System for nucleic acid analysis

Abstract

Using concepts of the present invention, a system is provided, the system comprising a test card receiver configured to receive a test card, the test card comprising a sample portion having a first side and a second side opposite the first side, the sample portion of the test card configured to hold a sample for PCR, a temperature regulator configured to regulate a temperature of the sample portion of the test card over a plurality of temperature cycles, each temperature cycle being between a first temperature and a second temperature different from the first temperature, the temperature regulator comprising a surface configured to contact the first side of the sample portion of the test card, the test card receiver comprising a spring member configured to urge the sample portion of the test card towards the surface of the temperature regulator, and monitoring the sample portion of the test card over the plurality of temperature cycles. and a monitoring device configured to monitor the temperature of the sample portion of the test card, the monitoring device comprising at least one optical sensor, an illumination system configured to illuminate the second side of the sample portion with light having a wavelength within at least three excitation wavelength ranges, an emission filter configured to transmit light having a wavelength within at least three emission wavelength ranges, and collection optics configured to collect light emanating from the second side of the sample portion of the test card and passing through the emission filter, the collection optics further configured to direct the collected light towards the optical sensor such that at least a portion of the collected light strikes the optical sensor, the optical sensor configured to generate at least one signal, each signal related to light striking the optical sensor after a unique number of temperature cycles of the plurality of temperature cycles.

Description

[Technical field]

[0001] The present invention relates to a system for nucleic acid analysis. [Background technology]

[0002] Nucleic acid testing is used to detect specific nucleic acid sequence analytes and is therefore a technique used to detect and identify specific species or subspecies of organisms, often viruses or bacteria, that act as pathogens, usually in samples such as blood, tissue, or urine. The amount of a given genetic DNA or RNA analyte may be negligible, and many nucleic acid tests include a step to amplify the genetic analyte.

[0003] Although several amplification methods exist, real-time polymerase chain reaction is the most established method for detecting and typing different nucleic acid analytes in the field of clinical diagnostics. Using PCR, single or multiple copies of a DNA or RNA sequence can be amplified to many copies, such as thousands or millions. The large number of copies generated by PCR makes PCR useful in qualitative and quantitative analysis of the presence of nucleic acid analyte(s) in a sample.

[0004] There are two strategies for visualization of amplified nucleic acid fragments: non-specific fluorescent dyes and fluorescently labeled oligonucleotide probes. The exponential production of PCR products during amplification causes a concomitant exponential increase in fluorescence. In a multiplex PCR setting, two or more target biomarker sequences within the same sample can be amplified in a qPCR reaction, with different fluorescently labeled sequence-specific oligonucleotides, e.g., probes or primers, allowing for simultaneous or sequential target detection. Detection of the fluorescent signal can be performed at any stage of the PCR reaction, e.g., at the final PCR stage (endpoint PCR) or in real time during the exponential phase of the amplification reaction.

[0005] Real-time reverse transcription polymerase chain reaction (RT-PCR) is a variant of PCR that allows for the use of RNA as a template. The RNA template is first converted to complementary (c)DNA, which then serves as a template for exponential amplification using PCR. Coronavirus disease 2019 (COVID-19) can be tested by RT-PCR amplification and detection of coronavirus SARS-CoV-2 in a sample from a test subject, for example, a sample obtained by nasopharyngeal or throat swab.

[0006] Rapid screening and low-cost diagnostics play a key role in choosing the right therapeutic intervention course and in controlling disease outbreaks. This is especially important when a disease has no effective treatment, as is the case for example with COVID-19 infection. In such cases, infection control and prevention depend inter alia on interrupting the transmission of the pathogen from infected animals or humans to new hosts or locations.

[0007] In laboratories, conventional PCR tests have a turnaround time of at least 3 hours. In certain situations, for example at the level of pre-departure testing at airports or pre-event testing, faster and more accurate test results are highly desirable.

[0008] Therefore, the long time required for the detection or quantitative analysis of certain infectious diseases, such as Covid-19, is problematic as it may increase waiting times for test takers and increase the risk of further human contamination.

[0009] Furthermore, detection or quantitative analysis of certain viral infections, such as coronaviruses, is often problematic due to the large laboratory equipment involved. Summary of the Invention

[0010] The object is to at least partially mitigate, alleviate or eliminate one or more of the above identified deficiencies and shortcomings in the art singly or in any combination and to solve at least the problems mentioned above.

[0011] A particular object is to provide a system for nucleic acid analysis that at least partially reduces, mitigates, or eliminates one or more of the above-identified deficiencies in the art.

[0012] A further specific object is to provide a system for PCR analysis that at least partially reduces, mitigates, or eliminates one or more of the above-identified deficiencies in the art.

[0013] In the following, nucleic acid analysis is described and exemplified in conjunction with PCR analysis, although other types of nucleic acid analysis may alternatively be used.

[0014] According to a first aspect, there is provided a system for PCR analysis, the system comprising: a test card receiver configured to receive a test card, the test card comprising a sample portion having a first side and a second side opposite the first side, the sample portion of the test card configured to hold a PCR sample; a temperature regulator configured to regulate a temperature of the sample portion of the test card over a plurality of temperature cycles, where each temperature cycle is between a first temperature and a second temperature different from the first temperature, the temperature regulator comprising a surface configured to contact a first side of the sample portion of the test card; the test card receiving portion includes a spring member configured to urge the sample portion of the test card toward a surface of the temperature regulator; a monitoring device configured to monitor the sample portion of the test card over a plurality of temperature cycles, the monitoring device comprising: at least one light sensor; an illumination system configured to illuminate a second side of the sample portion with light having a wavelength within at least three excitation wavelength ranges; an emission filter configured to transmit light having wavelengths within at least three emission wavelength ranges; and collection optics configured to collect light emanating from the second side of the sample portion of the test card and transmitted through the emission filter, the collection optics further configured to direct the collected light towards the light sensor such that at least a portion of the collected light strikes the light sensor; The light sensor is configured to generate at least one signal, each signal related to light impinging on the light sensor after a unique number of the plurality of temperature cycles.

[0015] As used herein, the term "sample" or "sample for PCR" should be broadly interpreted as any liquid that may contain an analyte of interest, which liquid may be provided in a test card and processed on said test card. As used herein, the analyte present in the liquid is usually a target nucleic acid that is detected with the aid of a test card that can be engaged in the present system for nucleic acid analysis. The sample may be a liquid biological sample obtained from a person, such as blood, but also a biological sample that has been pre-processed on the bench according to any sample processing protocol, such as a nucleic acid extraction protocol, such as a nasopharyngeal or throat swab sample. The liquid may further include all the reagents necessary to amplify and detect a specific nucleic acid analyte that may be present in such a biological sample. The nucleic acid in the liquid may then be directly analyzed for its presence using the system for nucleic acid analysis disclosed herein.

[0016] As used herein, the term "analyte" should be construed as synonymous with the terms "biomarker" or "target," which relate to a detectable nucleic acid fragment of interest, typically a nucleic acid fragment of a pathogen, such as viral DNA or RNA.

[0017] As used herein, the term "PCR" should be interpreted broadly as any technique that employs the polymerase chain reaction, and is understood to include RT-qPCR.

[0018] As used herein, the term "multiple" as in "multiple to", e.g., multiple temperature cycles, should be understood to refer to more than one. In the context of "multiple temperature cycles", the term "multiple" typically refers to more than one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or a range of multiples of 2, 5, or 10, or 5 to 60 cycles, or 45 to 55 cycles.

[0019] The test card receiver allows the system to be used with a test card. The test card comprises a sample portion, which may be a sample handling portion, such as a microfluidic chip, that allows the sample to be handled and subjected to PCR. A test card with a sample portion allows for desirable sample handling due to the sample portion, while at the same time allowing for a suitable shape and structure of the test card, and therefore desirable handling, such as handling before and during insertion of the test card into the system.

[0020] The temperature regulator allows for adjustment of the temperature of the sample portion of the test card, which is important in PCR. Adjusting the temperature of the sample portion of the test card over multiple temperature cycles, each temperature cycle being between a first temperature and a second temperature different from the first temperature, thus allowing denaturation and annealing in PCR.

[0021] A temperature regulator having a surface configured to contact a first side of the sample portion of the test card enables efficient thermal contact and heat transfer between the temperature regulator and the sample portion, thus providing benefits to PCR within the sample portion.

[0022] The test card receiver with a spring member configured to press the sample portion of the test card towards the surface of the temperature conditioner allows for efficient thermal contact and fast heating / cooling of the sample portion. Furthermore, the spring member allows for efficient insertion and removal of the test card into the system. The spring member further allows for a suitable and desired pressing force of the sample portion against the surface of the temperature conditioner, e.g., not only providing thermal contact but also providing a suitable force that does not cause damage to the sample portion. This may be beneficial, e.g., when the sample portion is a silica chip or other brittle material. The spring member further allows for optimization between high thermal contact while avoiding damage to the test card or the sample portion of the test card.

[0023] A monitoring device configured to monitor the sample portion of the test card over multiple temperature cycles allows for efficient monitoring of PCR occurring in or on the sample portion. For example, PCR can be monitored over time, providing real-time PCR monitoring. In an alternative setup, PCR may be monitored at an end-point stage or at a stage between PCR start and end-point.

[0024] Having at least one light sensor allows for sensing or detection of light from the PCR, such as fluorescent light produced during the PCR.

[0025] The illumination system configured to illuminate the second side of the sample portion while the temperature regulator comprises a surface configured to contact the first side of the sample portion provides a desirable design of the system, for example, the temperature regulator may be positioned such that illumination interference is reduced or avoided.

[0026] Light having wavelengths within at least three excitation wavelength ranges allows for efficient detection or monitoring of PCR. For example, at least three different markers or fluorophores can be used. Furthermore, simultaneous analysis or detection of at least three different DNA sequences can be achieved.

[0027] An emission filter configured to transmit light having a wavelength within at least three emission wavelength ranges allows for detecting at least three different emission signals. An emission filter configured to transmit light having a wavelength within at least three emission wavelength ranges can for example be realised by an emission filter consisting of at least three sub-emission filters.

[0028] The collection optics is configured to collect light emanating from a second side of the sample portion of the test card and passing through the emission filter, and is further configured to direct the collected light towards the light sensor so that at least a portion of the collected light strikes the light sensor, thereby enabling detection or sensing of light, such as fluorescence, from the sample portion, and thus, for example, monitoring of PCR within the sample portion.

[0029] An optical sensor configured to generate at least one signal, each signal associated with light striking the optical sensor after a unique number of temperature cycles of the plurality of temperature cycles, allows for efficient monitoring of PCR.

[0030] The system for PCR analysis may be a system for nucleic acid analysis. The system for PCR analysis may be a system for nucleic acid analysis using PCR or RT-PCR.

[0031] The nucleic acid can be DNA or RNA, or a sequence thereof.

[0032] The at least one optical sensor may be at least one image sensor, and the collection optics may be imaging optics configured to image the sample portion onto the image sensor.

[0033] This allows imaging of the sample portion and the PCR.

[0034] The monitoring device is At least one optical sensor; a first light source configured to illuminate the second side of the sample portion through a first excitation light filter, thereby providing light having a wavelength within a first excitation wavelength range at the second side of the sample portion of the test card; a first emission filter configured to transmit light having wavelengths within a first emission wavelength range; and a first collection optic configured to collect light emanating from the second side of the sample portion of the test card and transmitted through the first emission filter, where the collection optic is further configured to direct the collected light towards the light sensor such that at least a portion of the collected light strikes the light sensor; a second light source configured to illuminate a second side of the sample portion through a second excitation light filter, thereby providing light having a wavelength within a second excitation wavelength range at the second side of the sample portion of the test card; a second emission filter configured to transmit light having wavelengths within a second emission wavelength range; and a second collection optic configured to collect light emanating from a second side of the sample portion of the test card and transmitted through the second emission filter, where the collection optic is further configured to direct the collected light towards the light sensor such that at least a portion of the collected light strikes the light sensor; a third light source configured to illuminate the second side of the sample portion through a third excitation light filter, thereby providing light having a wavelength within a third excitation wavelength range at the second side of the sample portion of the test card; a third emission filter configured to transmit light having wavelengths within a third emission wavelength range; and and a third collection optical system configured to collect light emanating from a second side of the sample portion of the test card and passing through the third emission filter, the collection optical system further configured to direct the collected light towards the light sensor such that at least a portion of the collected light strikes the light sensor.

[0035] Such a system can be used to monitor three or more, i.e., a first, second, and third or more, excitation / emission reactions, allowing, for example, monitoring one or more of at least three different analyte sequences or portions of analyte sequences, or a plurality of sequences, e.g., DNA, e.g., one associated with the COVID virus, one associated with a "background reaction," and one associated with one or more other types of viruses or analytes.

[0036] The at least one optical sensor may be at least one imaging sensor, and each of the collection optics may be a respective imaging optic configured to collect light passing through the emission filter and image a second side of the sample portion of the test card onto the at least one image sensor.

[0037] Thereby, monitoring such as end-point or real-time monitoring of PCR within the sample portion can be achieved. Additionally, other types of reactions or occurrences within the sample portion can be tracked and monitored.

[0038] The at least one image sensor may be configured to capture at least one image of the second side of the sample portion, each image being captured after a unique number of temperature cycles of the plurality of temperature cycles.

[0039] Thereby, for example, all or part of a PCR can be monitored or imaged at a desired frequency of images.

[0040] The at least one light sensor may be at least one photodiode.

[0041] By using the photodiode(s), assembly tolerances can be relaxed. For example, the need to keep the focal plane above the sample can be avoided. Furthermore, systems with photodiode(s) can have a compact size. Furthermore, the analyzed data can be reduced in size, for example, compared to data relating to an image.

[0042] Each of the first light source, the second light source, and the third light source may be an LED.

[0043] This allows narrow bandwidth excitation light to be used with the system. Additionally, LEDs are readily available and require little maintenance.

[0044] The spring member may be configured to urge the sample portion of the test card towards the surface of the temperature regulator with a force of 5-15N, for example 8-12N.

[0045] This allows for efficient thermal contact between the sample portion and the temperature regulator while minimizing or reducing damage or the risk of damage to the sample portion or test card, particularly when a silicon chip is used as the sample portion.

[0046] The spring member may comprise a leaf spring. The leaf spring may be configured to have a slit or opening to allow transmission of light to and from the second side of the sample portion.

[0047] Thereby, even when the spring member is positioned at or adjacent to the second side of the sample portion, the spring member may be positioned such that it does not obscure the sample portion from the sensor system.

[0048] The temperature regulator may include a thermoelectric element. The thermoelectric element may be a Peltier element.

[0049] Heating and / or cooling can be accomplished efficiently using thermoelectric elements such as Peltier elements, whereby multiple successive temperature cycles can be achieved.

[0050] One of the first temperature and the second temperature may be in the interval from 60 to 70°C, and the other of the first temperature and the second temperature may be in the interval from 90 to 105°C.

[0051] Thereby, temperature cycles, or denaturation and annealing cycles, suitable for PCR can be achieved.

[0052] The temperature regulator may be configured to provide 1 to 100 temperature cycles, such as 5 to 60, or 45 to 50 temperature cycles.

[0053] This makes it possible to achieve a suitable number of denaturation and annealing cycles for PCR.

[0054] Each temperature cycle may have a duration of 20 seconds or less, or 15 seconds or less.

[0055] Such temperature cycle durations can be used to achieve PCR in the system according to the first aspect or embodiment.

[0056] The optical sensor may be configured to generate at least one signal per temperature cycle over at least 5 temperature cycles, or 10 temperature cycles, or 45-55 temperature cycles.

[0057] Thereby, PCR can be monitored over time and real-time PCR is achievable.

[0058] The first excitation wavelength range, the second excitation wavelength range, and the third excitation wavelength range may be different wavelength ranges.

[0059] Thereby, for example, a first, a second and a third sequence or unique DNA may be monitored. Further, for example, three different viruses may be analyzed or three different types of fluorophores may be monitored.

[0060] The first emission wavelength range, the second emission wavelength range, and the third emission wavelength range are different wavelength ranges.

[0061] Thereby, for example, a first, a second and a third sequence or unique DNA may be monitored. Further, for example, three different viruses may be analyzed or three different types of fluorophores may be monitored.

[0062] The first and second sides of the sample portion can be parallel to the major sides of the test card.

[0063] Thus, for example, a flat test card may be used.

[0064] According to a second aspect, there is provided a method for performing a PCR analysis using a system according to the first aspect, comprising: receiving a test card in the test card receiving portion, the test card comprising a sample portion having a first side and a second side opposite the first side, the sample portion of the test card holding a PCR sample; adjusting a temperature of the sample portion of the test card (6) over a plurality of temperature cycles, where each temperature cycle is between a first temperature and a second temperature different from the first temperature, and using the temperature regulator includes contacting a surface with a first side of the sample portion of the test card; the test card receiving portion includes a spring member that urges the sample portion of the test card toward the surface of the temperature regulator; and monitoring the sample portion of the test card over a plurality of temperature cycles, wherein using the monitoring device comprises: At least one optical sensor; an illumination system for illuminating a second side of the sample portion with light having a wavelength within at least three excitation wavelength ranges; an emission filter that transmits light having wavelengths within at least three emission wavelength ranges; and collection optics for collecting light emanating from the second side of the sample portion of the test card and passing through the emission filter, the collection optics further directing the collected light towards a light sensor, at least a portion of the collected light impinging on the light sensor; The light sensor generates at least one signal, each signal related to light impinging on the light sensor after a unique number of the plurality of temperature cycles.

[0065] According to a third aspect there is provided the use of a system according to any one of the claims of the first aspect for PCR analysis or nucleic acid analysis.

[0066] Features described above with respect to one of the aspects also apply to the other of the aspects, when relevant: in order to avoid undue repetition, reference is made to the above.

[0067] Further scope of applicability of the present disclosure will become apparent from the detailed description given below. However, it should be understood that the detailed description and examples, while showing preferred variations of the inventive concept, are given by way of example only, since various changes and modifications within the scope of the inventive concept will become apparent to those skilled in the art from the detailed description. Thus, it should be understood that the inventive concept is not limited to the specific steps of such methods or component parts of such systems, since the described methods and systems may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting. It should be noted that, as used in this specification and the appended claims, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of an element, unless the context clearly dictates otherwise. Thus, for example, reference to "a unit" or "the unit" may include several devices, and the like. Furthermore, the words "comprising," "including," "containing," and similar phrases do not exclude other elements or steps.

[0068] The above and other aspects of the inventive concept will now be described in detail with reference to the accompanying drawings, which show variations of the inventive concept. The drawings should not be considered as limiting the inventive concept to a particular variation, but are used to explain and understand the inventive concept. As illustrated in the drawings, the sizes of layers and regions are exaggerated for illustrative purposes and are therefore provided to illustrate the general structure of the variations of the inventive concept. Like reference numbers refer to like elements throughout. [Brief description of the drawings]

[0069] [Figure 1] 1 illustrates a system according to one embodiment. [Diagram 2] 1 illustrates a system according to one embodiment. [Diagram 3] 1 illustrates a system according to one embodiment. [Figure 4] 1 illustrates an embodiment comprising a leaf spring. [Diagram 5] 1 illustrates a system including a housing according to one embodiment. [Figure 6] 1 illustrates an example temperature profile during temperature cycling according to one embodiment. [Figure 7] 1 illustrates a method according to one embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] The inventive concepts will now be described more fully hereinafter with reference to the accompanying drawings illustrating presently preferred versions of the inventive concepts. However, the inventive concepts may be embodied in many different forms and should not be construed as limited to the variations set forth herein, but rather these variations are provided for thoroughness and completeness and to fully convey the scope of the inventive concepts to those skilled in the art.

[0071] The system described herein can be used to perform and monitor nucleic acid analysis. Furthermore, PCR can be performed and monitored. How this can be achieved will be understood from the following. The temperature regulator allows the necessary heating and cooling, for example used for DNA denaturation and annealing. Thereby, DNA and RNA can be analyzed, and RNA can be analyzed via preceding transcription into cDNA. The light source, light filter, light collection optics, and light sensor allow monitoring of PCR, for example by using fluorophores on DNA or RNA, which can be excited and emit light, and the emitted light can be detected by the light sensor. Thus, PCR can be monitored using the system. The actual PCR takes place on the sample portion of the test card. It is therefore envisaged that the sample portion can contain or receive, for example, a sample containing RNA or DNA, reagents for PCR, and any necessary liquids or buffers, etc.

[0072] PCR chemistries and detection formats compatible with the inventive system herein are described. These include non-specific detection formats such as intercalating dyes, such as double-stranded DNA binding dyes, e.g., CYBR Green. The use of hairpin primers that emit a fluorescent signal upon unfolding during incorporation into the amplification product has been described, e.g., Amplifluor technology. Sequence-specific detection formats that result in measurable fluorescence emission include probes homologous to the internal region amplified by the two PCR primers. They can be divided into two main groups: hydrolysis probes, e.g., TaqMan probes, TaqMan MGB probes, and hybridization probes, e.g., FRET hybridization probes, Molecular Beacons, Scorpion primers, etc. The fluorescent signal intensity can be related to the amount of PCR product by (i) a product-dependent decrease in quenching of the reporter fluorophore, or (ii) an increase in fluorescence resonance energy transfer (FRET) from the donor to the acceptor fluorophore. Commonly used fluorescent reporter dyes are FAM, TET (tetrachloro-6-car-boxyfluorescein), JOE (2,7-dimethoxy-4,5-dichloro-6-carboxy-fluorescein), or HEX (hexacholoro-6-carboxyfluorescein), and commonly used quenchers are TAMRA, DABCYL, and black hole quencher (BHQ). Other techniques use nucleic acid analogs, such as peptide nucleic acids (PNAs), in place of natural nucleic acid sequence recognition elements.

[0073] PCR and RT-PCR are exemplary nucleic acid amplification methods best known in the art, although alternative methods such as, for example, LAMP, NASBA, etc., may be used as compatible with different embodiments of the systems described herein.

[0074] A system 1 for PCR analysis will now be described with reference to FIG. 1. It should be understood that FIG. 1 is a schematic diagram illustrating components of the system 1. The system 1 comprises a test card receiving portion 4 configured to receive a test card 6. The test card 6 is illustrated with a dotted line to illustrate that it is not part of the system 1 in the illustrated embodiment. The test card 6 comprises a sample portion 8 having a first side 10 and a second side 12 opposite the first side, the test card sample portion 8 being configured to hold a PCR sample. The PCR sample may be a sample comprising DNA or RNA, or a sequence or sequences thereof. The system 1 further comprises a temperature regulator 14 configured to regulate a temperature of the sample portion 8 of the test card 6 over a plurality of temperature cycles, each temperature cycle being between a first temperature and a second temperature different from the first temperature, the temperature regulator 14 comprising a surface 16 configured to contact the first side 10 of the sample portion 8 of the test card 6.

[0075] The test card receiver 4 includes a spring member 18 configured to urge the sample portion 8 of the test card 6 toward a surface 16 of the temperature regulator 14. The system 1 further includes a monitoring device 20 configured to monitor the sample portion 8 of the test card 6 over a number of temperature cycles. The monitoring device 20 comprises at least one optical sensor 22, an illumination system 24 configured to illuminate the second side 12 of the sample portion 8 with light having a wavelength within at least three excitation wavelength ranges, an emission filter 26 configured to transmit light having a wavelength within at least three emission wavelength ranges, and collection optics 28 configured to collect light emanating from the second side 12 of the sample portion 8 of the test card 6 and passing through the emission filter 26, the collection optics 28 being further configured to direct the collected light towards the optical sensor 22 such that at least a portion of the collected light strikes the optical sensor 22, and the optical sensor 22 is configured to generate at least one signal S, each signal S related to light striking the optical sensor 22 after a unique number of temperature cycles among the plurality of temperature cycles.

[0076] In addition to the sample portion 8 of the test card being configured to hold a sample for PCR, such as at least one DNA or RNA molecule, the sample portion may be configured to hold reagents for PCR, as well as any necessary liquids and / or buffers.

[0077] For example, it will be understood that the positioning of the emission filter 26 and the collection optics 28 relative to each other, and relative to the test card 6 and the light sensor 22, may differ from that illustrated. The positions of the emission filter 26 and the collection optics 28 may be swapped. The positioning or order from the test card 6 to the light sensor 22 may be test card 6-collection optics 28-emission filter 26-light sensor 22 as illustrated, or test card 6-emission filter 26-collection optics 28-light sensor 22.

[0078] Test card receiver 4 may be any suitable means for receiving and / or holding test card 6. Test card 6 may be received in a fixed position relative to monitoring device 20, or in a fixed position relative to monitoring device 20 and temperature regulator 14.

[0079] The test card receiver 4 may be any suitable space within the system that is large enough to hold the test card 6. For example, the system may include rails or guides, or other suitable means, that are used to allow insertion and retention of the test card 6, so that the sample portion of the test card may be guided and aligned with the surface of the temperature regulator, so that suitable or optimal contact between the two may be achieved. Furthermore, the sample portion may be suitably positioned, for example, relative to the focal plane of the camera, such as where camera imaging is used.

[0080] The test card receiver 4 allows the system 1 to be used with a test card 6. The test card 6 comprises a sample portion, which may be a sample handling portion, such as a microfluidic chip, that allows the sample to be handled and subjected to PCR.

[0081] According to one example, the test card 6 may be, for example, 10-100 mm long, 5-50 mm wide and 0.1-10 mm thick. Such a test card 6 may be efficiently handled during loading of the sample and insertion into the system. The sample portion 8, for example in the form of a chip, may be 15 2 mm 2 Smaller, e.g. 15x15mm 2 Below, 12 x 12 mm 2 For example, 5 x 10 mm 2 It may have major surfaces parallel to the major surfaces of the test card, such as:

[0082] In an alternative embodiment, the monitoring device may comprise at least one optical sensor, an illumination system 24 configured to illuminate the second side of the sample portion with light having a wavelength within at least two or at least one excitation wavelength range, and an emission filter 26 configured to transmit light having a wavelength within at least two or at least one emission wavelength range. Such an illumination system may comprise, for example, two or one light sources.

[0083] In order to improve the understanding of the system and the monitoring device 20, FIG. 2 will now be described. FIG. 2 illustrates a part of the system 1, some of which are omitted to improve the clarity and understanding of the following description. The system 1 may be identical to the system 1 illustrated and described with reference to FIG. 1. In FIG. 2, an illumination system 24, which may comprise at least three light sources, such as light emitting diodes (LEDs), is configured to provide three different excitation wavelength ranges, illustrated using three arrows 30, 32, 34, which are simplified in a schematic manner, and illuminate the second side 12 of the sample portion 8 of the test card 6. For example, the light sources of the illumination system 24 may be directed towards the second side 12 of the sample portion 8. The light sources of the illumination system may be configured to illuminate the sample portions sequentially or simultaneously.

[0084] The collection optics 28 collects light emanating from the second side 12 of the sample portion 8 of the test card 6, and the light (schematically simplified and illustrated using three arrows 30', 32', 34') is transmitted through the emission filter 26. The emission filter 26 can thereby filter out light that is not of interest, such as light that is not associated with a fluorescent reaction of interest associated with a corresponding excitation wavelength. The collection optics 28 directs the collected light towards the image photosensor 22 such that at least a portion of the collected light strikes the photosensor 22. For example, the collection optics 28 may comprise at least three lenses, one for each emission wavelength range, which may direct each emission wavelength range towards a respective portion of the photosensor 22. It should therefore be appreciated that the system 1 is suitable for monitoring PCR occurring within the sample portion 8 of the test card 6. For example, each excitation wavelength range may act to excite fluorophores of a unique DNA / RNA sequence associated with, for example, a unique virus or a unique portion of a virus. The light emitted by each fluorophore may then be filtered and directed towards and sensed by light sensor 22, thereby allowing monitoring of three unique sequences, or PCR of, for example, three different viruses.

[0085] The emission filter 26 configured to transmit light having wavelengths within at least three emission wavelength ranges may for example be realised by an emission filter consisting of or comprising at least three sub-emission filters.

[0086] Collection optics 28 configured to collect light emanating from second side 12 of sample portion 8 of test card 6 and transmitted through emission filter 26 allows the collection optics to be associated with each emission wavelength range individually. Collection optics 28 further configured to direct the collected light towards light sensor 22 such that at least a portion of the collected light strikes light sensor 22 allows each emission wavelength range to be associated with an individual portion of light sensor 22.

[0087] The signal S generated by the light sensor 22 may relate to the intensity of light impinging on the light sensor. Suitable software and / or controllers, which may be integrated with the system 1 or may be external to the system, may be used for handling the sensor, analyzing at least one signal, and / or handling the imaging.

[0088] If the light sensor is an image sensor and the collection optics are imaging optics, the signal may relate to an image of at least a portion of the second side 12 of the sample portion 8. The image may be a colour image or a greyscale image.

[0089] A sample for PCR can be a sample that comprises or consists of at least one nucleic acid molecule, such as DNA or RNA.

[0090] A sample portion configured to hold a sample for PCR can be achieved by a sample portion comprising a channel, such as a microfluidic or capillary channel, or a sample well or compartment capable of holding a sample, the sample may comprise DNA or RNA, or at least a fragment thereof, and optionally any liquids, such as buffers, and reagents, required for PCR, for example. For example, the sample portion 8 may be a chip, such as a silica chip, which may comprise a channel, or a compartment, or a well, or a combination of two or more thereof.

[0091] The RNA of the sample can be converted to cDNA for analysis.

[0092] The at least one optical sensor may be at least one image sensor, and the collection optics may be imaging optics configured to image the sample portion onto the image sensor.

[0093] This allows imaging of the sample portion and the PCR.

[0094] Next, referring to Fig. 3, the system 100 according to an example or embodiment will be described in further detail. The system 100 comprises the components described with reference to Fig. 1, but will be described in further detail as explained below. The temperature regulator 14, the test card receiver 4, and the spring member 18 are omitted from the view of Fig. 3 for clarity, but it should be understood that they may be positioned, for example, as illustrated in Fig. 1. A monitoring device 120 is illustrated, comprising at least one optical sensor 122. The monitoring device 120 further comprises an illumination system 124 comprising a first light source 150, a second light source 152, and a third light source 154, and an emission filter 126 comprising a first emission filter 160, a second emission filter 162, and a third emission filter 164. The light collection optics 128 of the monitoring device 120 comprises a first light collection optics 170, a second light collection optics 172, and a third light collection optics 174.

[0095] The first light source 150 is configured to illuminate the second side 112 of the sample portion 108 through the first excitation light filter 180, thereby providing light having a wavelength within a first excitation wavelength range to the second side 112 of the sample portion 108 of the test card 106. The first emission filter 160 is configured to transmit light having a wavelength within the first emission wavelength range, and the first collection optics 170 is configured to collect light emanating from the second side 112 of the sample portion 108 of the test card 106 and transmitted through the first emission filter 160, the first collection optics 170 being further configured to direct the collected light towards the light sensor 122 such that at least a portion of the collected light strikes the light sensor 122.

[0096] The second light source 152 is configured to illuminate the second side 112 of the sample portion 108 through the second excitation light filter 182, thereby providing light having a wavelength within a second excitation wavelength range to the second side 112 of the sample portion 108 of the test card 106. The second emission filter 162 is configured to transmit light having a wavelength within the second emission wavelength range, and the second collection optics 172 is configured to collect light emanating from the second side 112 of the sample portion 108 of the test card 106 and transmitted through the second emission filter 162, the collection optics further configured to direct the collected light towards the light sensor 122 such that at least a portion of the collected light strikes the light sensor 122.

[0097] The third light source 154 is configured to illuminate the second side 112 of the sample portion 108 through the third excitation light filter 184, thereby providing light having a wavelength within a third excitation wavelength range to the second side 112 of the sample portion 108 of the test card 106. The third emission filter 164 is configured to transmit light having a wavelength within the third emission wavelength range, and the third collection optics 174 is configured to collect light emanating from the second side 112 of the sample portion 108 of the test card 106 and transmitted through the third emission filter 164, and the third collection optics 174 is further configured to direct the collected light towards the light sensor 122 such that at least a portion of the collected light strikes the light sensor 122.

[0098] Such a system can be used to monitor three or more, i.e., a first, second, and third or more, excitation / emission reactions, allowing, for example, monitoring one or more of three different sequences or portions of sequences, or DNA, e.g., one associated with the COVID virus, one associated with a control sample, and one associated with one or more other types of viruses.

[0099] Emission filters such as the first, second and third emission filters, in addition to being configured to transmit light having wavelengths within the first, second and third emission wavelength ranges, respectively, may be further configured to block a portion of light having wavelengths outside the respective wavelength ranges.

[0100] The light sensor 122 may comprise multiple light sensors. For example, there may be a first, second, and third light sensor, one for each of the first, second, and third collection optics. Additionally, the light sensor 122 may be a single light sensor divided into one or more portions corresponding to respective emission wavelength ranges.

[0101] The system 100 may be used to image the sample portion 8 of the test card 6, or a portion of the sample portion 8, and any PCR within the sample portion 8 of the test card 6. In such a system 100, similar to the system 100 described with reference to FIG. 3, the at least one light sensor 122 may be at least one imaging sensor 122, and each of the collection optics 128 may be a respective imaging optic configured to collect light transmitted through the emission filter 126 and image the second side 112 of the sample portion 108 of the test card 106 onto the at least one image sensor 122.

[0102] Thereby, monitoring such as end-point or real-time monitoring of PCR within the sample portion can be achieved. Furthermore, other types of reactions or occurrences within the sample portion can be efficiently tracked and monitored.

[0103] The at least one imaging sensor 122 may be, for example, a CMOS sensor or a CCD sensor.

[0104] The at least one image sensor may be configured to capture at least one image of the second side of the sample portion, each image being captured after a unique number of temperature cycles of the plurality of temperature cycles.

[0105] This shall be understood as each image of the at least one image being captured after a certain number of temperature cycles, which number is unique for each of the at least one image. In other words, for example, if three images, namely a first, second and third image, are captured, each of the three images is captured after a respective number of temperature cycles. For example, the first image may be captured after one temperature cycle, the second image may be captured after ten temperature cycles, and the third image may be captured after twenty temperature cycles. According to another example, one image may be captured after one temperature cycle, another image may be captured after two temperature cycles, and the third image may be captured after three temperature cycles. To provide further examples, an image may be captured after each temperature cycle, every ten temperature cycles, etc.

[0106] For one temperature cycle, at least one image may be captured either sequentially or simultaneously, for example when two or more image sensors are used.

[0107] When the at least one optical sensor is at least one image sensor, each signal of the at least one signal may be an image of the second side of the sample portion.

[0108] Thereby, all or part of the PCR can be monitored or imaged at a desired frequency of images. It may be a decision to monitor at the beginning and end of a PCR having a predetermined number of cycles. It may be that the beginning of the PCR is monitored closely to determine when the copy number is increasing rapidly, or the end of the PCR is monitored to determine when the PCR has terminated and / or a sufficient number of copies have been obtained to allow for the desired analysis.

[0109] The at least one light sensor can be at least one photodiode. Using collection optics, each emission wavelength range can be directed towards a respective portion of the light sensor or image sensor, thus still allowing separate analysis of each emission wavelength range.

[0110] Each of the first light source, the second light source, and the third light source may be an LED.

[0111] This allows narrow bandwidth excitation light to be used with the system. Additionally, LEDs are readily available and require little maintenance.

[0112] Alternatively, each of the first light source, the second light source, and the third light source may be a LASER.

[0113] Additionally, the light source may include both an LED and a LASER.

[0114] The spring member may be configured to urge the sample portion of the test card towards the surface of the temperature regulator with a force of 5-15N, for example 8-12N.

[0115] This allows efficient heat transfer between the sample portion and the temperature regulator while minimizing damage or the risk of damage to the sample portion or test card, particularly when a silicon chip is used as the sample portion.

[0116] According to one embodiment, a silica or glass chip comprises the sample portion 8. The parts of the test card that do not constitute the chip or sample portion 8 may be manufactured from plastic.

[0117] The spring member may contact the sample portion 8 or test card 6 at a side adjacent or overlapping the second side of the sample portion.

[0118] The tension or force between the sample portion or tip of the test card and the temperature regulator affects the thermal transition. The force on the test card can be adjusted using a leaf spring, as described above.

[0119] The leaf springs may be manufactured as necessary to withstand force differences due to manufacturing or assembly processes. For example, the leaf springs may provide a nominal value of 10 N with a force of 0.6 N / mm stored in the leaf spring. The tips of the leaf springs may be bent to prevent deflection of the test card. The bent tips may also allow the test card to slide efficiently over the leaf springs, thus obtaining a pretension when the test card is inserted.

[0120] The leaf springs may be plasma polished to minimize friction and debris from the test card, thereby reducing the risk of fouling the system, such as the optical path.

[0121] Thus, a force may be presented to the sample part or test card in two steps, which may for example follow examples 1) and 2) below. 1) The temperature regulator is in the up position (e.g., by being fastened to the lid of the system housing and the lid being in the open position) and the test card is slid into the test card receptacle, resulting in a deflection of 0.9 mm in the spring, which equates to 5.6 N in the spring. 2) The thermoregulator is in the down position (e.g., by being clipped to the lid of the system housing and the lid is in the closed position). The thermoregulator then pushes the sample part or test card towards the leaf spring, resulting in a deflection of, e.g., 1.6 mm, which is equivalent to 10 N in the leaf spring.

[0122] The contact area between the temperature regulator and the sample part, in this example in the form of a silicon chip, is 5.75 mm × 5 mm, i.e. 28.75 mm 2 It has a size of 2.88N / mm 2 This results in a contact force of

[0123] Thus, the movement of the temperature regulator in contact with the sample part or tip can be limited and the force on the test card can be controlled so that it is not too high, as the leaf spring flexes under pressure.

[0124] The spring member 18 may comprise a leaf spring configured to have a slit or opening or gap to allow transmission of light to and from the second side of the sample portion. FIG. 4 illustrates an example of such a spring member 18, which is a leaf spring, in a portion of the system 1. FIG. 4(A) illustrates a bottom view and FIG. 4(B) illustrates a side view. The test card 6 with the sample portion 8 is clamped between the spring member 18 and the temperature regulator 14. The slit or opening in the spring member 18 is illustrated and indicated by brackets 19. It is clear that the slit or opening 19 allows light to enter, exit, or illuminate the sample portion 8 of the test card 6 from the side of the test card where the spring member 18 is located.

[0125] Thereby, even when the spring member is positioned on or adjacent to the second side of the sample portion, as illustrated, the spring member can be positioned so as not to obscure the sample portion from the sensor system.

[0126] The temperature regulator may include a thermoelectric element. The thermoelectric element may be a Peltier element.

[0127] The temperature regulator may further comprise a heat sink that may be combined with a fan, thereby reducing the cooling time of the sample portion, thereby reducing the overall time of the temperature cycle and PCR. The heat sink may be any heat sink known in the art, such as those that use flanges or other types of protrusions, and optionally a fan.

[0128] Using thermoelectric elements such as Peltier elements, heating and / or cooling can be efficiently achieved and controlled, and multiple successive temperature cycles can be achieved.

[0129] The thermoelectric or Peltier element may have hot and cold sides made of aluminum, which allows for high power throughput and provides good moldability for good thermal contact. When combined with a silicon or glass chip with the sample portion, heating cycles can be rapid since the silicon or glass chip allows for high temperatures.

[0130] One of the first temperature and the second temperature may be in the interval from 60 to 70°C, and the other of the first temperature and the second temperature may be in the interval from 90 to 105°C.

[0131] Thereby, temperature cycles, or denaturation and annealing cycles, suitable for PCR can be achieved. In particular, these temperature ranges are suitable for PCR for COVID analysis.

[0132] Heating cycles do not exclude that other heating and / or cooling steps are used in addition to the heating cycles. For example, for PCR analysis of COVID, etc., a first reverse transcription (RT) step may be performed on the nucleic acids present in the sample at a temperature suitable for reverse transcriptase activity, for example 60-62°C, e.g. 61°C, followed by a heating cycle. This principle is known as RT-PCR.

[0133] For example, prior to temperature cycling, any RNA present in the sample may be converted to DNA, which may be carried out, for example, for a time period of about 1 minute, i.e. within an interval of 30 to 90 seconds.

[0134] In the temperature cycle, the first temperature may be a higher temperature and the second temperature may be a lower temperature, the first higher temperature may correspond to or result in denaturation of the nucleic acid in PCR, and the second lower temperature may correspond to or result in annealing of the nucleic acid in PCR.

[0135] A temperature cycle is described using two temperatures, namely a first temperature and a second temperature. It will be understood that a cycle implies not only going from a first temperature to a second temperature, but also going back to the first temperature. However, it is not excluded that a cycle may be preceded or followed by a non-complete cycle, as long as the temperature regulator of the system regulates the temperature over multiple temperature cycles. For example, PCR may be within the scope of the embodiments herein according to the following examples of thermal regulation: T1-T2-T1-T2-T1 (2 cycles), or T2-T1-T2-T1-T2-T1 (2 cycles preceded by T2), or T1-T2-T1-T2-T1-T2 (2 cycles followed by regulation to a second temperature).

[0136] It is understood that the cycle profile does not have to be symmetrical, but may be, and thus the first and second temperatures do not have to be held for equal lengths of time, but do not exclude that they must be held. For example, the higher temperature can be held for a shorter time than the lower temperature. This can be beneficial, as denaturation can occur more quickly than annealing, and can accelerate cycling time. Alternatively, the higher temperature can be held for a longer time than the lower temperature, or for an equal time.

[0137] The temperature regulator configured to regulate the temperature of the sample portion may be by heating a surface of the temperature regulator to 1-5° C. above the first and second temperatures, respectively. It has been found that a desired temperature within the sample portion of the test card can thereby be rapidly achieved. The control settings of the temperature regulator may be determined, for example, using experiments and measurements incorporating the sample portion and the temperature regulator.

[0138] The temperature regulator may be configured to provide 1-100 temperature cycles, for example 5-60 or 45-50 temperature cycles. Further, the temperature regulator may be configured to provide 5-60, 10-60, 15-60, 20-60, 25-60, 30-60, 40-60, 40-55, 41-54, 42-53, 43-52, 44-51, 45-50 temperature cycles.

[0139] This makes it possible to achieve a suitable number of denaturation and annealing cycles for PCR.

[0140] Each temperature cycle may have a duration of 20 seconds or less, or 15 seconds or less.

[0141] Such temperature cycle durations can be used to achieve PCR in the system according to the first aspect or embodiment.

[0142] Each temperature cycle may have a duration of, for example, 5 to 20 seconds, or 5 to 15 seconds.

[0143] Thus, it may be possible to use the system to provide rapid nucleic acid analysis using PCR.

[0144] The optical sensor may be configured to generate at least one signal per temperature cycle over at least 5 temperature cycles, or 10 temperature cycles, or 45-55 temperature cycles.

[0145] Further, the optical sensor may be configured to generate at least one signal per temperature cycle over at least 1-100 temperature cycles, e.g., 5-60, 10-60, 15-60, 20-60, 25-60, 30-60, 40-60, 40-55, 41-54, 42-53, 43-52, 44-51, 45-50 temperature cycles, etc.

[0146] Thereby, PCR can be monitored over time and real-time PCR is achievable.

[0147] The first excitation wavelength range, the second excitation wavelength range, and the third excitation wavelength range may be different wavelength ranges.

[0148] Thereby, for example, a first, a second and a third sequence or unique DNA may be monitored. Further, for example, three different viruses may be analyzed or three different types of fluorophores may be monitored.

[0149] The first emission wavelength range, the second emission wavelength range, and the third emission wavelength range are different wavelength ranges.

[0150] Thereby, for example, a first, a second and a third sequence or unique DNA may be monitored. Further, for example, three different viruses may be analyzed or three different types of fluorophores may be monitored.

[0151] The first and second sides of the sample portion can be parallel to the major sides of the test card.

[0152] Thus, for example, a flat test card may be used.

[0153] The system may be contained in a housing or the system may comprise a housing. Now, with reference to FIG. 5, a system 500 is described which comprises a housing 590. The housing may comprise all the details of the system as described with reference to FIG. 1 or as described with reference to other embodiments. Thereby, the housing may comprise, for example, a test card receiving portion 504 configured to receive a test card 6 (not shown). The first side 10 of the test card faces upwards in FIG. 5. The system 501 further comprises a temperature regulator 514 which comprises, for example, a Peltier element 514a and a heat sink 514b. The temperature regulator 514 comprises a surface 516 configured to contact the first side 10 of the sample portion 8 of the test card 6.

[0154] The test card receiver 504 comprises a spring member 518 configured to urge the sample portion 8 of the test card 6 towards a surface 516 of the thermal regulator 514. The system 1 further comprises a monitoring device 520 as previously described, for example with reference to FIG. 3. According to this example, the system is provided with an image sensor and the collection optics are imaging optics configured to image the sample portion onto the image sensor. The housing further comprises electronics 599 adapted to send and receive signals and / or control the thermal regulator 514 and the imaging sensor. The electronics 599 is further adapted to receive and provide power within the system.

[0155] A hinge mechanism 594 allows the housing lid 596 to be opened as indicated by arrow 595. The housing 590 in FIG. 5 is illustrated in a closed position. A spring member 518 provides a force urging the sample portion 8 of the test card 6 towards a surface 516 of the temperature regulator 514. The lid 596 further provides a light-tight seal to enhance the sensitivity of the system. The test card receiver 504 is arranged to receive the test card when the housing lid 596 is in an open position. The temperature regulator 514 is provided in a fixed position relative to the lid and the test card receiver is provided in a fixed position relative to a bottom 597 of the housing. The system is thereby arranged such that when the lid is closed, an additional force is provided by the surface 516 of the temperature regulator in addition to the force provided by the spring member 518 urging the sample portion 8 of the test card 6 towards the surface 516 of the temperature regulator 514. In this example, the force from spring member 518 and surface 516 with lid 596 in its closed position is about 9-11 N, or about 10 N. This force has been found to be suitable for test cards 6 having silica chips with sample portions 8. Desired thermal contact was achieved, allowing for rapid temperature cycling and therefore rapid PCR, and the test card was not damaged. Higher temperatures were possible with the silica chips compared to chips made from plastic materials.

[0156] Although not illustrated in FIG. 5, the system may further include a barcode reader used to identify individual test cards and / or a USB hub used for transfer of information from the system.

[0157] With further reference to the above description, a spring member, such as the leaf spring used in the example above with reference to FIG. 5, may be fixedly attached relative to the housing. The spring member or leaf spring provides at least a portion of the force when the test card is received by the system and positioned in contact with the leaf spring. The lid of the housing may be configured to provide additional force to the sample portion when the lid is in a closed position. The surface of the temperature regulator may be considered to be fixedly attached relative to the lid of the housing.

[0158] Other standard and / or useful features added to the system may further include additional features in the test card receiving portion, i.e. features that allow the test card to be introduced into the system at a slight angle of less than 2 degrees upwards and to the side, features that allow for rough and precise alignment across the width of the system in direct relation to the pins in the monitoring device, and features to prevent the test card from hitting the temperature regulator while receiving the test card in the system.

[0159] According to a second aspect, there is provided a method 70 for performing a PCR analysis using the system according to the first aspect. The method 70 is illustrated in Figure 7. The method 70 comprises: receiving S700 a test card in the test card receiving portion, where the test card comprises a sample portion having a first side and a second side opposite the first side, the sample portion of the test card holding a PCR sample; adjusting S702 a temperature of the sample portion of the test card (6) over a plurality of temperature cycles, where each temperature cycle is between a first temperature and a second temperature different from the first temperature, and using the temperature regulator includes contacting a surface with a first side of the sample portion of the test card; the test card receiving portion includes a spring member that urges the sample portion of the test card toward the surface of the temperature regulator; and monitoring the sample portion of the test card over a plurality of temperature cycles S704, wherein using the monitoring device comprises: At least one optical sensor; an illumination system for illuminating a second side of the sample portion with light having a wavelength within at least three excitation wavelength ranges; an emission filter that transmits light having wavelengths within at least three emission wavelength ranges; and collection optics for collecting light emanating from the second side of the sample portion of the test card and passing through the emission filter, the collection optics further directing the collected light towards a light sensor, at least a portion of the collected light impinging on the light sensor; The light sensor generates at least one signal, each signal related to light impinging on the light sensor after a unique number of the plurality of temperature cycles.

[0160] The method 70 may further comprise, prior to receiving the test card S700, loading the test card with a sample for PCR S706.

[0161] According to a third aspect there is provided the use of a system according to any one of the claims of the first aspect for PCR analysis or nucleic acid analysis.

[0162] [Example] Nucleic acid analysis using PCR analysis was performed using a system for imaging detection using three LEDs as light sources. The system was installed in a housing illustrated in FIG. 5. A first light source (red) and corresponding optics and filters were dedicated to one sequence of COVID-19. A second light source (green) and corresponding optics and filters were dedicated to another sequence of COVID-19. A third light source (orange) and corresponding optics and filters were dedicated to a reference sample. A first, second, and third different fluorophore were provided for each sequence. The imaging optics and sensor were used for real-time imaging. The sample was added to the silica chip of the test card.

[0163] Fifty heating cycles, each lasting about 10 seconds, were performed, summing to a total analysis time of about 8 minutes. The temperatures of the cycles were set according to embodiments herein and as illustrated in FIG. 6, which illustrates an example temperature cycle. T is temperature and t is time. In this example, the rate of temperature increase is similar to the rate of temperature decrease, but other profiles of temperature cycles can be realized and used. Thus, the rate of temperature increase can be equal to the rate of temperature decrease, or can be greater or less. In this example, the temperature increase occurs for about 2 seconds and the temperature decrease occurs for about 2 seconds. In this example, the maximum temperature T2 was maintained for about 1 second Δt2, and the minimum temperature T1 was maintained for about 5 seconds Δt1. In this example, T2 is about 100° C., T1 is about 63° C., t1 is 0 seconds, and t2 is about 10 seconds. Images were captured for each of the cycles. Due to the optical system, an image associated with each of the light sources was imaged onto a respective portion of the image sensor.

[0164] Despite the short total analysis time, the presence of COVID-19 in the samples could be successfully detected and quantified. This is believed to be due, at least in part, to the use of relatively high temperatures made possible by a combination of the chip material and the force applied between the chip and the surface of the temperature regulator. In this example, the higher temperatures were held for shorter times than the lower temperatures. This was beneficial and accelerated cycling times, which could be achieved at least in part due to the more rapid denaturation compared to annealing.

Claims

1. A system for PCR analysis (1), A test card receiving section (4) configured to receive a test card (6), wherein the test card (6) comprises a sample portion (8) having a first side surface (10) and a second side surface (12) opposite to the first side surface (10), and the sample portion (8) of the test card (6) is configured to hold a PCR sample. A temperature controller (14) configured to adjust the temperature of the sample portion (8) of the test card (6) over a plurality of temperature cycles, wherein each temperature cycle is between a first temperature and a second temperature different from the first temperature, and the temperature controller (14) has a surface (16) configured to contact the first side surface (10) of the sample portion (8) of the test card (6), The test card receiving section (4) includes a spring member (18) configured to push the sample portion (8) of the test card (6) toward the surface (16) of the temperature controller (14), A monitoring device (20) configured to monitor the sample portion (8) of the test card (6) over the aforementioned multiple temperature cycles, The monitoring device (20) is equipped with, At least one light sensor (22) and An illumination system (24) configured to illuminate the second side surface (12) of the sample portion (8) with light having wavelengths within at least three excitation wavelength ranges, A light-emitting filter (26) configured to transmit light having wavelengths within at least three emission wavelength ranges, A focusing optical system (28) is configured to collect light emitted from the second side surface (12) of the sample portion (8) of the test card (6) and transmitted through the light-emitting filter (26), The focusing optical system (28) is further configured to direct the focused light toward the light sensor (22) such that at least a portion of the focused light strikes the light sensor (22). The system (1) is configured such that the light sensor (22) generates at least one signal (S), each signal (S) relating to light hitting the light sensor (22) after a specific number of temperature cycles among the plurality of temperature cycles.

2. The system according to claim 1, wherein the at least one light sensor is at least one image sensor, and the focusing optical system is an imaging optical system configured to form an image of the sample portion on the image sensor.

3. The monitoring device is The at least one light sensor, A first light source is configured to illuminate the second side surface of the sample portion through a first excitation light filter, thereby providing light having a wavelength within a first excitation wavelength range to the second side surface of the sample portion of the test card, A first light-emitting filter configured to transmit light having wavelengths within a first emission wavelength range, A first focusing optical system is configured to focus light emitted from the second side of the sample portion of the test card and transmitted through the first light-emitting filter, wherein the first focusing optical system is further configured to direct the focused light toward the light sensor such that at least a portion of the focused light strikes the light sensor. A second light source is configured to illuminate the second side surface of the sample portion through a second excitation light filter, thereby providing light having a wavelength within a second excitation wavelength range to the second side surface of the sample portion of the test card, A second light-emitting filter configured to transmit light having wavelengths within a second emission wavelength range, A second focusing optical system is configured to focus light emitted from the second side of the sample portion of the test card and transmitted through the second light-emitting filter, wherein the second focusing optical system is further configured to direct the focused light toward the light sensor such that at least a portion of the focused light strikes the light sensor. A third light source is configured to illuminate the second side of the sample portion through a third excitation light filter, thereby providing light having a wavelength within a third excitation wavelength range to the second side of the sample portion of the test card, A third light-emitting filter configured to transmit light having wavelengths within a third emission wavelength range, A third focusing optical system is configured to focus light emitted from the second side of the sample portion of the test card and transmitted through the third light-emitting filter, wherein the third focusing optical system is further configured to direct the focused light toward the light sensor such that at least a portion of the focused light strikes the light sensor. The system according to claim 1, comprising:

4. The aforementioned at least one light sensor is at least one imaging sensor, Each of the aforementioned focusing optical systems is an imaging optical system configured to focus light transmitted through the light-emitting filter and to image the second side surface of the sample portion of the test card onto at least one image sensor. The system according to claim 3.

5. The system according to claim 4, wherein the at least one image sensor is configured to capture at least one image of the second side of the sample portion, and each image is captured after a specific number of temperature cycles among the plurality of temperature cycles.

6. The system according to claim 3, wherein the at least one light sensor is at least one photodiode.

7. The system according to any one of claims 2 to 6, wherein each of the first light source, the second light source, and the third light source is an LED.

8. The system according to any one of claims 1 to 6, wherein the spring member is configured to press the sample portion of the test card toward the surface of the temperature controller with a force of 5 to 15 N, for example, 8 to 12 N.

9. The system according to any one of claims 1 to 6, wherein the spring member comprises a leaf spring configured to allow light to and from the second side surface of the sample portion by having a slit or opening.

10. The system according to any one of claims 1 to 6, wherein the temperature controller comprises a thermoelectric element such as a Peltier element.

11. The system according to any one of claims 1 to 6, wherein one of the first temperature and the second temperature is within the range of 60 to 70°C, and the other of the first temperature and the second temperature is within the range of 90 to 105°C.

12. The system according to any one of claims 1 to 6, wherein the temperature controller is configured to provide 1 to 100 temperature cycles, for example, 5 to 60 or 45 to 50 temperature cycles.

13. The system according to any one of claims 1 to 6, wherein each temperature cycle has a duration of 20 seconds or less, or 15 seconds or less.

14. The system according to any one of claims 1 to 6, wherein the optical sensor is configured to generate at least one signal for each temperature cycle over at least five temperature cycles, or ten temperature cycles, or forty-five to fifty-five temperature cycles.

15. The system according to any one of claims 3 to 6, wherein the first excitation wavelength range, the second excitation wavelength range, and the third excitation wavelength range are different wavelength ranges.

16. The system according to any one of claims 3 to 6, wherein the first emission wavelength range, the second emission wavelength range, and the third emission wavelength range are different wavelength ranges.

17. The system according to any one of claims 1 to 6, wherein the first and second sides of the sample portion are parallel to the main side of the test card.

18. A method (70) for performing PCR analysis using the system described in any one of claims 1 to 6, The test card is received in the test card receiving section (4) (S700), wherein the test card (6) has a sample portion (8) having a first side surface (10) and a second side surface (12) opposite to the first side surface (10), and the sample portion (8) of the test card (6) holds the PCR sample. Adjusting the temperature of the sample portion (8) of the test card (6) over multiple temperature cycles (S702), where each temperature cycle is between a first temperature and a second temperature different from the first temperature, and using a temperature controller (14) includes the surface (16) being in contact with the first side surface (10) of the sample portion (8) of the test card (6), and the test card receiving portion (4) includes a spring member (18) that pushes the sample portion (8) of the test card (6) toward the surface (16) of the temperature controller (14), Monitoring the sample portion (8) of the test card (6) over the aforementioned multiple temperature cycles (S704), The monitoring device (20) is provided and is used At least one light sensor (22) and An illumination system (24) that illuminates the second side surface (12) of the sample portion (8) with light having wavelengths within at least three excitation wavelength ranges, A light-emitting filter (26) that transmits light having wavelengths within at least three emission wavelength ranges, A focusing optical system (28) that collects light emitted from the second side surface (12) of the sample portion (8) of the test card (6) and transmitted through the light-emitting filter (26), The focusing optical system (28) further directs the focused light toward the light sensor (22), and at least a portion of the focused light strikes the light sensor (22). The method (70) wherein the light sensor (22) generates at least one signal (S), each signal (S) relating to light hitting the light sensor (22) after a specific number of temperature cycles among the plurality of temperature cycles.

19. Use of the system according to any one of claims 1 to 6 for PCR analysis or nucleic acid analysis.

Images