Method and apparatus for detecting several nucleic acids side by side
By employing fluorescent dyes with different melting curve temperatures and molecular beacons, the method addresses the challenge of cross-talk in nucleic acid detection, enabling accurate multiplexed detection of multiple nucleic acids in a shared reaction cavity.
Patent Information
- Authority / Receiving Office
- DE · DE
- Patent Type
- Applications
- Current Assignee / Owner
- ROBERT BOSCH GMBH
- Filing Date
- 2024-12-13
- Publication Date
- 2026-06-18
AI Technical Summary
Existing methods for simultaneously detecting multiple nucleic acids face challenges in distinguishing between multiple fluorescent dyes due to overlapping excitation and emission wavelengths, leading to cross-talk and reduced spectral distinguishability.
The method employs fluorescent dyes with distinct melting curve temperatures, using molecular beacons with different stem-loop structures and chemical modifications to minimize cross-talk, and performs selective detection by assigning optical channels to specific temperature ranges.
This approach allows for reliable and efficient multiplexed detection of multiple nucleic acids by reducing cross-talk and optimizing image acquisition, enabling accurate identification of pathogens in a shared reaction cavity.
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Abstract
Description
[0001] The present invention relates to a method for detecting several nucleic acids simultaneously. Furthermore, the present invention relates to a device configured to carry out the steps of the method. State of the art
[0002] In molecular diagnostics, diseases can be detected based on nucleic acid biomarkers. The aim is to detect as many pathogens as possible simultaneously via their specific nucleic acids. Since the relevant biomarkers are often present only in very small quantities in the patient sample, detection usually takes place during or after amplification of the sample. One way to detect multiple targets simultaneously is to perform individual assays in several cavities of a microarray in parallel. This requires the presence of reagents in the corresponding cavities that allow for the specific detection of the pathogens.
[0003] Alternatively, multiplexing can be performed within a reaction cavity. For this, several template-specific nucleic acid-binding fluorescent dyes are used as probes, which can be operated at different excitation and detection wavelengths. However, the more probes are used, the more difficult it becomes to distinguish them spectrally. Disclosure of the invention
[0004] A method for the simultaneous detection of multiple nucleic acids, such as DNA or RNA, is proposed, suitable for identifying several different pathogens in a patient sample. The simultaneous detection of multiple nucleic acids specifically refers to their presence in a shared spatial region, such as a cavity of a microarray. This method involves nucleic acid amplification, which can be performed, for example, using qPCR. Amplification is carried out in the presence of several nucleic acid-binding fluorescent dyes. These dyes can be excited to fluorescence at specific excitation wavelengths and emit fluorescent light at specific emission wavelengths. Following amplification, a melting curve analysis of the amplification product is performed.Melting curve analysis is performed using a first set of optical channels. An optical channel is defined as a combination of an excitation wavelength and a detection wavelength. The excitation wavelength is the wavelength of light used to irradiate the amplification product and stimulate it to fluoresce. The detection wavelength is the wavelength at which light emitted by the excited amplification product is detected. An optical channel can be configured by combining an excitation wavelength with a detection wavelength at which light emission is expected for one of the fluorescent dyes after excitation with the excitation wavelength. The difference between these wavelengths is called the Stokes shift.Theoretically, multiplexing within a local area encompassing at least some of the amplified nucleic acids, particularly within a reaction cavity, allows for the selective detection of any fluorescent dye specific to a particular nucleic acid. However, in practice, the problem arises that when using many fluorescent dyes, a single excitation wavelength often excites several of them. The wavelength ranges of the fluorescence become so close or even overlap that cross-talk between multiple fluorescent dyes can occur within a single optical channel.
[0005] The present method provides for several first fluorescent dyes to have melting curve temperatures that are lower than the melting curve temperatures of several second fluorescent dyes. This makes it possible, after reaching the melting curve temperatures of the group of first fluorescent dyes, to exclude them from further fluorescence analysis and thus reliably perform multiplexing with a larger number of fluorescent dyes than would be possible without selecting different melting curve temperatures.
[0006] Typically, nucleic acid-binding fluorescent dyes exhibit melting curve temperatures so close that they are indistinguishable within the error range of a melting curve analysis. Fluorescent dyes with significantly different melting curve temperatures can be molecular beacons coupled to a fluorophore. Molecular beacons are hybridization probes containing single-stranded DNA with a stem loop or hairpin structure. The fluorescent dye is located at their stem-associated 3' and 5' ends. The loop or hairpin structure contains a DNA sequence complementary to the target nucleic acid, particularly that of the pathogen to be detected.
[0007] Molecular beacons can exhibit chemical modifications in addition to the fluorophore, for example, to minimize background fluorescence in the absence of templates. They can also be designed as sloppy molecular beacons.
[0008] Preferably, at least two first fluorescent dyes and / or at least two second fluorescent dyes have the same fluorophore but different molecular beacons with different melting curve temperatures. In this way, the emission wavelength of one fluorophore can be used to detect several target nucleic acids, particularly pathogens, whereby different nucleic acids binding to the same fluorophore can be distinguished by the different melting curve temperatures of the molecular beacons used.
[0009] To optimally exploit the advantage of the different melting curve temperatures of the fluorescent dyes, it is preferred that the melting curve analysis below the melting curve temperatures of the first fluorescent dyes is performed only in optical channels assigned to those dyes. Above the melting curve temperatures of the first fluorescent dye, it is performed only in optical channels assigned to the second fluorescent dyes. All first fluorescent dyes are preferably selected such that no or only minimal crosstalk from the second fluorescent dyes is expected in their assigned optical channels. Once the melting curve temperatures of the first fluorescent dyes are reached, they are thermally excluded.Even though cross-referencing from a first fluorescent dye would have been expected in the optical channels assigned to the second fluorescent dye, this is prevented above the melting curve temperatures of the first fluorescent dye. Besides reducing cross-referencing, this approach has the advantage that in each of the two temperature ranges, excitation and detection only need to be performed in a subset of the optical channels of the first group, allowing more images to be acquired in each optical channel than would be possible if all optical channels of the first group were used.
[0010] In a melting curve analysis, melting curve temperatures are defined for each fluorescent dye. It is preferred that the melting curve temperatures of the first fluorescent dyes are at least 2 °C higher than the melting curve temperatures defined for them in the melting curve analysis. The melting curve temperatures of the second fluorescent dyes are preferably at least 2 °C lower than the melting curve temperatures defined for them in the melting curve analysis. This compensates for the uncertainty regarding the actual temperature in a reaction cavity, particularly in microfluidic applications. Furthermore, this approach allows the temperature offset in each experiment to be determined after the actual melting temperatures of the first fluorescent dyes (those that melt first) have been established, and the subsequent temperatures or image acquisitions during the melting curve analysis to be adjusted accordingly.
[0011] During melting curve analysis, for the quantitative determination of target nucleic acids, especially pathogens, only monitoring of those optical channels where fluorescence is actually expected is necessary. Therefore, it is advantageous to precede the melting curve analysis with a qualitative determination. This can preferably be achieved by recording a fluorescence image in at least one optical channel of a second group of optical channels after each amplification cycle. Based on these images, the first group is then selected as a subset of the second group. If all fluorescent dyes used have bound to targets, the first group corresponds to the second group. Otherwise, the excitation in some optical channels is not answered by fluorescence, and these optical channels do not need to be included in the second group.
[0012] To avoid delaying amplification by acquiring fluorescence images, it is still preferred that a fluorescence image be acquired after each cycle in a maximum of two optical channels of the second group of optical channels.
[0013] The device for detecting multiple nucleic acids side by side is designed to perform the steps of the procedure. In particular, the device is a microfluidic device in which a sample can be subjected to amplification and melting curve analysis in one or more reaction cavities on a microarray.
[0014] The device for carrying out the method is set up, on the one hand, by implementing the process steps, for example, in a computer program. Components of the device that can be controlled in the method include, in particular, at least one heat source that can change the temperature of a biological sample to carry out amplification cycles and to perform a melting curve analysis, at least one light source by means of which the sample can be irradiated with excitation light, and at least one optical sensor that can detect fluorescence light.To represent multiple optical channels, either multiple light sources and multiple optical sensors, each designed for different wavelengths, can be used, or one or more light sources and sensors can be equipped with multiple optical filters, or other adjustability of the light sources with respect to the emitted light and of the sensors with respect to the detected light can be provided. Brief description of the drawings
[0015] Exemplary embodiments of the invention are shown in the drawings and are explained in more detail in the following description. Fig. Figure 1 schematically shows a device according to an embodiment of the invention. Fig. Figure 2 shows a flowchart of a method according to an embodiment of the invention. Exemplary embodiments of the invention
[0016] A device 10 according to an embodiment of the invention is in Fig. Figure 1 shows a microcavity array 11 arranged on a heat source 12. A biological sample 20 to be examined can be placed in a cavity of the microcavity array 11. A light source 13 is arranged so that it can shine light 31 of different wavelengths onto the sample 20 to excite it to fluorescence. An optical sensor 14, for example a camera, is arranged so that it can detect fluorescence light 32 emitted by the excited sample 20 at different wavelengths.
[0017] The sequence of a method according to an embodiment of the invention is described in Fig. Figure 2 illustrates this. For example, the method includes the detection of several pathogens via their specific nucleic acids, also referred to as target nucleic acids. After starting the method, the sample is first placed on the microcavity array together with twelve nucleic acid-binding fluorescent dyes. These were obtained by coupling three fluorophores to two different molecular beacons each, and three further fluorophores to two other different molecular beacons each. These, along with their excitation wavelength ranges λ, are described below. ex and their fluorescence emission wavelength ranges λ em listed in Table 1: Table 1 Kanal Fluorophor l ex [nm] l em [nm] T m1 [°C] T m2 [°C] 1 FAM 451,5 - 486,5 500,0 - 530,0 74 83 2 CAL Fluor Orange 560 533,0 - 543,0 544,0 - 575,0710,0 - 790,0 74 83 3 CAL Fluor Red 610 577,0 - 573,0675,0 - 705,0 597,0 - 623,0 57 66 4 Quasar 670 630,0 - 645,0 652,0 - 678,0 57 66 5 Pulsar 650 451,5 - 486,5 652,0 - 678,0 57 66 6 Alexa Fluor 700 577,0 - 583,0675,0 - 705,0 544,0 - 575,0710,0 - 790,0 74 83
[0018] The combination of an excitation wavelength λ ex with an emission wavelength λ emEach fluorescent dye forms an optical channel. These channels are numbered 1 to 6 in Table 1. The fluorescent dyes of channels 3 to 5 have a first molecular beacon, and the fluorescent dyes of channels 1, 2, and 6 have a second molecular beacon. Due to the use of different molecular beacons, the fluorescent dyes of channels 3 to 5 have a first melting curve temperature Tm. m1 and a second melting curve temperature T m2 on, which below a first melting curve temperature T m1 and a second melting curve temperature T m2 the fluorescent dyes of channels 1, 2 and 6.
[0019] The nucleic acids in the provided biological sample are now amplified in, for example, 40 amplification cycles 42 using qPCR. After each amplification cycle 42, a fluorescence image is recorded in one of the optical channels 43. This results in a fluorescence response, which is shown in Table 2: Table 2 1 2 3 4 5 6 1 1,00 0,00 0,00 0,00 0,01 0,00 2 0,00 1,00 0,01 0,00 0,03 0,02 3 0,00 0,00 1,00 0,00 0,01 0,00 4 0,00 0,00 0,00 1,00 0,00 0,02 5 0,00 0,00 0,01 0,00 1,00 0,00 6 0,00 0,02 0,07 0,04 0,01 1,00
[0020] Each row corresponds to the excitation in an optical channel. Each column indicates the fluorescence response of the fluorescent dye assigned to that optical channel. The tabulated values have been normalized such that a value of 1.00 corresponds to the expected fluorescence response of the fluorescent dye assigned to that optical channel. If other fluorescent dyes also produce a fluorescence response in this optical channel, this is referred to as cross-talk.
[0021] After a test 44 has shown that all planned amplification cycles have been completed, a temperature step 45 follows, in which all nucleic acids contained in the amplification product are denatured, for example, for two minutes at a temperature of 95 °C. Subsequently, the amplification product is cooled 46 to a temperature of 55 °C in 10 °C increments, with each increment being held for thirty seconds. This temperature, which is to serve as the starting temperature for the subsequent melting curve analysis, is below the first melting curve temperature T. m1 of 57 °C of the fluorescent dyes of channels 3 to 5.
[0022] In the following melting curve analysis, twelve different pathogens can be distinguished using the six optical channels, by assigning each optical channel to the detection of two or more nucleic acids. This utilizes the distinguishability of the different fluorescent dyes in the melting curve analysis. First, a selection of the optical channels to be used in the melting curve analysis is made. If a fluorescence response is observed in each of the optical channels during the acquisition of the fluorescence images, as shown in Table 2, then all optical channels are used. If no fluorescence had been detected in some of the optical channels, these optical channels would not have been used. In the melting curve analysis, the temperature is then gradually increased to a value of 85 °C, which is above the second melting curve temperature T. m2The temperature of the fluorescent dyes in channels 1, 2, and 6 is [missing information]. Since all fluorescent dyes are thermally excluded upon reaching this temperature, a further increase in temperature would provide no further information about the presence of pathogens. The temperature increase occurs, for example, in steps of 0.1 °C per second. Due to the time required to switch the light source 13 and the optical sensor 14 to a different optical channel, a fluorescence image can be acquired every 1.5 °C. Initially, this is done sequentially only in optical channels 3 to 5. The result of these image acquisitions 52 during the melting curve analysis is shown in Table 3. Table 3 2 3 4 5 6 3 0,00 0,00 1,00 0,00 0,01 0,00 0,01 4 0,00 0,00 0,00 1,00 0,00 0,02 0,02 5 0,00 0,00 0,01 0,00 1,00 0,00 0,01
[0023] The total column on the right-hand side of Table 3 indicates the sum of the cross-referencing that occurs in addition to the desired fluorescence signals with an intensity of 1.00 in the respective optical channel. It is evident that by omitting the acquisition of fluorescence images in channels 1, 2, and 6, the high level of cross-referencing visible in Table 2 for these optical channels does not impair a quantitative analysis of the pathogens present, and only a lower level of cross-referencing remains. As soon as a test 53 shows that the melting curve analysis reveals a temperature above the second melting curve temperature T m2 Once the fluorescent dyes in channels 3 to 5 have reached their operating temperature, i.e., once a temperature of 66 °C has been exceeded, further fluorescence images are acquired in optical channels 1, 2, and 6. The result of this further melting curve analysis is shown in Table 4: Table 4 1 2 6 Σ 1 1,00 0,00 0,00 0,00 2 0,00 1,00 0,02 0,02 6 0,00 0,02 1,00 0,02
[0024] Since the fluorescent dyes of optical channels 3 to 5 have already melted, they no longer produce a fluorescence response to the excitation light on channels 1, 2, and 6 and are therefore not listed in the columns of Table 4. Contrary to what might have been expected based on Table 2, the total column on the right-hand side of Table 4 shows only a small amount of crosstalk in addition to the expected fluorescence response of 1.00 in each optical channel. This method thus enables reliable detection of the target nucleic acids of the pathogens to be detected even in these optical channels.
[0025] After a further test 55 shows that a temperature of 85 °C has been reached, the melting curve analysis is terminated, and a calculation 56 is performed to determine which pathogens could be detected in an analytically significant quantity in the patient sample. After this information is output, the procedure is terminated 57.
Claims
Method for detecting several nucleic acids side by side, comprising the following steps: - Performing an amplification (42 - 44) of the nucleic acids in the presence of several nucleic acid-binding fluorescent dyes, and - Performing a melting curve analysis (52 - 55) of an amplification product resulting from the amplification (42 - 44) with a first group of optical channels, characterized in that several first fluorescent dyes have melting curve temperatures which are below the melting curve temperatures of several second fluorescent dyes. Method according to claim 1, characterized in that the fluorescent dyes each have a molecular beacon coupled to a fluorophore. The method according to claim 2, characterized in that at least two first fluorescent dyes and / or at least two second fluorescent dyes have the same fluorophore, but different molecular beacons with different melting curve temperatures. Method according to one of claims 1 to 3, characterized in that the melting curve analysis (52 - 55) below the melting curve temperatures of the first fluorescent dyes is carried out only in optical channels that are assigned to the first fluorescent dyes and the melting curve analysis above the melting curve temperatures of the first fluorescent dyes is carried out only in optical channels that are assigned to the second fluorescent dyes. Method according to claim 4, characterized in that the melting curve temperatures of the first fluorescent dyes are at least 2°C above the melting curve temperatures applied for them in the melting curve analysis (52 - 55) and the melting curve temperatures of the second fluorescent dyes are at least 2°C below the melting curve temperatures applied for them in the melting curve analysis (52 - 55). Method according to one of claims 1 to 5, characterized in that during amplification (42 - 44) after each cycle (42) a fluorescence image is recorded in at least one optical channel of a second group of optical channels (43) and the first group is selected as a subset of the second group based on the images (51). Method according to claim 6, characterized in that during amplification (42 - 44) a fluorescence image is recorded in a maximum of two optical channels of the second group of optical channels after each cycle (43). Device (10) for detecting several nucleic acids side by side, characterized in that it is configured to carry out the steps of a method according to one of claims 1 to 7. Device according to claim 8, characterized in that it is designed as a microfluidic device. Device according to claim 8 or 9, characterized in that it has at least one heat source (12), at least one light source (13) and at least one optical sensor (14).