Method and kit for simultaneous detection of plurality of different pathogens in sample

Abstract

The invention relates to a method for detecting a plurality of different pathogens in a sample, in which in a pre-enrichment step (1) the sample is supplied to a pre-enrichment medium for pre-enrichment, in a lysis step (2) cell walls of the pathogens in the pre-enriched sample are disrupted and thus lysates are produced, in a detection step (3) cell walls of the pathogens in the pre-enriched sample are disrupted and thus lysates are produced. The plurality of pathogens are detected by means of a multiplex PCR test, in which the pre-enrichment step (1), the cleavage step (2) and the detection step (3) are carried out in this order, and in which the cleavage is suspended without increasing the temperature prior to the detection step (3). Furthermore, a kit (100) for detecting a plurality of different pathogens is proposed.

Description

[0001] The present invention relates to a method for the simultaneous detection of a plurality of different pathogens in a sample by means of a multiplex PCR test. Furthermore, the present invention relates to a kit for the simultaneous detection of a plurality of different pathogens in a sample by means of a multiplex PCR test.

[0002] From the prior art, tests for detecting samples for pathogens are known. Thus, for example, the publication WO 2016 / 164 407 A2 discloses a method for detecting a plurality of pathogens in a sample. The pathogens are enriched in a pre-enrichment medium. Then, in a first lysis step and in a second lysis step, the cell walls are broken in order to prepare a lysate suitable for testing by means of a multiplex PCR method. In this case, the second lysis step is carried out at a higher temperature than the first step.

[0003] The object of the present invention is to provide a method which exhibits an improved procedure compared to the prior art, and to provide a kit with which such a method can be carried out.

[0004] This object is achieved by the method according to claim 1 and the kit according to claim 10.

[0005] For carrying out the method, a sample is preferably taken from the substance to be tested. It is conceivable that the substance is a food. For example, but not limiting, it is further conceivable that the substance is a meat product or a dairy product. The meat product may be, for example, minced meat, cuts of meat, sausages, ham or any other food in which meat has been processed. The dairy product may be, for example, milk, curd, yogurt or any other food in which milk has been processed. It is further conceivable that the substance is an animal feed.

[0006] Preferably, it is provided that the weight of the sample is greater than 1 g and less than 100 g, particularly preferably greater than 10 g and less than 50 g, and in particular 25 g.

[0007] In order to detect the multiple different pathogens, according to the present invention, the sample first undergoes a pre-enrichment step to be enriched in a pre-enrichment medium. Subsequently, in a lysis step, the cell wall of the pathogen in the pre-enriched sample is broken. This helps to expose the DNA of the pathogen. The breaking of the cell wall of the pathogen is also referred to as lysis. The lysis can be caused by different means. Therefore, in the case of thermal lysis, the cell wall can be broken with an elevated temperature. In the case of chemical lysis, cell decomposition occurs, for example, by adding chaotropic salts, detergents or other chemical aids. Enzymatic lysis uses enzymes, such as proteinase K and / or lysozyme, for cell decomposition. In addition, proteinase K also contributes to the elimination of nucleases released during cell decomposition. Mechanical lysis is also possible, in which case cell decomposition is caused, for example, by mechanical stress on the cell wall with so-called beads. Beads are small balls made of hard materials (e.g., based on ceramics, glass or zirconium). In particular, it is pre-specified that in the lysis step, thermal lysis is first used, followed by chemical lysis, for cell decomposition. Additionally, enzymatic lysis and / or mechanical lysis during the lysis step may contribute to cell disruption.

[0008] If the lysis step is over, the multiple pathogens are detected in the detection step by means of a multiplex PCR test. To this end, the lysate is added to a PCR reagent. For each target organism to be detected, i.e., for each pathogen to be detected, the PCR reagent comprises at least one specific oligonucleotide primer pair (in short: "primer"), at least one labeled oligonucleotide (which can be used as a probe for detecting pathogens) and a thermostable DNA polymerase (e.g., Taq polymerase). Further, the PCR reagent comprises deoxynucleotides, cofactors for the polymerase, and a polymerase buffer solution. In a thermal cycler, the lysate is first heated to about 96° C. so that the double-stranded DNA is separated into two single strands. Then, the primers hybridize with the single strands at a lower temperature and each determines the starting point for the subsequent DNA synthesis. For DNA synthesis, the temperature is increased again. In this case, the Taq polymerase complements the free chain with nucleotides. However, it is also conceivable that the temperature for DNA synthesis is not increased and that DNA synthesis occurs at the same temperature as primer hybridization. The described process is repeated for a determined number of cycles. If no pathogen is detected after a certain number of cycles, it is assumed that the sample is free of the pathogen to be detected.

[0009] In order to protect the Taq polymerase and to be able to perform the PCR test, the cleavage step is interrupted before the detection step. It is known from the prior art to interrupt the enzymatic cleavage by increasing the temperature. If the cleavage is carried out with an enzyme, for example with proteinase K, the enzyme can be inactivated by increasing the temperature. The disadvantages associated with the increase in temperature, in particular the waiting time, are circumvented in the case of the method according to the invention.

[0010] It is conceivable that the method is carried out as a pool test, i.e. the multiplex PCR test is carried out on a PCR sample containing several lysates. For this purpose, it is conceivable that the lysates are prepared from different samples. Preferably, it is pre-specified for this purpose that the different samples are samples from different foods. However, it is also conceivable that the different samples are samples from different batches of the same food.

[0011] Advantageous embodiments and developments of the invention are apparent from the dependent claims and from the description with reference to the drawings.

[0012] According to a preferred embodiment of the present invention, it is pre-defined that the suspension of cleavage is supported and / or caused by diluting the lysate, and the multiplex PCR test is performed for the diluted lysate. By this, it is possible to stop cleavage at a constant or even falling temperature in an advantageous manner. It is conceivable that, in order to dilute, water without nucleic acids and nucleases is pipetted into the lysate with a pipette. Within the scope of the present invention, pipetting with a pipette is understood as adding water without nucleic acids and nucleases to the lysate or adding lysate to water without nucleic acids and nucleases. Preferably, it is pre-defined that the dilution is 1:3 to 1:100, particularly preferably 1:10 to 1:75, and particularly 1:20 to 1:40. It is also conceivable that the PCR reagent present in a lyophilized form is dissolved in water without nucleic acids and nucleases, and then the lysate is pipetted with a pipette to achieve the mentioned lysate dilution. Surprisingly, it is shown that dilution causes the suspension of cleavage.

[0013] Alternatively or additionally, preferably pre-determine that the cessation of cleavage (particularly cleavage facilitated or supported by protease) is supported and / or caused by adding protease inhibitors to the lysate, such as 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). Surprisingly, it has been shown that adding protease inhibitors to the lysate stops the cleavage. If the cleavage step is carried out with the help of an enzyme, then the enzyme is chemically inactivated to stop the cleavage by adding protease inhibitors. Preferably, pre-determine that a waiting time of 1 minute to 40 minutes is followed between the cleavage step and the detection step. It is thereby ensured that the cleavage is stopped before the lysate is contacted with the PCR reagents.

[0014] According to a further preferred embodiment of the present invention, it is provided that the suspension of cleavage is supported and / or caused by adding a protease inhibitor (e.g., 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF)) and dilution to the lysate. Preferably, it is provided that the dilution follows the addition of the protease inhibitor in time. The multiplex PCR test is then carried out on the diluted lysate. The dilution is preferably 1:3 to 1:100, particularly preferably 1:10 to 1:75, and in particular 1:20 to 1:40. The addition of the protease inhibitor and the subsequent dilution lead to a particularly reliable suspension of cleavage. If the cleavage step is carried out with the aid of enzymatic cleavage with an enzyme, the enzyme is chemically inactivated by adding a protease inhibitor to stop the cleavage.

[0015] For this purpose, it is preferably provided that a waiting time is followed between the addition of the protease inhibitor and the dilution. Thus, the protease inhibitor is given sufficient time to chemically terminate the enzymatic cleavage.

[0016] According to a particularly preferred embodiment of the present invention, it is predetermined that the lysate is a crude lysate. A crude lysate is a lysate in which no separation of DNA and residual material from the lysate is performed. In this case, the concentration of the DNA is not performed. Thereby, the purification of the lysate is cancelled in an advantageous manner. Therefore, compared to the method wherein the purification or concentration with the help of a centrifugal column or magnetic beads is adopted, the method is significantly more time-saving and less expensive and more reagent-saving. Separation can mean physical separation. However, it is also conceivable that separation means that the DNA is bound to a surface, such as the surface of a bead or a column matrix.

[0017] In order to improve the efficiency of the lysis, according to a preferred embodiment of the present invention, it is provided that during the lysis step, thermal lysis is first carried out at a first temperature. That is, the cell wall of the pathogen is first broken by heating. Subsequently, chemical lysis and / or enzymatic lysis and / or mechanical lysis are carried out at a second temperature. The second temperature is lower than the first temperature, i.e. the temperature is lowered after thermal lysis to start chemical or enzymatic or mechanical lysis. The cell wall of the pathogen is very reliably broken by a combination of thermal lysis, chemical and enzymatic or mechanical lysis. It is conceivable that the enzymatic lysis is carried out with proteinase K. It is conceivable that the mechanical lysis is carried out with beads (e.g., based on ceramic, glass or zirconium).

[0018] According to a further preferred embodiment of the present invention, it is pre-specified that the pre-enrichment medium is a non-selective pre-enrichment medium and the pre-enrichment is a non-selective pre-enrichment. The non-selective pre-enrichment medium, for example, does not have an antibiotic. Therefore, the different pathogens and the pathogenic or non-pathogenic accompanying flora optionally contained in the sample increase simultaneously. Within the scope of the present invention, the accompanying flora includes microorganisms that do not belong to the multiple pathogens.

[0019] Alternatively thereto, it is preferably provided that the preenrichment medium is a selective preenrichment medium and that the preenrichment is a selective preenrichment.

[0020] In particular, it is provided that the plurality of different pathogens comprises at least two pathogens from the following pathogen group: Enterobacteriaceae, Salmonella, Listeria, Staphylococcus and Shiga toxin-producing Escherichia coli (E. coli) and Cronobacter. Preferably, it is provided that the plurality of different pathogens comprises Enterobacteriaceae, Salmonella, Listeria, Staphylococcus and Shiga toxin-producing Escherichia coli. It is thus possible to carry out the legally required tests for foods with respect to pathogens simultaneously in a time-saving manner.

[0021] A further object of the invention for solving the task set out at the outset is a kit according to claim 10. The kit according to the invention for detecting a plurality of different pathogens is intended for use with the method according to the invention. The kit according to the invention has a pre-enrichment medium for pre-enrichment in a pre-enrichment step. In addition, the kit according to the invention has a lysis reagent for disrupting the cell walls in a lysis step. The lysis reagent preferably has proteinase K. Furthermore, the kit according to the invention comprises a PCR reagent for carrying out the multiplex PCR test in the detection step.

[0022] According to a preferred embodiment of the present invention, it is provided that the kit contains a protease inhibitor, such as 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), for stopping the lysis. Further, it is conceivable that the kit contains a lysis buffer.

[0023] Preferably, it is provided that the kit contains nucleic acid-free and nuclease-free water for diluting the lysate.

[0024] Preferably, it is predetermined that the plurality of different pathogens include Salmonella species, Listeria species, Listeria monocytogenes, Enterobacteriaceae. Preferably, it is predetermined that the plurality of different pathogens include Cronobacter, coagulase-positive Staphylococcus, STX / STC and EAC.

[0025] All details, features and advantages disclosed above in connection with the method according to the invention also relate to the kit according to the invention.

[0026] Further details, features and advantages of the invention are apparent from the drawings and from the following description of preferred embodiments according to the drawings. Nevertheless, the drawings illustrate only exemplary embodiments of the invention and do not limit the inventive concept.

[0027] The attached figure shows:

[0028] Figure 1 : A schematic diagram shows a method flow according to a preferred embodiment of the present invention, with a kit according to a preferred embodiment of the present invention.

[0029] Figure 1 The method according to a preferred embodiment of the present invention is schematically shown. In addition, Figure 1 A kit (100) according to a preferred embodiment of the present invention is shown in a highly schematic diagram.

[0030] In the method shown for detecting a plurality of different pathogens in a sample, first in a pre-enrichment step (1) the sample is provided to a pre-enrichment medium for pre-enrichment. The sample is a food sample weighing 25 g, which is incubated in 225 ml of pre-enrichment medium at 37° C. for 18 to 30 hours, in particular 22 + / - 2 hours. The pre-enrichment medium is a non-selective pre-enrichment medium for non-selective pre-enrichment of several pathogens.

[0031] After sufficient pre-enrichment, in a lysis step (2), the cell walls of the pathogens in the pre-enriched sample are disrupted. To this end, the pre-enriched sample is first heated to a temperature of 95°C + / - 5°C and subjected to thermal lysis. Then, the temperature is lowered and a reagent for chemical lysis is added. It is conceivable that an enzyme, such as proteinase K, is further added to cause enzymatic lysis. In addition, it is conceivable that lysis is additionally or alternatively caused or supported by mechanical lysis. Enzymatic lysis is terminated after an incubation time of 5 to 30 minutes. This occurs while the temperature is maintained or reduced. For this purpose, a protease inhibitor is added to the lysate to inactivate proteinase K. In addition, after the addition of the protease inhibitor and the waiting time, the lysate is diluted with nucleic acid-free and nuclease-free water. In this case, the dilution is preferably 1:20 to 1:40.

[0032] In the exemplary embodiment shown, it is provided that the lysate is a crude lysate.

[0033] If the lysis is terminated, i.e. the lysis step (2) is terminated, then the pathogen is detected in the detection step (3) by means of a multiplex PCR test. For this purpose, the diluted crude lysate is added to a PCR reagent. For each target organism to be detected, i.e. for each pathogen to be detected, the PCR reagent comprises at least one specific oligonucleotide primer pair (in short: "primer"), at least one labeled oligonucleotide (which can be used as a probe for detecting the pathogen) and a thermostable DNA polymerase (e.g. Taq polymerase). Further, the PCR reagent comprises deoxynucleotides, cofactors for the polymerase and a polymerase buffer solution. In a thermal cycler, the lysate is first heated to approximately 96° C. so that the double-stranded DNA separates into two single strands. The primers then hybridize to the single strands at a lower temperature and each determine the starting point for the subsequent DNA synthesis. For DNA synthesis, the temperature is increased again. In this case, the Taq polymerase complements the free strands with nucleotides. However, it is also conceivable that the temperature for DNA synthesis is not increased and that DNA synthesis occurs at the same temperature as the primer hybridization. The described process is repeated for a determined number of cycles. If no pathogen is detected after a certain number of cycles, it is assumed that the sample is free of the pathogen to be detected.

[0034] In this case, the kit (100) according to the invention comprises a non-selective pre-enrichment medium; a lysis reagent, for example with proteinase K; preferably a lysis buffer; a protease inhibitor for stopping the enzymatic lysis; nucleic acid-free and nuclease-free water for diluting the crude lysate; and PCR reagents.

[0035] Reference Mark List :

[0036] 1 Pre-enrichment step

[0037] 2. Lysis Step

[0038] 3. Detection steps

[0039] 100 test kits

Claims

1. A method for detecting a plurality of different pathogens in a sample, wherein In the pre-enrichment step (1), the sample is provided to a pre-enrichment medium for pre-enrichment, In the lysis step (2), the cell walls of the pathogens in the pre-enriched sample are disrupted and a lysate is thus prepared, In the detection step (3), the plurality of pathogens are detected by means of a multiplex PCR test, The pre-enrichment step (1), the lysis step (2) and the detection step (3) are performed in this order, and the lysis is stopped before the detection step (3) without increasing the temperature.

2. The method according to claim 1, characterized in that The suspension of lysis is supported and / or caused by diluting the lysate and carrying out the multiplex PCR test on the diluted lysate, wherein the dilution is preferably 1:3 to 1:100, particularly preferably 1:10 to 1:75 and in particular 1:10 to 1:

40.

3. The method according to any one of the preceding claims, characterized in that The cessation of cleavage is supported and / or caused by the addition of a protease inhibitor to the lysate, preferably 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride.

4. The method according to claim 1, characterized in that: The suspension of lysis is supported and / or caused by adding a protease inhibitor to the lysate, preferably 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, and subsequent dilution, and the multiplex PCR test is performed on the diluted lysate, wherein the dilution is preferably 1:3 to 1:100, particularly preferably 1:10 to 1:75, and in particular 1:10 to 1:

40.

5. The method according to claim 4, characterized in that A waiting time was followed between the addition of the protease inhibitors and the dilution.

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

7. The method according to any one of the preceding claims, characterized in that During the cleavage step (2), thermal cleavage is first performed at a first temperature, followed by chemical cleavage and / or enzymatic cleavage and / or mechanical cleavage at a second temperature, wherein the second temperature is lower than the first temperature.

8. The method according to any one of the preceding claims, characterized in that The pre-enrichment medium is a non-selective pre-enrichment medium, and the pre-enrichment is a non-selective pre-enrichment.

9. The method according to any one of the preceding claims, characterized in that The plurality of different pathogens comprises at least two pathogens from the following group of pathogens: Enterobacteriaceae, Salmonella, Listeria, Staphylococcus, Shiga toxin-producing Escherichia coli (E. coli) and Cronobacter, wherein the plurality of different pathogens preferably comprises Enterobacteriaceae, Salmonella, Listeria, Staphylococcus and Shiga toxin-producing E. coli.

10. A kit (100) for detecting a plurality of different pathogens using the method according to one of the preceding claims, wherein the kit has the following components: - a pre-enrichment medium for pre-enrichment in the pre-enrichment step (1), - a lysis reagent for disrupting the cell walls in the lysis step (2), - PCR reagents for performing the multiplex PCR test in the detection step (3).

11. The kit (100) according to claim 10, characterized in that: The kit (100) contains a protease inhibitor, such as 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), for stopping the cleavage.

12. The kit (100) according to any one of claims 10 to 11, characterized in that: The kit contains nucleic acid-free and nuclease-free water for dilution of the lysate.

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