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DE-102024132689-A1 - System for LAMP amplification of DNA and/or RNA

DE102024132689A1DE 102024132689 A1DE102024132689 A1DE 102024132689A1DE-102024132689-A1

Abstract

The invention relates to a system 400 for performing LAMP amplification and for detecting the presence of DNA and/or RNA in a sample. The system comprises the elements of a sample preparation device 200, a reaction vessel 100, and optionally a heating device 300. The system 400 allows for the simple and rapid detection of the presence of DNA and/or RNA in a sample, for example, to detect the presence of a virus in the sample.

Inventors

  • Robin BAYER
  • Reza Esmaillie

Assignees

  • Detechgene GmbH

Dates

Publication Date
20260513
Application Date
20241108

Claims (20)

  1. Sample preparation device (200) for preparing a sample for performing a LAMP analysis to detect the presence of DNA and/or RNA in the sample, comprising: • a sample receiving chamber (202), which preferably includes a lysis buffer; • a purification unit (214); and • an eluate reservoir (212), wherein the purification unit (214) is part of a flask (216), the sample receiving chamber (202) being detachably connectable to the flask (216) so that, upon connection of the sample receiving chamber (202) to the flask (216), pressure can be built up in the flask (216), by which a lysate, preferably formed in the sample receiving chamber (202) by reaction of the sample with the lysis buffer, can be conveyed through the purification unit (214) into the eluate reservoir (212) to form an eluate.
  2. Sample preparation device (200) according to Claim 1 , characterized in that the piston (216) is displaceable relative to the eluate reservoir (212) in an actuation direction (217).
  3. Sample preparation device (200) according to Claim 2 , characterized in that a release unit (232) is provided which then allows a displacement of the piston (216) in the actuation direction (217) towards the eluate reservoir (212) when the release unit (232) has been moved into an unlocking position.
  4. Sample preparation device (200) according to Claim 3 , characterized in that the release unit (232) can be moved into the unlocking position by a rotational movement (244) of the piston (216).
  5. Sample preparation device (200) according to one of the Claims 2 until 4 , characterized in that , during the movement of the piston (216) against the eluate reservoir (212), a cylinder (220) can be slid externally over the eluate reservoir (212).
  6. Sample preparation device (200) according to one of the preceding claims, characterized in that the purification unit (214) comprises a filtration column (208) and a filter which are adaptable to the sample and the DNA and/or RNA to be detected.
  7. Reaction vessel (100) for providing a sample for LAMP analysis to detect the presence of DNA and/or RNA in the sample, comprising an access port (104) for adding the sample, which is present as an eluate, into a fluid channel system (108) connected to the access port (104), which connects the access port (104) to at least two reaction chambers (114), wherein each reaction chamber (114) contains a reaction solution (116) in lyophilized form specific for the DNA and/or RNA to be detected, wherein at least the reaction chambers (114) are closed at a top (120) of the reaction vessel (100) with a second membrane (118) which is permeable to air but not to the eluate.
  8. reaction vessel (100) after Claim 7 , characterized in that the access port (104) is closed by a first membrane (106) which is self-closing.
  9. reaction vessel (100) after Claim 7 or 8 , characterized in that the reaction chambers (114) are conically shaped.
  10. reaction vessel (100) according to one of the Claims 6 until 8 , characterized in that the reaction vessel (100) has a transparent bottom (124) on a bottom side (122) opposite the top side (120).
  11. reaction vessel (100) after Claim 10 , characterized in that the bottom (124) in the area of the reaction chambers (114) has a sight cone (126) in each reaction chamber (114) which tapers conically towards the top (120).
  12. Heating device (300) for heating a reaction vessel for isothermal heating for LAMP analysis to detect the presence of DNA and/or RNA in the sample, in particular a reaction vessel (100) according to one of the Claims 7 until 11 , with at least two reaction chambers (114), comprising a receptacle (304) for the reaction vessel (100), which can be inserted into the receptacle (304) in an insertion direction (305) so that the reaction vessel (100) can be fixed in a defined position, wherein an electrical heating element (308) is formed in the area of the reaction chambers (114) of the reaction vessel (100), which is suitable and intended for heating to and maintaining a predeterminable temperature by establishing an electrical connection with a voltage supply (310), wherein a control device (312) is formed, by which the heating process can be started automatically when the reaction vessel (100) is inserted into the receptacle (304) and can be terminated after a predeterminable time.
  13. Heating device (300) according to Claim 12 , characterized in that the electrical heating element (308) is made of a PTC material, the final temperature of which is appropriate for the process corresponds to the temperature required for LAMP analysis.
  14. Heating device (300) according to one of the Claims 12 until 13 , characterized in that a contact switch (306) is provided which starts the heating process upon contact with the reaction vessel (100).
  15. Heating device (300) according to one of the Claims 12 until 14 , characterized in that an optical status indicator (314) is provided by which the status of the heating process can be visually indicated.
  16. System (400) for performing a LAMP analysis to detect the presence of DNA and/or RNA in the sample, comprising a sample preparation device (200) according to one of the Claims 1 until 6 and a reaction vessel (100) after one of the Claims 7 until 10 .
  17. System (400) according to Claim 16 , further comprising a heating device (300) according to one of the Claims 11 until 15 .
  18. System (400) according to Claim 16 or 17 , characterized in that the access port (104) of the reaction vessel (100) has a centering aid (107) by which, in conjunction with a positioning collar (222) of the sample preparation device (200), a hollow cannula (224) of the sample preparation device (200) connected to the eluate reservoir (212) can be positioned relative to the first membrane (106) of the access port (104).
  19. System (400) according to one of the Claims 16 until 18 , characterized in that the access port (104) of the reaction vessel (100) has a stop (105) by which, when the sample preparation device (200) is placed on the access port (104), the eluate reservoir (212) of the sample preparation device (200) can be deformed to build up pressure in the eluate reservoir (212).
  20. Method for LAMP analysis for the detection of the presence of DNA and/or RNA in a sample using a system (400) according to one of the Claims 16 until 19 , comprising the following steps: • Providing the sample; • Contacting the sample with the lysis buffer in the transfer vessel (202) of the sample preparation device (200); • Building up pressure by which the formed lysate is conveyed through the purification unit (214) into the eluate reservoir (212); • Transferring the eluate under pressure from the sample preparation device (200) through the access port (104) into the reaction vessel (100) so that the eluate enters the reaction chambers (114); • Heating the reaction chambers (114) to a temperature adapted for the LAMP reaction of the DNA or RNA to be tested; and • Maintaining the reaction chambers (114) at the adapted temperature for a duration required for the LAMP reaction to proceed.

Description

Field of invention The invention relates to a system for LAMP amplification of DNA and/or RNA, as well as elements of said system comprising a sample preparation device, a reaction vessel, and a heating device. Furthermore, a method for LAMP analysis is described, with which the presence of a specific DNA and/or RNA in a sample can be detected using the system presented here. Background of the invention For the detection of pathogenic material, such as viruses or bacteria, the so-called LAMP method (loop-terminated isothermal amplification), as described, for example, in Notomi et al., "Loop-mediated isothermal amplification of DNA", Nucleic Acids Research, 2000, Vol. 28, No. 12, No. E63, is known as a variant of isothermal DNA amplification. This method is successfully used, for example, to detect viral infections with the SARS-CoV-2 virus. In the LAMP method, the target sequence to be detected, which can be DNA or RNA, is isothermally amplified in a sample at a constant temperature, usually between 60°C and 85°C, using at least one primer and one polymerase. Typically, four different primers are used to amplify six different regions of the target gene, which increases specificity. The additional use of so-called loop primers can further accelerate the reaction. In most cases, isothermal heating of the sample for 15 to 30 minutes is sufficient. After completion of the isothermal heating, the amplified DNA or RNA can be detected using optical methods, for example, photometric methods or by the naked eye by exploiting a color change upon reaction of the DNA or RNA with another reagent (for example, complementary gold-bound ss-DNA). In the US 10,968,493 B1 A method is described for processing large numbers of samples in parallel. While such methods are helpful in the event of an epidemic, they are not useful for the early detection of viruses or bacteria, or generally in non-epidemic situations, because they require complex equipment and necessitate the collection of numerous samples for parallel processing. Consequently, the time between sample collection from the patient and the availability of the test result is statistically comparatively long. It would therefore be desirable to improve upon these disadvantages. Summary of the invention Based on this, the present invention aims to at least improve the disadvantages known from the prior art and, in particular, to simplify LAMP analysis using equipment and to shorten the time between taking a sample from the patient and obtaining the test result. The task is solved by a sample preparation device for preparing a sample for performing a LAMP analysis to detect the presence of DNA and/or RNA in the sample, comprising • a sample reception chamber, preferably comprising a lysis buffer; • a purification unit; and • an eluate reservoir wherein the purification unit is part of a flask, wherein the sample receiving chamber is detachably connectable to the flask, so that when the sample receiving chamber is connected to the flask a pressure can be built up in the flask, by which a lysate which is preferably formed in the sample receiving chamber by reaction of the sample with the lysis buffer can be conveyed through the purification unit to the eluate reservoir, forming an eluate. In this document, LAMP analysis refers to the analysis of whether a sample contains detectable DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). This analysis is performed using the LAMP method. In the LAMP method, the target sequence, which can be DNA or RNA, is isothermally amplified (multiplied) in a sample at a constant temperature, typically between 60°C and 85°C, using at least one primer and a polymerase. Generally, four different primers are used to amplify six different regions of the target gene, which increases specificity. The additional use of loop primers can further accelerate the reaction. The presence or absence of the target DNA or RNA is detected optically after amplification, preferably by a color change reaction with a reagent specific to the DNA or RNA being detected. Preferably, the sample receiving chamber includes a corresponding lysis buffer through which the sample is lysed to form a lysate. Alternatively, it is also preferred that, instead of providing a lysis buffer within the sample receiving chamber, the lysis is carried out outside the chamber and the resulting lysate is introduced into the chamber. The sample receiving chamber is detachably connected to the piston and, for filling the sample or lysate into the chamber, is detached from the piston and then reconnected to the piston under pressure. The flask contains the purification unit. This unit serves to purify the lysate, which is preferably generated in the sample receiving chamber. Impurities, such as other cell components that arise as unwanted byproducts during lysis, are retained in the purification unit, for example, by size exclusion processes. The sample preparation device is preferably part