WO2015150113A1

WO2015150113A1 - Method for molecular diagnostics for concentrating a nucleic acid from a biological sample

Info

Publication number: WO2015150113A1
Application number: PCT/EP2015/055873
Authority: WO
Inventors: Oliver Hayden; Gerald Warnat; Michael Johannes Helou; Mathias Reisbeck; Lukas RICHTER
Original assignee: Siemens Aktiengesellschaft
Priority date: 2014-04-03
Filing date: 2015-03-20
Publication date: 2015-10-08
Also published as: EP3092319A1; DE102014206444A1

Abstract

The invention relates to a method for molecular diagnostics, for concentrating nucleic acids (11) from microorganisms (12) from a biological sample (10) of an organism, wherein at least a portion of the microorganisms (12) are marked (S1) by opsonins (16), which are each coupled to a marker (18), followed by a removal (S3) of the marked microorganisms (12) from the biological sample (10) by means of a cell-sorting device (34), whereby the marked microorganisms (12) are concentrated and thus the nucleic acids (11) of the microorganisms (12) are concentrated, in order to provide a fast, simple-to-perform routine for laboratory diagnostics close to the patient, in particular for sepsis diagnostics.

Description

description

Method for molecular diagnostics; for enriching a nucleic acid from a biological sample

The invention relates to a method for molecular diagnostics for enriching a nucleic acid from a biological sample.

So far, in molecular diagnostics, when there is little time available, two methods are the most common

Nucleic acid diagnostics used. On the one hand, Roche's Septifast product can be used to extract a nucleic acid directly from at least 1.5 milliliters of whole blood by means of an initial cell disruption, ie an initial cell lysis. On the other hand, nucleic acid extraction can be carried out by means of a product of Molzym via selective lysis. There, first a mild lysis followed by a

Centrifugation takes place. This is followed by hard lysis and purification of the nucleic acid.

For a particularly rapid diagnosis, in particular a near-patient laboratory diagnostics, which can also be performed by ordinary medical staff without laboratory training, there is currently no suitable method. This applies in particular to rapid sepsis diagnostics, in which bacteria or resistances are to be diagnosed.

One object of the present invention is to provide a method for molecular diagnostics for enriching nucleic acid from a biological sample, which is particularly quick and easy to perform, so that it can be performed as a routine diagnostic of patient-related laboratory diagnostics by personnel without laboratory training.

This object is achieved by the method according to the independent claim. Further advantageous embodiments result from the dependent claims, the description and the figure.

The invention relates to a method for the molecular diag- nostic, for enriching, in particular for concentrating, nucleic acids from microorganisms from a biological sample of an organism. The microorganisms are in particular organism-foreign, e.g. organism damaging microorganisms. An organism is preferably to be understood here as meaning a human or an animal, but also a plant, and in particular a living organism. In particular, the organism can also be a foodstuff, for example a beverage containing living microorganisms, such as e.g. Beer with yeast or milk with mushroom act. In particular, the nucleic acids can be a multiplicity of specimens with an identical nucleic acid structure. It can then be used, for example, nucleic acids from e.g. Enriched pathogenic cells. The biological sample may in particular be an unlysed biological sample with which lysis has not yet been carried out after removal from the organism. In particular, this may also be a chemically untreated biological sample.

In order for the process to be carried out simply and quickly, at least one fraction of the microorganisms of the biological sample is initially marked

Opsonins, wherein the opsonins are each coupled to a marker. Since only at least a proportion of the microorganisms is labeled, it is also possible, for statistical reasons, for example, to leave unmarked microorganisms in the biological sample after labeling. Marking is followed by separation, ie separation, of the labeled microorganisms from the biological sample by means of a cell sorting device, which enriches the labeled microorganisms and thus enriches the nucleic acids of the microorganisms. In order to enrich the nucleic acids of the microorganisms so are not the nucleic acids self-labeled. Rather, there is a nonspecific marking of the microorganisms, in which a coupling of opsonins to cell structures, which occur in particular in a variety of different types of microorganisms. The microorganisms may be, for example, microorganisms floating freely in the biological sample and / or formerly phagocytosed microorganisms. The cell sorting device may be, for example, a flow sorter, which, for example, sorts cells and / or microorganisms with different optical markings into different vessels. Such flux sorters are also known as "FACS" devices, where FACS stands for "fluorescence-activated cell sorting". The cells and / or microorganisms can be separated, for example mechanically or via an electrostatic force. The nucleic acids may in particular comprise single- or double-stranded deoxyribonucleic acids (DNA) or ribonucleic acids of the phagocytosed cells. The method is thus in particular as

To designate pre-analysis methods.

The method has the advantage that the enrichment takes place without an initial, that is to say previous, complicated lysis, so that considerable time savings occur. Furthermore, by separating the marked microorganisms from the biological sample for subsequent further process steps a reduced background compared to conventional methods before disturbing, especially because hemoglobin, free DNA and other cells of the biological sample early on of the nucleic acids containing parts of the biological Sample, so the labeled microorganisms are separated.

In a particularly advantageous embodiment it is provided that the opsonins are each coupled to at least one magnetic marker, in particular to at least one para- or superparamagnetic marker, and the separation is effected by means of a magnetic field applied by the cell sorting device. The use of magnetic markers allows a combination of the enrichment process with a qualitative detection or a quantification step for the microorganisms. This allows a labor and time-saving multiple use of the markers. Accordingly, it is particularly easy to detect the marked cells, preferably between the marking and the separation.

In a further advantageous embodiment it is provided that, prior to separation, detection of the marked microorganisms is effected by a detection device, in particular a magnetic and / or optical flow cytometer. In particular, the detection comprises a quantification of the microorganisms, which in turn comprises in particular a determination or determination of a number of the labeled microorganisms, in particular per by the

Flow cytometer flowing sample volume as a concentration of the respective labeled microorganisms, and / or determining a diameter of the respective labeled microorganisms and / or a volume of the respective labeled microorganisms and / or a flow rate of the respective labeled microorganisms, in particular a diameter distribution of the microorganisms. This has the advantage that the multiple use of the markers results in a combination of the enrichment and the detection method, so that time and additional work steps, which may require training of a person carrying out the method, are again saved here.

By quantifying, for example, via the detected diameters of the microorganisms, a specific detection is possible, for example, whether microorganisms of a known diameter are even contained in the biological sample. By way of example, a simple volume measurement with a, in particular magnetic, running time measurement, a concentration of the labeled microorganisms can be determined here, for example. This can be done very quickly and completed within a few minutes. The result is a very pure, defined sample with known eigen- Stems that can already be used to verify that the sample is suitable for further and sometimes time-consuming and expensive diagnostic procedures.

A defined sample is to be understood here and below as meaning a sample whose composition is known. In particular, it is thus known which types of microorganisms and / or other constituents are present in which respective proportions and / or in which particular number in the sample, and which accuracy this information has in each case.

In a further embodiment, it is provided that the separation of the marked microorganisms from a volume flow of the biological sample in a cell sorting device, in particular by means of an applied, preferably already used in the preceding method steps, applied magnetic field, in a sorting pot of the cell sorting and / or washing or cleaning the separated microorganisms in the sorting pot by means of a flowing through the cell sorting buffer solution following the separation, in particular in the presence of a, preferably already used in the preceding method steps, applied magnetic field. This has the advantage that a particularly efficient removal of the non-labeled sample components, such as in the case of a whole blood sample erythrocytes, free hemoglobin or other constituents of the blood, takes place. The purified separated microorganisms can then be removed from the sorting pot in a simple manner, for example by pipetting out. In addition, in this case, in comparison with known purification methods, an efficient or almost complete reduction of interfering components of the biological sample which are unnecessary for accumulating the nucleic acids is achieved. Otherwise, these unnecessary components form, in particular, for later amplification, if appropriate, of the nucleic acids by means of, for example, one

Polymerase chain reaction has a disturbing background. Also Thus, a very well-defined sample is generated, which is highly concentrated and of which only a few microliters are sufficient for further diagnostics. Such a sample may be required for, for example, newer and more expensive Next Generation Sequencing (NGS) technologies.

The cell sorting device can comprise, for example, a cartridge or a cartridge into which the respective sample can be introduced, in particular injected. The cartridge may in particular be a replaceable cartridge for a holding device and / or for a cell sorting device and / or for a detection device, in particular a magnetic and / or optical flow cytometer. In particular, in the respective device, a magnetic field with a predetermined orientation and strength can act on the respective sample contained in the cartridge and in particular flowing through the cartridge.

In an advantageous embodiment it is provided that a lysing, in particular a specific lysing, of at least a portion of the separated microorganisms takes place after the separation and in particular after the washing of the microorganisms. For example, the portion which is lysed may be pipetted out of the previously separated microorganisms. The lysing can be carried out in particular after a washing according to one of the preceding method steps. This has the advantage of a very well-defined sample, namely high titer and purified nucleic acids, of microorganisms for molecular diagnostics. This defined sample was generated without the need for expensive lysing of cells of all cell types in the biological sample. The precisely defined sample can be generated quickly with just a few simple process steps. This makes the method suitable for molecular diagnostics in time-critical scenarios, for example in a trauma room of a hospital. In a further embodiment, it is provided that, in particular following the lysing, if this has taken place, an amplification of the nucleic acids of the microorganisms is carried out, in particular by means of a polymerase chain reaction. This has the advantage of increasing the amount of nucleic acids, making detection easier.

The biological sample may comprise a urine sample and / or lymph sample and / or a CSF sample, ie in particular a sample of a cerebrospinal fluid, and / or plant extract and / or a food sample and / or a blood sample, in particular a whole blood sample. The food sample may be, in particular, the sample of a liquid food, e.g. Beer with a yeast or milk with a mushroom. This has the advantage that the sample can be taken directly from an organism, in particular from a patient, as is desirable for near-patient laboratory diagnostics. Blood, especially whole blood, has the advantage that it can be very directly recognized whether a dangerous disease or infection exists. Here, the use of whole blood has the particular advantage that no dilution is required, which not only saves time, but also reduces required laboratory knowledge of the operating staff.

In a further embodiment, it is provided that the opsonins bind nonspecifically to cells foreign to the organism, that is to say foreign to the organism, and in particular the opsonins are complement factors and / or a mixture of a plurality of mutually different antibodies, in particular complement factors. The different antibodies then belong to different types of antibodies. In particular, in the case of opsonins, immunoglobulin G can also be covalently bound to one molecule each, superparamagnetic beads can each be covalently bound to immunoglobulin G or mixtures thereof. This has the advantage that any organism-foreign microorganisms nism is marked and can be separated from the sample in a row.

It is especially envisaged that the microorganisms are pathogens, in particular bacteria and / or fungi, and in particular the nucleic acids which are enriched are fractions of a genome of the microorganisms or entire genomes of the microorganisms. This has the advantage of providing a rapid method for accumulating nucleic acids from pathogens, which is desirable in an infected human patient. Especially in the case of sepsis, rapid identification and therefore also enrichment is highly relevant because the mortality of sepsis patients increases rapidly. Rapid detection of sepsis or sepsis of underlying pathogens thus provides life-saving emergency measures. Enriching partial or total pathogen genomes is beneficial in identifying the pathogen and performing resistance testing.

Further features will become apparent from the description of a preferred embodiment according to the figure. It shows

1 shows schematically an exemplary embodiment of the method according to the invention.

In the exemplary process sequence shown in FIG. 1, a very rapid enrichment and detection of nucleic acids 11 of microorganisms 12, for example bacterial pathogens, on a fresh, for example chemically untreated, biological sample 10 is used, which in the present example a few minutes before carrying out the process an organism, for example a human patient with eg a sepsis. This is done to simultaneously detect the microorganisms 12 and, for example, to identify triggers of sepsis and any inhibitors present in the biological sample 10 for a polymerase chain reaction such as hemoglobin. in, free DNA or other blood components 22 need not be considered in the further process. By combining detection and enrichment of, for example, pathogens as microorganisms 12, time is saved. Furthermore, such a nucleic acid diagnosis can be provided a standardized enriched sample without said inhibitors. This is advantageous because newer and more expensive Next Generation Sequencing (NGS) technologies require a sample standardized in concentration and purity. By clarifying in the pre-analytical enrichment procedure, whether a sample is suitable for an NGS method, costs and time can be saved in acute diagnostics.

In the exemplary embodiment illustrated in FIG. 1, general cell membrane structures 20 of microorganisms 12, in this case bacterial pathogens in, for example, an undiluted whole blood sample as biological sample 10, are shown in FIG. 1 in a sample vessel 5 in order to carry out marking (method step S1). The organism's own cells, for example the other blood components 22, do not have the general cell membrane structures 20, for example certain sugar molecules or certain protein structures. The labeling (method step S1) takes place here by opsonins 16, which bind only to cell membrane proteins 20 of the organism-foreign microorganisms 12 and not to the other blood components 22, and with e.g. superparamagnetic markers 18 as a label.

Here, after marking (method step S1), quantification (method step S2) takes place, for example via specific detection of the labeled microorganisms 12, eg pathogens, by measuring, for example, volume and flow rate of the labeled microorganisms 12. In the present case, this is achieved by magnetic transit time measurement executed by means of a detection device 30, for example a flow cytometer. The run-time measurement makes it possible, for example, to determine the diameter of the labeled microorganisms 12. By quantifying (process step S2), in the present case in the form of a magneto-sensitive detection, of the microorganisms 12 present as bacterial pathogens, therefore, a concentration of the microorganisms 12 can be determined and all labeled and detected microorganisms 12 can be quantified.

Here follows a separation (step S3) in which the enriched and quantified microorganisms 12 are separated from the other blood components 22, e.g. Erythrocytes, for example, immediately after the magneto-sensitive detection, are separated from the biological sample 10, for example in a microfluidic chamber 32 of a cell sorting device 34, through which the blood flows as a volume flow. This separation (step S3) can be done in a few minutes. After completion of the separation, for example, a washing of the separated microorganisms 12 can be carried out by unmarked other blood components 22, which may also be accidentally mixed with the separated microorganisms 12. For example, the washing can be carried out by means of a buffer solution which is known to the person skilled in the art and suitable for a washing step in a sorting pot 36 of the cell sorting device 34 in the presence of a magnetic field. The magnetic field then stops e.g. During washing, the labeled microorganisms 12 in the sorting pot 36. The so enriched and purified microorganisms 12, here bacterial pathogens, may subsequently for the follow-up diagnostics or for further enrichment from the cell sorting device 34 or a particular belonging to the cell sorting device 34 cartridge or cartridge be removed. Since the sample of e.g. bacterial pathogens are very clean and in the case of a quantification carried out also accurately determined, even a small proportion of the biological sample 10 of a few microliters is sufficient for further diagnostics.

With the separation (step S3) and washing it is achieved that a polymerase chain reaction interferes with hemoglobin, free DNA and other unlabeled blood components. Nenten 22 were removed, without it would have been necessary to perform, for example, a selective lysis of erythrocytes and / or other complex purification steps. This can be compared to commercial

Purification method a reduction of the polymerase chain reaction disturbing background by up to 99 percent based on the total number of cells in the blood can be achieved.

A proportion of the purified microorganisms 12, here the bacterial pathogens, is now taken from the cell sorting device 34, for example. For molecular diagnostics, a lysing (method step S4) of the microorganisms 12 can then be provided, to which an amplification (method step S5), for example of pathogenic nucleic acids 11 or of the pathogenic genome, for example by means of a polymerase chain reaction can take place.

Claims

claims

A method for molecular diagnostics, for enriching nucleic acids (11) from microorganisms (12) from a biological sample (10) of an organism, in which

- Marking (step Sl) of at least a proportion of the microorganisms (12) by opsonins (16), which are each coupled to a marker (18), followed by

- Separating (step S3) of the labeled microorganisms (12) from the biological sample (10) by means of a cell sorting device (34), whereby an accumulation of the labeled microorganisms (12) and thus enrichment of the nucleic acids (11) of the microorganisms (12) he follows.

2. The method according to claim 1, characterized in that the marking (method step Sl) is in each case coupled to a magnetic, in particular paramagnetic, marker (18) coupled opsonins (16) and the separation (step S3) by means of a by the cell sorting device (34 ) applied magnetic field.

3. The method according to any one of the preceding claims, characterized in that prior to the separation (step S3) detecting (step S2) of the marked microorganisms (12) by a detection device (30), in particular a magnetic and / or optical

Flow cytometer, done.

4. The method according to claim 3, characterized in that the detection (method step S2) comprises a quantification, and the quantification in particular a determination of a number of the labeled microorganisms (12), in particular per sample volume flowed through the detection device (30), and or determining a diameter and / or a volume and / or a flow rate of respective labeled microorganisms (12) and / or a Diameter distribution of the labeled microorganisms (12) encompassed.

5. The method according to any one of the preceding claims, character- ized in that

- The separation (step S3) of the labeled microorganisms (12) from a volume flow of the biological sample

(10) in the cell sorting device (34), in particular by means of an applied magnetic field, in a sorting pot (36) of the cell sorting device (34) takes place and / or

- A washing of the separated microorganisms (12) in the sorting pot (36) by means of a cell sorting device (34) flowing buffer solution to the separation (step S3) follows, in particular in the presence of a, preferably already used in the preceding method steps, applied magnetic field ,

6. The method according to any one of the preceding claims, characterized in that

- Lysing (step S4) of the separated microorganisms (12), in particular after washing of the microorganisms (12) takes place.

7. The method according to any one of the preceding claims, character- ized in that

- Amplifying (step S5) of the nucleic acids

(11) the microorganisms (12) is carried out, in particular by means of the polymerase chain reaction, and that the amplification (method step S5) takes place, in particular after lysing (method step S4).

8. The method according to any one of the preceding claims, characterized in that the biological sample (10) comprises a urine sample and / or lymph sample and / or CSF sample and / or herbal extract and / or a food sample and / or blood sample, in particular a whole blood sample.

9. The method according to any one of the preceding claims, characterized in that the opsonins (16) nonspecific bind a foreign cells with respect to the organism and in particular in the opsonins (16) to complement factors and / or a mixture of a plurality of mutually different antibodies, especially with complement factors.

10. Method according to one of the preceding claims, characterized in that the microorganisms (12) are bacteria and / or fungi.