Ready-to-use functionalised microtiter plate and detection method

Abstract

The invention relates to a ready-to-use functionalised microtiter plate (10) for detecting nucleic acids by means of reverse transcription (RT) and / or polymerase chain reaction (PCR), the plate having been isolated from an examination material and cleaned. According to the invention – the microtiter plate (10) contains, in a strip or a plate, a plurality of vessels (20) which have an opening (22) and of which one or more are functionalised; – the functionalised vessels (20) are liquid-free and contain only functional reaction chemicals (30, 32, 34), for detecting the nucleic acid, and optionally one or more wax layers (40), which cover at least some of the functional reaction chemicals in order to protect them from light and oxygen; – the functional reaction chemicals (30, 32, 34) contain specific primers and probes, dNTPs, MgCl2, buffer, salt, polymerase, in the event that RNA is to be detected, additionally reverse transcriptase; and – the probes are each marked with a first fluorescent reporter dye and are provided with a second dye which serves as quencher and suppresses the fluorescence signals of the intact probes.

Description

[0001] Ready-to-use functionalized microtiter plate and detection method

[0002] The invention relates to a ready-to-use, functionalized microtiter plate for the detection of nucleic acids, in particular by reverse transcription (RT) and / or polymerase chain reaction (PCR). The invention also relates to a corresponding detection method.

[0003] From the utility model DE 202020 004407, a functionalized microtiter plate is known which is used for the direct detection of pathogens, especially viruses.

[0004] The object of the present invention is to provide advantageous further developments of this known microtiter plate, in particular microtiter plates which are simpler and more cost-effective to produce and which can be used easily and flexibly in the detection of nucleic acids.

[0005] This object is achieved by the features of the independent claims. Further developments of the invention are the subject of the dependent claims.

[0006] The invention provides a ready-to-use, functionalized microtiter plate for the detection of nucleic acids. The detection of the nucleic acid, in particular a pathogen-encoding nucleic acid isolated and purified from a test material, takes place using reverse transcription (RT) and / or polymerase chain reaction (PCR). The microtiter plate contains, in a strip or plate, a plurality of wells with an opening, one or more of which are functionalized. Advantageously, even all wells of the microtiter plate are functionalized.

[0007] The functionalized vessels are liquid-free and contain only functional reaction chemicals for the detection of the nucleic acid and optionally one or more wax layers that cover at least part of the functional reaction chemicals to protect them from light and oxygen. In other words, the functionalized vessels either contain only the functional reaction chemicals mentioned or contain the functional reaction chemicals mentioned and one or more, in particular exactly one or exactly two, wax layers. The functionalized vessels do not contain any other solid and especially liquid materials.

[0008] The functional reaction chemicals contain specific primers and probes, dNTPs, MgCh, buffer, salt, polymerase, and, in case RNA is to be detected, additionally reverse transcriptase.

[0009] The probes are each labeled with a first fluorescent reporter dye and are provided with a second dye that acts as a quencher and suppresses the fluorescence signals of the intact probes.

[0010] Due to the liquid-free, functionalized wells, the microtiter plate has a particularly long shelf life. Microtiter plates with functionalized wells containing a liquid often require refrigerated storage to prevent or slow bacterial growth in the liquid or other deterioration processes. The liquid-free microtiter plate of the invention, in contrast, has a virtually unlimited shelf life even at room temperature.

[0011] In an advantageous embodiment, the functionalized wells of the microtiter plates are sealed with a well cover. The well cover can be formed, in particular, from an opaque film to ensure particularly high long-term stability of the functional reaction chemicals. The well cover preferably seals all wells of the microtiter plate, but can also seal only some of the wells.

[0012] The functional reaction chemicals are particularly advantageous when air-dried. This air-drying process makes microtiter plates easy to produce, requires little energy, and is cost-effective. Furthermore, practical experience has shown that air-dried reaction chemicals adhere better to the vessel surfaces than freeze-dried reaction chemicals, which typically have a more powdery consistency.

[0013] Compared to the commonly used lyophilization, air drying offers the advantages of a simple process that requires no special equipment, speed with a process time in the range of minutes instead of hours, and lower costs, in addition to the aforementioned improved adhesion to the vessel surfaces. According to an advantageous embodiment, the functional reaction chemicals contain chelating agents such as ETDA (ethylenediaminetetraacetic acid) and / or agents for reducing potential inhibitors of transcription and / or polymerase reactions and / or agents for stabilizing the enzymes.

[0014] The functional reaction chemicals advantageously further contain an internal control substance, in particular an artificial or non-naturally occurring RNA sequence, or an artificial or non-naturally occurring DNA sequence.

[0015] In an advantageous embodiment, the functionalized vessels contain only functional reaction chemicals for the detection of the nucleic acid, but no other solid or liquid materials.

[0016] In another advantageous embodiment, the functionalized vessels contain only functional reaction chemicals for detecting the nucleic acid and precisely one wax layer that completely covers the functional reaction chemicals. The functionalized vessels in this embodiment do not contain any other solid or liquid materials.

[0017] In a further advantageous embodiment, the functionalized vessels contain only functional reaction chemicals for detecting the nucleic acid and precisely one wax layer covering only a portion of the functional reaction chemicals. The functionalized vessels in this embodiment do not contain any other solid or liquid materials. Preferably, the functional reaction chemicals comprise PCR chemicals for performing PCR and RT chemicals for performing reverse transcription, with the PCR chemicals being covered by said wax layer and the RT chemicals being present on said wax layer without a wax coating.

[0018] With such a design, a two-step reaction can be easily carried out in a functionalized vessel. After introducing a test material, a first reaction, for example, reverse transcription, can be carried out using the portion of the functional reaction chemicals not covered by the wax layer. The functional reaction chemicals covered by the wax layer, in particular specific primers and probes, cannot therefore interfere with or adversely influence the first reaction. After melting the wax layer, a second reaction, for example, PCR, can then be carried out using the portion of the functional reaction chemicals originally covered by the wax layer.

[0019] In a further advantageous embodiment, the functionalized vessels contain only functional reaction chemicals for detecting the nucleic acid and precisely two wax layers, each covering a portion of the functional reaction chemicals, wherein the two wax layers advantageously have different melting points. The functionalized vessels in this embodiment do not contain any other solid or liquid materials. In particular, all functional reaction chemicals of the functionalized vessels are covered by the two wax layers. Preferably, in this embodiment, the functional reaction chemicals comprise PCR chemicals for performing PCR and RT chemicals for performing reverse transcription, wherein the PCR chemicals are covered by a first of the two wax layers and the RT chemicals are covered by the second of the two wax layers.

[0020] Particularly advantageously, the two wax layers are formed by a higher-melting wax layer, preferably with a melting temperature of 60 °C or more, and a lower-melting wax layer, preferably with a melting temperature of 50 °C or less. In the functionalized vessels, the PCR chemicals are covered by the higher-melting wax layer, while the RT chemicals are present on the higher-melting wax layer and are in turn covered by the lower-melting wax layer.

[0021] The lower-melting wax layer advantageously has a melting point between 40°C and 50°C, for example, approximately 45°C. The higher-melting wax layer advantageously has a melting point between 60°C and 75°C, for example, approximately 65°C.

[0022] Even with such a design, a two-step reaction can be carried out in a simple and targeted manner in a functionalized vessel. After the introduction of a test material, a first reaction, for example a reverse transcription, can be carried out with the part of the functional reaction chemicals originally covered by the lower-melting wax layer after the melting of the lower-melting wax layer. The functional reaction chemicals covered by the higher-melting wax layer, in particular specific primers and probes, cannot interfere with or adversely influence the first reaction. After the melting of the higher-melting wax layer, a second reaction, for example a PCR, can then be carried out with the part of the functional reaction chemicals originally covered by the higher-melting wax layer.The nucleic acid transcribed from RNA into cDNA during reverse transcription serves as a template for PCR.

[0023] The invention also includes a method for detecting nucleic acids, in which a ready-to-use functionalized microtiter plate of the type described above is provided, any vessel cover present is removed, a test material containing one or more nucleic acids to be detected is introduced into the opening of one or more of the functionalized vessels, the microtiter plate filled with test material is introduced into a qPCR device and a quantitative real-time (RT-)PCR is carried out, and a fluorescence signal of the functionalized vessels is measured in order to detect the presence of one or more of the nucleic acids to be detected in the test material.

[0024] Advantageously, the method involves extracting nucleic acids from a patient sample through isolation and purification to obtain the test material. Since the functionalized microtiter plate is ready-to-use, no further reagents are added to the functionalized vessels for nucleic acid detection other than the test material.

[0025] In an advantageous process variant, the functionalized vessels contain exactly one wax layer that partially or completely covers the functional reaction chemicals, whereby in a (RT-)PCR step the wax layer is melted, the test material is thereby brought into contact with the functional reaction chemicals, and a real-time (RT-)PCR is carried out.

[0026] If the functional reaction chemicals are completely covered by the wax layer, they conveniently contain PCR chemicals and RT chemicals. If the functional reaction chemicals are only partially covered by the wax layer, the covered part conveniently contains PCR chemicals and the uncovered part RT chemicals.

[0027] In another, equally advantageous process variant, the functionalized vessels contain exactly two wax layers with different melting points, whereby in a RT step

[0028] — the lower melting wax layer is melted,

[0029] — the test material is thereby brought into contact with a first part of the functional reaction chemicals, and

[0030] — reverse transcription is carried out, and in a subsequent PCR step — the higher melting wax layer is melted,

[0031] — the test material is thereby brought into contact with a second part of the functional reaction chemicals, and

[0032] — real-time PCR is performed.

[0033] Conveniently, the first part of the functional reaction chemicals contains RT chemicals and the second part of the functional reaction chemicals contains PCR chemicals.

[0034] Advantages of the invention as well as an embodiment are explained below with reference to the figures, in which a true-to-scale and true-to-proportion reproduction has been omitted in order to increase clarity.

[0035] They show:

[0036] Fig. 1 shows a section of a microtiter plate according to the invention in cross section,

[0037] Fig. 2 a ready-to-use functionalized microtiter plate with a plurality of wells, and

[0038] Fig. 3 to 6 each show a section of microtiter plates according to the invention according to further embodiments of the invention in cross section.

[0039] The invention will now be explained using the example of ready-to-use, functionalized microtiter plates for RNA tests. Figure 2 shows a perspective view of a microtiter plate 10 with a base plate 12 containing a plurality, for example 96, of similar vessels 20, each connected to the plate 12 at its upper edge. Figure 1 shows three of the vessels 20 of the microtiter plate 10 schematically in cross-section. The vessels 20 each have an opening 22 accessible from above and a downwardly extending body 24, which is closed at the bottom by a base 26.

[0040] Some or even all of the wells 20 of the microtiter plate are functionalized, meaning they contain air-dried, functional reaction chemicals 30, 32, 34 for the detection of nucleic acids. The wells 20 of the microtiter plate 10 can contain different functional reaction chemicals 30, 32, 34, for example, for a multiplex test, or they can each contain the same reaction chemicals.

[0041] To protect against light and oxygen, the air-dried, functional reaction chemicals 30, 32, 34 are covered with a wax layer 40. In the exemplary embodiment, no other solid or liquid materials are present in the functionalized vessels 20 beyond the reaction chemicals and the wax layer. The functionalized vessels 20 are thus liquid-free, and in particular, they do not contain any buffer solution.

[0042] For printing a desired RNA assay, the functional reaction chemicals contain specific primers and probes, dNTPs, MgCl, buffer, salt, polymerase, and, since the example is intended to detect RNA from pathogens, reverse transcriptase. Optionally, the functional reaction chemicals contain chelating agents, agents for reducing potential inhibitors of transcription and / or the polymerase reaction, and agents for stabilizing the enzymes.

[0043] The probes contained in the reaction chemicals are each labeled with a first, fluorescent reporter dye and are additionally provided with a second dye that serves as a quencher and suppresses the fluorescence signals of the intact probes. In the embodiment of Figures 1 and 2, the wells 20 of the microtiter plate 10 are also sealed with a well cover in the form of an opaque film 50 to ensure particularly high long-term stability of the functional reaction chemicals in the wells 20.

[0044] The test principle is explained in more detail below using the example of a respiratory RT-PCR panel that simultaneously detects SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus (RSV) A / B. Such an RT-PCR test can be used as an assay for the in vitro investigation of viral RNA in nasal, nasopharyngeal, or oral / oropharyngeal swabs to obtain information to support the diagnosis of patients with suspected respiratory diseases.

[0045] In this case, the functional reaction chemicals contain detection probes specific for SARS-CoV-2 and the subgenus Sarbecovirus, Influenza A, Influenza B, RSV A, and RSV B, as well as an internal control. The probes are each labeled with fluorescent reporter dyes, for example, FAM for SARS-CoV-2, N gene, and Sarbecovirus, E gene; Cy5 for Influenza A, MP gene; HEX for Influenza B, NS gene; CalFluor Red 610 for RSV A and RSV B, N gene each; and Cy5.5 or Cyan500 for the internal control. Each probe also contains a second dye that acts as a quencher and suppresses the fluorescent signals of the intact probes.

[0046] For pathogen detection, nucleic acids are extracted from a patient sample by isolation and purification. The resulting sample is introduced into the opening of one or more of the functionalized vessels 20 after removing the cover film 50 from the functionalized microtiter plate 10. Since the microtiter plate 10 is already a ready-to-use system, the addition of additional reagents is not necessary.

[0047] The microtiter plate filled with the test material is then placed in a qPCR device, for example, a Roche LightCycler® System (96 / 480 II) or a Bio Rad CFX96™ Dx System, and a quantitative real-time RT-PCR is performed. The elevated temperature required for RT or PCR in the qPCR device melts the wax layer 40 in the tubes 20, allowing the test material to mix with the functional reaction chemicals 30, 32, 34, and the desired PCR or RT-PCR to be performed.

[0048] During PCR amplification, the probes hybridize to a specific target sequence, if present in the sample. This leads to cleavage of the probe by the 5'-to-3' exonuclease activity of the DNA polymerase, thus separating the reporter and quencher dyes, which in turn leads to an increase in the fluorescence signal.

[0049] With each PCR cycle, more and more cleaved probes are generated and the signal continues to increase.

[0050] Each reporter dye is measured at a defined wavelength, allowing the simultaneous detection and differentiation of various pathogen-specific nucleic acids as well as the internal control. The analysis is performed semi-quantitatively by comparing Ct (cycle threshold) values. The Ct value describes the cycle in which the signal first rises above a certain threshold. The more target copies (here, for example, SARS-CoV-2 virus RNA) present in the sample, the lower the value.

[0051] To ensure that a patient sample does not contain RT-PCR-inhibiting substances, an internal control substance is added to each reaction. In this test, an artificial RNA target with no known homologies and included in the same RT-PCR reaction is transcribed into cDNA, amplified, and detected. This eliminates false-negative test results due to RT-PCR inhibition.

[0052] Figure 3 shows a section of a microtiter plate 10' according to another embodiment of the invention. In the microtiter plate 10', the functionalized vessels 20' contain only air-dried, functional reaction chemicals 30, 32, 34, but no other solid or liquid materials, in particular no wax layer or buffer solution. Figures 4 and 5 show sections of microtiter plates according to further embodiments of the invention, in which the vessels are not sealed with a vessel cover.

[0053] In the microtiter plate 10" of Fig. 4, the air-dried, functional reaction chemicals 30, 32, 34 are covered with a wax layer 40, as in the embodiment of Fig. 1, to protect them from the influence of light and oxygen. Apart from the reaction chemicals and the wax layer, no other solid or liquid materials are present in the functionalized vessels 20".

[0054] In the microtiter plate 10"' of Fig. 5, the functionalized vessels 20"' contain, as in the embodiment of Fig. 3, only air-dried, functional reaction chemicals 30, 32, 34, but no other solid or liquid materials.

[0055] Figure 6 shows a section of a microtiter plate according to a further embodiment of the invention.

[0056] In the microtiter plate 110 of Fig. 6, the air-dried, functional reaction chemicals 30, 32, 34 in each functionalized reaction vessel 120 are divided into two portions, with a first portion 30A, 32A, 34A containing reaction chemicals used to perform a PCR (PCR chemicals) and a second portion 30B, 32B, 34B containing reaction chemicals used to perform reverse transcription (RT chemicals). The vessels 120 can contain different PCR chemicals 30A, 32A, 34A, for example, for a multiplex test, or they can each contain the same PCR chemicals. The RT chemicals 30B, 32B, 34B in the vessels 120 can also be the same or different and, in the latter case, can contain, for example, sequence-specific primers.

[0057] The PCR chemicals 30A, 32A, 34A are each covered with a higher-melting wax layer 40 with a melting temperature above 60°C, for example, 65°C. The RT chemicals 30B, 32B, 34B are present on the wax layer 40 and are in turn covered by a lower-melting wax layer 140 with a melting temperature below 50°C, for example, 45°C.

[0058] Apart from the reaction chemicals 30, 32, 34 and the wax layers 40, 140, no other solid or liquid materials are present in the functionalized vessels 120.

[0059] With such a design, a two-step reaction can be carried out in a simple manner in a functionalized vessel, as already described above.

[0060] In some embodiments, the wax layer 140 may also be omitted, so that the RT chemicals 30B, 32B, 34B are present on the wax layer 40 without a wax covering.

[0061] Alternatively or additionally, the vessel cover 50 can be omitted.

Claims

Patent claims 1. Ready-to-use functionalized microtiter plate for the detection of nucleic acids by reverse transcription (RT) and / or polymerase chain reaction (PCR), which has been isolated and purified from a test material, wherein the microtiter plate contains in a strip or a plate a plurality of vessels having an opening, one or more of which are functionalized, the functionalized vessels are liquid-free and contain only functional reaction chemicals for the detection of the nucleic acid and optionally one or more wax layers that cover at least part of the functional reaction chemicals to protect against light and oxygen, the functional reaction chemicals contain specific primers and probes, dNTPs, MgCl, buffer, salt, polymerase, in the case that RNA is to be detected, additionally reverse transcriptase, and the probes are each provided with a first,fluorescent reporter dye and are provided with a second dye that acts as a quencher and suppresses the fluorescence signals of the intact probes.

2. Microtiter plate according to claim 1, characterized in that the functionalized vessels of the microtiter plates are closed with a vessel cover.

3. Microtiter plate according to claim 2, characterized in that the vessel cover is formed by an opaque film.

4. Microtiter plate according to at least one of claims 1 to 3, characterized in that the functional reaction chemicals are air-dried.

5. Microtiter plate according to one of claims 1 to 4, characterized in that the functional reaction chemicals contain chelating agents such as ETDA (ethylenediaminetetraacetic acid) and / or agents for reducing potential inhibitors of the transcription and / or polymerase reaction and / or agents for stabilizing the enzymes.

6. Microtiter plate according to claim 5, characterized in that the functional reaction chemicals further contain an internal control substance, in particular an artificial or non-naturally occurring RNA sequence, or an artificial or non-naturally occurring DNA sequence.

7. Microtiter plate according to one of claims 1 to 6, characterized in that the functionalized vessels contain only functional reaction chemicals for the detection of the nucleic acid.

8. Microtiter plate according to one of claims 1 to 6, characterized in that the functionalized vessels contain only functional reaction chemicals for the detection of the nucleic acid and exactly one wax layer completely covering the functional reaction chemicals.

9. Microtiter plate according to one of claims 1 to 6, characterized in that the functionalized vessels contain only functional reaction chemicals for the detection of the nucleic acid and exactly one wax layer covering only a part of the functional reaction chemicals.

10. Microtiter plate according to claim 9, characterized in that the functional reaction chemicals comprise PCR chemicals for carrying out a PCR and RT chemicals for carrying out a reverse transcription, wherein the PCR chemicals are covered by said wax layer and the RT chemicals are present on said wax layer without wax covering.

11. Microtiter plate according to one of claims 1 to 6, characterized in that the functionalized vessels contain only functional reaction chemicals for the detection of the nucleic acid and exactly two wax layers, each covering a part of the functional reaction chemicals, wherein the two wax layers advantageously have different melting points.

12. Microtiter plate according to claim 11, characterized in that the functional reaction chemicals comprise PCR chemicals for carrying out a PCR and RT chemicals for carrying out a reverse transcription, wherein the PCR chemicals are covered by a first of the two wax layers and the RT chemicals are covered by the second of the two wax layers.

13. Microtiter plate according to claim 12, characterized in that the two wax layers are formed by a higher-melting wax layer, preferably with a melting temperature of 60 °C or more, and a lower-melting wax layer, preferably with a melting temperature of 50 °C or less, and the PCR chemicals are covered by the higher-melting wax layer, the RT chemicals are present on the higher-melting wax layer and are covered by the lower-melting wax layer.

14. A method for detecting nucleic acids, in which a ready-to-use functionalized microtiter plate according to one of claims 1 to 13 is provided, any vessel cover present is removed, a test material containing one or more nucleic acids to be detected is introduced into the opening of one or more of the functionalized vessels, the microtiter plate filled with test material is introduced into a qPCR device and a quantitative real-time (RT-)PCR is carried out, and a fluorescence signal of the functionalized vessels is measured in order to detect the presence of one or more of the nucleic acids to be detected in the test material.

15. The method according to claim 14, characterized in that nucleic acids are extracted from a patient sample by isolation and purification in order to obtain the test material.

16. Method according to claim 14 or 15, characterized in that no further reagents are added to the functionalized vessels other than the test material.

17. Method according to one of claims 14 to 16, characterized in that the functionalized vessels contain exactly one wax layer which partially or completely covers the functional reaction chemicals, and in that in a (RT-)PCR step the wax layer is melted, the test material is thereby brought into contact with the functional reaction chemicals, and a real-time (RT-)PCR is carried out.

18. Method according to one of claims 14 to 16, characterized in that the functionalized vessels contain exactly two wax layers with different melting points, and that in a RT step — the lower melting wax layer is melted, — the test material is thereby brought into contact with a first part of the functional reaction chemicals, and — reverse transcription is carried out, and in a subsequent PCR step — the higher melting wax layer is melted, — the test material is thereby brought into contact with a second part of the functional reaction chemicals, and — real-time PCR is performed.

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