PCR method can also be carried out several times, with different primers, to search for a multitude of potential biochemical elements. On the other hand, it is advantageous in all cases to use at least 45 thermocyles.

This third step E3 therefore uses at least partially a known method of reducing, in other words amplification, traces of DNA. The PCR method is based on a heat-resistant polymerase, which makes it possible to copy sequences of DNA strands. The recognition of this sequence very rapidly among a large number of sequences is a particular phenomenon that has sometimes been explained by Brownian motion, which is controversial. The method is here successfully implemented in an original manner from a highly diluted plasma, which suggests the potential implementation of phenomena other than Brownian motion. It turns out that this particular implementation allows to obtain unexpectedly greater sensitivity. This effect is explained by a polymerase operation different from that traditionally accepted, not involving a simple copy of a sequence of a model but a transmission of information of a different nature. As a remark, the PCR method is not implemented on DNA extracted from the plasma, as traditionally in the state of the art, but directly on the highly diluted plasma.

Naturally, the method may use different variants of PCR amplifications, or any equivalent or alternative amplification method.

At the end of this third step E3, the amplification makes it possible to reach a sufficient quantity of DNA sequences for their detection and analysis. We will call simply amplified solution the solution resulting from this third step.

The method according to the embodiment then implements a fourth electrophoresis step E4. 5 μl of each sample from the previous step is mixed with 1 ml of a buffer known by its trade name 6X, forming a marker, and subjected to electrophoresis on a 1.2% aragose gel, in the presence of a fluorescent dye of DNA, known by its English name of SybrSAFE sold by InVitrogen. A scale of markers, for example known by its name 100 bp ladder, marketed by InVitrogen, is used in parallel to estimate the size of the fragments. This step allows the separation of the biochemical elements according to their molecular weight and the visualization of the DNA fragment present in the amplified highly diluted plasma (amplicon). In particular, it can be verified that the height of the band corresponds to that of a positive control. In certain cases, depending on the biochemical element sought, this fourth step E4 proves to be sufficient, that is to say it transmits sufficient information for the identification of a biochemical element during the analysis step. E6. The method can implement a fifth step E5, in addition or replacement of the fourth step E4, sequencing amplicons produced by the PCR method of the third step. For this, the amplicons are purified with a purification kit, for example that called "QIAquick PCR purification kit" marketed by the company QIAGEN. The purified amplicons are then sequenced directly by the Sanger method, using the primers of the PCR method.

Alternatively, particularly if it turns out that the number of amplicons is small, the purified amplicons are cloned in a plasmid vector pCR2.1, for example using the cloning kit known by its trade name TOPO TA marketed by InVitrogen, which is used to transform competent bacteria that are cultured on LB agar medium selection boxes containing ampicillin. Colonies are isolated and cultured from a single bacterium in 4 ml LB culture broths also containing ampicillin and plasmid clones are purified and screened by EcoRI restriction endonuclease digestion to check for the presence of inserts. Positive plasmids are sequenced by the Sanger method using M13 direct and reverse universal primers.

The method then implements a sixth step E6 of result analysis. This step may comprise the simple comparison of the result of the fourth step E4 with respect to controls. It may also, or alternatively, include an analysis of the sequences obtained by the fifth step E5, which can be done by searching a database, for example with the Blast tool. The nucleotide sequence of the purified or cloned amplicons is analyzed with this tool to compare the elements obtained by the method with stored data, and identify significant approximations that allow to conclude the identification of the sequences of the bacterial species involved.

The process described above thus ultimately makes it possible to detect any biochemical element, in particular of a bacterial or viral nature. It allows for example to detect the bacteria Borrelia burgdorferi, or the neighboring species, or the bacteria Sutterella. As a remark, the process will naturally be adapted to the desired biochemical element. In particular, as explained above, the amplification step E3 uses suitable primers. In addition, the filtrations will be similarly adapted to the size of the desired element, so as to eliminate the maximum number of elements from the solution, while retaining traces of the microbial nucleic acid type, in order to be able to detect them thereafter. Thus, in the case of a search for a biochemical element of the virus type, the method described according to this embodiment can be implemented identically by replacing the filtering at 100 nm with a filtering at 20 nm (sub- filtration stage E12), to take into account the smallest dimension of the biochemical element.

The method has been described on the basis of blood plasma, but can be implemented from any solution.

As a remark, an essential characteristic of the process consists in carrying out a high dilution of the test solution. We call strong dilution any dilution of at least three or four times the decimal dilution, a dilution rate of less than or equal to 10 ³ or 10 ⁴ . As a variant, the dilution ratio may be less than or equal to 10 ⁸ , or even 10 ¹⁰ , or even 10 ¹³ , even 10 ¹⁵ , that is to say a dilution equivalent to a dilution greater than or equal to 8, even 10 or 13 or 15, decimal dilutions. Preferably, the dilution rate is between 10 ³ and 10 ³ inclusive, or even of between 10 ¹⁰ and 10 ^20, inclusive. This high dilution brings the unexpected effect of a higher sensitivity of the detection of a biochemical element than the same process implemented without high dilution, as mentioned above. Note that this effect is verified regardless of the biochemical element concerned, and more specifically regardless of the DNA trace of the biochemical element in the solution.

As a remark, the process is advantageously repeated with several samples of the highly diluted solution, characterized by several different dilution ratios. It can then be concluded in the sixth step E6 of result analysis that the biochemical element was indeed present in the initial solution, since at least one, or even or at least two or three, sample (s) allows ( try) its detection. This approach thus makes it possible to obtain a positive result even when the biochemical element is present in a very small quantity in the solution. An example of application of the method described above to a 14-year-old patient suffering from chronic Lyme disease, manifested by fatigue, joint pain, as well as intestinal inflammation, will now be described, with reference to the Figure 2a.

If the PCR method is conventionally applied for this patient, which is not shown, no detection is obtained as a result of the fourth E4 electrophoresis step. In other words, the method of the state of the art does not detect the presence of the bacterium Borrelia burgdorferi.

FIG. 2a represents the results obtained for this patient, by applying the method according to the embodiment of the invention, from the highly diluted plasma. The different columns numbered from 1 to 20 represent the various decimal dilutions applied to the plasma already diluted to one-hundredth in substep E1 1. Thus, column 1 corresponds to a third dilution and a dilution ratio of 10 ³ , and and so on. Figure 2a shows that a 499 bp band is detected for column 3, then for columns 14-20. Sanger sequencing of the purified amplicon (fifth step E5) and Blast analysis ( sixth step E6) finally indicate the strongest homology of the sequence of Borrelia ribosomal 16s expected at +/- 1 bp in comparison with the reference sequence of the Borrelia burgdorferi B31 sequence provided by ATCC, which corresponds to the disease of Lyme.

It thus appears that the diagnosis of the disease can not be established or confirmed with traditional techniques, especially with the traditional PCR method, which does not demonstrate the presence of the bacterium Borrelia burgdorferi. On the contrary, with the method according to the embodiment of the invention, an infection with the bacterium Borrelia burgdorferi has been shown, which has led to the conclusion that it is Lyme disease. The method according to the embodiment of the invention thus makes it possible to improve the diagnosis of a disease, and then of course the associated medication.

In addition, it should be noted that the patient's mother also developed substantially the same symptoms as her son. FIG. 2b illustrates the results obtained by the fourth electrophoresis step for several dilution rates on the mother's plasma, during the implementation of the method according to the embodiment of the invention in a manner similar to the method of the invention. Figure 2a. Low band detection at 499 bp is obtained with a dilution ratio of 10 ³ , followed by detection for many other dilution levels. Note, if the PCR method is conventionally applied for this patient, which is not shown, no detection is obtained as a result of the fourth electrophoresis step E4.

This example illustrates that the detection method of the invention surprisingly reaches a very high sensitivity using high dilution rates, which allows the detection of the presence of a biochemical element that was not possible before. This results in a new and more reliable diagnosis, as illustrated in this example with Lyme disease, to define a suitable medication, contrary to what was previously proposed.

As a result of the invention, there are many applications, including of course a method for understanding disease and improving disease detection. This method of the invention makes it possible, for example, to highlight elements of a bacterial or viral nature in relation to different diseases, demonstrating that these diseases are of an infectious nature, whereas they were considered non-infectious. previously because of the impossibility of detecting the microorganisms at the origin of the infection. By way of examples, mention may be made of autism, rheumatoid arthritis and Alzheimer's disease. The method is therefore also used as a method of diagnosing a disease.

The invention also relates to a method of analyzing a certain disease. This process comprises the following steps:

- Application of the method for detecting biochemical elements as described above on body fluids of several people suffering from the disease to be analyzed. This process may be repeated on all samples so as to search for a multitude of potential biochemical elements, in particular of microbial nature;

- comparison of the results obtained for each person, and detection of the common biochemical element (s) for these different people, to conclude that the disease is of infectious type, caused by this (or these) common biochemical element.

Thus, the invention makes it possible to identify the biochemical elements responsible for a certain disease that may be new or poorly understood to date. By way of examples, it is demonstrated by the method according to the embodiment of the invention that people suffering from autism carry the bacteria Sutterella.

This improvement of the diagnosis and the knowledge of the diseases naturally makes it possible to define a medication more adapted than it is currently the case. Thus, the method for detecting biochemical elements according to the invention is also useful for defining a medication, particularly based on an antibiotic, in the case of diseases identified as a bacterial source.

The invention also relates to a device for detecting a biochemical element for carrying out the method for detecting at least one biochemical element as described above, in a manner that is at least partially automatic, which comprises:

a diluter robot, which implements the dilution of a solution in a tube and a stirring of a tube comprising the diluted solution; and

a thermocycler using amplification by polymerase chain reaction PCR.

This device may also comprise a device for analyzing results by electrophoresis and / or sequencing, in known manner.

Claims

1. A method for detecting a biochemical element in a solution, characterized in that it comprises the following steps:

- High dilution (E2) of the solution, with a dilution ratio of less than or equal to 10 ³ ;

- amplification (E3) of a certain trace of a biochemical element in the solution strongly diluted by polymerase chain reaction PCR;

- search for the presence of a biochemical element in the amplified solution by an electrophoresis step (E4) and / or by sequencing (E5) of a fragment obtained, in particular by the Sanger method, and analysis (E6) of the result.

2. Method for detecting a biochemical element according to the preceding claim, characterized in that the high dilution of the solution comprises a dilution at a dilution ratio of less than or equal to 10 ⁴ , or 10 ⁸ , or 10 ¹ °, or ¹³ , or even at a dilution ratio of less than or equal to 1 0 ¹⁵ and / or at a dilution ratio of between 10 ³ and 10 ³ inclusive, or even between 10 ¹ and 10 ² inclusive.

3. A method for detecting a biochemical element according to one of the preceding claims, characterized in that the method is repeated, from the amplification step (E3), with several samples of the solution corresponding to several strong different dilutions.

4. A method for detecting a biochemical element according to one of the preceding claims, characterized in that the method comprises a substep of strong agitation of the highly diluted solution before the amplification step (E3).

5. A method for detecting a biochemical element according to one of the preceding claims, characterized in that the step of high dilution (E2) comprises a substep dilution (E22) which comprises a series of successive dilutions of the solution, in uncontaminated water, with stirring for at least 10 seconds, or even at least 15 seconds, between all or part of the dilutions of the series of successive dilutions.

6. A method for detecting a biochemical element according to one of the preceding claims, characterized in that the amplification step

(E3) uses a polymerase chain reaction PCR comprising at least 45 thermocycles, and / or using as polymerase that known by its trade name PerFecTa.

7. A method for detecting a biochemical element according to one of the preceding claims, characterized in that the high dilution step (E2) comprises a substep (E21) of filtration of sterile water.

8. A method for detecting a biochemical element according to one of the preceding claims, characterized in that it comprises one or more filtration steps (E12) of the solution, in particular by 450 nm and / or 100 nm filters. before the amplification step (E3).

9. A method for detecting a biochemical element according to one of the preceding claims, characterized in that the biochemical element is of bacterial or viral nature.

10. A method for detecting a biochemical element according to one of the preceding claims, characterized in that the solution is a blood plasma.

1. A method for detecting a biochemical element according to the preceding claim, characterized in that it comprises a step of preparation (E1) of the solution by adding an anticoagulant to a blood sample, and then the separation. plasma by centrifuging the blood at a rate of about 3000 rpm for about five minutes at a controlled temperature of about 20 ° C.

12. A method for detecting a biochemical element according to one of claims 9 to 11, characterized in that the biochemical element is the bacterium Borrelia burgdorferi or the bacteria Sutterella.

13. A method for detecting a biochemical element according to one of the preceding claims, characterized in that the solution comprises said biochemical element at a level less than or equal to 100 pg / l, or even less than or equal to 50 pg / l, even less than or equal to 10 pg / l.

14. A method for analyzing a patient, in particular suffering from Lyme disease or autism, characterized in that it comprises a step of analyzing a patient's plasma by implementing a method for detecting a biochemical element in this plasma according to one of the preceding claims.

15. A method of analyzing a disease, characterized in that it comprises the following steps:

- Method of detecting at least one biochemical element according to one of claims 1 to 13 on body fluids of several people suffering from the disease to be analyzed;

16. Definition of an antibiotic-based medication applied to Lyme disease, autism, rheumatoid arthritis, or Alzheimer's disease, after it has been detected by a method according to one of claims 9 to 13.

17. Antibiotic treatment applied to a disease analyzed by an analysis method according to claim 15.

18. A device for detecting a biochemical element for carrying out the method for detecting at least one biochemical element according to one of claims 1 to 13, characterized in that it comprises:

a thermocycler using amplification by polymerase chain reaction PCR.