Search

EP-4739789-A1 - SAMPLE LYSIS PROCEDURE FOR DIRECT PCR MOLECULAR DIAGNOSTIC

EP4739789A1EP 4739789 A1EP4739789 A1EP 4739789A1EP-4739789-A1

Abstract

A method for the preparation of a nucleic acid sample, suspected of comprising pathogenic nucleic acids, in particular fungal nucleic acids, suitable for PCR experiments from a sample characterized in that the method applies a combination of bead-free mechanical, chemical, thermal and/or enzymatic conditions.

Inventors

  • VAN ACKER, Koen
  • MESNIERES, Marion
  • SPEDALE, Gianpiero

Assignees

  • miDiagnostics NV

Dates

Publication Date
20260513
Application Date
20240705

Claims (17)

  1. 1. A method for the preparation of a nucleic acid sample, suspected of comprising pathogenic nucleic acids, characterized in that the method comprises the following steps: - providing a sample suspected of comprising pathogenic nucleic acids; - applying a chemical treatment to said sample; wherein the chemical treatment comprises a treatment with a composition comprising Tetrabutylammonium hydroxide 30-hydrate; - applying a bead-free mechanical treatment to said sample; - applying a thermal treatment to said sample, to obtain a sample lysate; and - subjecting said sample lysate to a PCR reaction allowing nucleic acid amplification from the sample lysate.
  2. 2. The method according to claim 1 wherein the sample lysate is one or more of the following: - an extraction-free sample lysate; - a purification-free crude sample lysate; and/or - a bead-free sample lysate.
  3. 3. The method according to any of the previous claims wherein the steps of chemical treatment and bead-free mechanical treatment are performed within a time period taking no longer than 5 minutes or 30 seconds.
  4. 4. The method according to any of the previous claims wherein the bead-free mechanical treatment is selected from squeezing or shearing the sample, preferably squeezing the sample.
  5. 5. The method according to any of the previous claims wherein the bead- free mechanical treatment is performed at the same time as or after the chemical treatment.
  6. 6. The method according to any of the previous claims wherein the thermal treatment is performed after the chemical treatment and/or the bead-free mechanical treatment.
  7. 7. The method according to any of the previous claims wherein said sample suspected of comprising pathogenic nucleic acids is a vaginal discharge sample; in particular a vaginal discharge sample suspected of comprising fungal nucleic acids.
  8. 8. The method according to any of the previous claims wherein the chemical treatment comprises a treatment with a composition further comprising any of components Sodium Dodecyl Sulfate, GenapolX-80, Digitonin, Tergitol, Saponin, l,5-Diazabicyclo[4.3.0]non-5- ene, 1,1,3,3-Tetramethylguanadin, l,5,7-Triazabicyclo[4.4.0]dec-5-ene, Phospazene base Pl- t-Bu-tris(tetramethylene), Cinnamaldehyde, Ocimene, Camphene, 1 -Butyl- 1- methylpyrrolidinium chloride, Graphene Oxide nanocolloids, Lithium Acetate, Octenidine, Genapol X-100, Curcumin, Tetrabutylphosphonium hydroxide, and polyacrylic acid (PAA) ; or any combination thereof; in particular a composition comprising a combination of components chosen from the combinations - 1,1,3,3-Tetramethylguanadin, SDS and Ocimene; - Phospazene base Pl-t-Bu-tris(tetramethylene) and SDS; - Tetrabutylammonium hydroxide 30-hydrate and SDS - Tetrabutylammonium hydroxide 30-hydrate and polyacrylic acid (PAA), and - Tetrabutylammonium hydroxide 30-hydrate, poly aery lie acid (PAA) and SDS.
  9. 9. The method according to any of the previous claims wherein the chemical treatment comprises a treatment with a composition further comprising one or more buffering agents, selected from the list comprising: tripotassium phosphate (K3PO4), 4-(cyclohexylamino)-l- butanesulfonic acid (CABS), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) ortetrabutylammonium chloride (TBAC1)).
  10. 10. The method according to any of the previous claims wherein the method further comprises enzymatic treatment.
  11. 11. The method according to claim 10, wherein said enzymatic treatment comprises a treatment with a composition comprising any of components Lyticase, Zymolyase, Chitinase and/or (Hemi)Cellulase.
  12. 12. The method according to any one of claims 1 to 11 wherein the thermal treatment is a heat treatment and the heat treatment and PCR reaction are performed in two separate instruments or in one and the same instrument.
  13. 13. The method according to claim 12 wherein the one and the same instrument is the only electrical appliance used in the method.
  14. 14. A composition for producing a bead-free sample lysate as defined in any of claims 2 to 12, which composition comprises Tetrabutylammonium hydroxide 30-hydrate any optionally one or more components selected from the list comprising Sodium Dodecyl Sulfate, GenapolX-80, Digitonin, Tergitol, Saponin, l,5-Diazabicyclo[4.3.0]non-5-ene, 1,1,3,3-Tetramethylguanadin, l,5,7-Triazabicyclo[4.4.0]dec-5-ene, Phospazene base Pl-t-Bu-tris(tetramethylene), Cinnamaldehyde, Ocimene, Camphene, 1 -Butyl- 1-methylpyrrolidinium chloride, Graphene Oxide nanocolloids, Lithium Acetate, Octenidine, Genapol X-100, Curcumin, Tetrabutylphosphonium hydroxide and poly acrylic acid (PAA); or any combination thereof; and wherein said bead-free lysate is directly transportable through a microfluidic assembly for DL-PCR.
  15. 15. The composition according to claim 14, wherein the composition further comprises any of the components Lyticase, Zymolase, Chitinase and/or (Hemi)Cellulase.
  16. 16. The composition according to claim 14 or 15, further comprising one or more buffering agents, selected from the list comprising: tripotassium phosphate (K3PO4), 4- (cyclohexylamino)-l -butanesulfonic acid (CABS), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) ortetrabutylammonium chloride (TBAC1)).
  17. 17. Use of a method according to claims 1 to 13 or a composition according to claims 14-16 for the detection of pathogenic nucleic acids in a sample obtained from a subject suspected of having a pathogenic infection; in particular from a subject suspected of having a fungal infection.

Description

SAMPLE LYSIS PROCEDURE FOR DIRECT PCR MOLECULAR DIAGNOSTIC TECHNICAL FIELD The field of the invention generally relates to a fast multi-conditional sample preparation for nucleic-acid based molecular diagnostic testing. In particular, compositions and a combination of mechanical, chemical, thermal and/or enzymatic conditions are provided for fast lysis of pathogens comprised in a sample, thereby allowing amplification of the lysate, preferably by a protocol using thermocycling. In particular, the present invention provides a method for the preparation of a nucleic acid sample, suspected of comprising pathogenic nucleic acids, in particular fungal nucleic acids, suitable for PCR experiments from a sample characterized in that the method applies a combination of bead- free mechanical, chemical, thermal and/or enzymatic conditions, and comprises the following steps: applying a chemical treatment condition to a sample, suspected of comprising pathogenic nucleic acids, in particular fungal nucleic acids; - applying a bead-free mechanical treatment condition to said sample - incubating said sample with an enzyme lysis composition; to obtain a sample lysate - subjecting the sample lysate to a PCR reaction allowing nucleic acid amplification from the sample lysate. Alternatively, the method of the invention comprises the following steps: - applying a chemical treatment to a sample, suspected of comprising pathogenic nucleic acids, in particular fungal nucleic acids; - applying a bead-free mechanical treatment to said sample; - applying a thermal treatment to said sample to obtain a sample lysates; and - subjecting the sample lysate to a PCR reaction allowing nucleic acid amplification from the sample lysate. In particular, wherein the chemical treatment comprises a treatment with a composition comprising Tetrabutylammonium hydroxide 30-hydrate. BACKGROUND Diagnostic technologies have improved and expanded substantially over the last several decades. Foremost, most advanced diagnostic laboratory technologies are centralized, and require highly trained staff and specialized facilities. With the continuous advancement of molecular biology and biosensor technology, new clinical diagnostic tools that can function in settings with limited access to a central laboratory are emerging. Medical testing that is performed outside of a laboratory setting is known as point-of-care (PoC) testing. The driving notion behind point of care testing (PoCT) is to bring the test conveniently and immediately to or near the point-of-need and receive instant results near the patient. This allows for better immediate clinical management decisions and timely medical intervention. Current existing PoC diagnosis tests can be categorized as immunological PoCT, biomarkerbased PoCT, and nucleic-acid-based PoCT. Devices for these PoCT come in various forms. They may use, for instance, rapid dipsticks as with urinalysis, small handheld instruments like glucose meters, and compact benchtop analyzers to detect diseases or infections (Price CP. Point of care testing. BMJ. 2001 May 26;322(7297): 1285-8. doi: 10.1136/bmj.322.7297.1285. PMID: 11375233; PMCID: PMC1120384.). Compact benchtop analyzers are smaller versions of the laboratory analyzers and have some or all workflow steps automated. One category uses cartridges or cassettes that operate in conjunction with the bench-top analyzer. More recently, microfluidic assemblies and compatible herewith instruments for performing on a miniaturized scale with low reagent consumption requirement and shorter sample-to-result turnaround times provide efficient tools for performing nucleic-acid-based testing (NAT) (D. Bos el al. Prospective Performance Evaluation of the miDiagnostics COVID- 19 PCR Test for Rapid SARS-CoV-2 Detection on Nasopharyngeal Swabs. J Clin Microbiology 2023. https://doi.org/10.1128/jcm.01871-22). The general workflow for NAT generally consists of three steps: sample preparation, in which nucleic acid is released in and extracted from the testing sample; amplification, in which the extracted target nucleic acid is amplified via various methods; and detection, in which the amplified product is qualitatively or quantitatively analyzed. The fundamental assurance for successful nucleic acid amplification is the quality and efficiency of the sample preparation. Several approaches to lysis of pathogens and cells have been developed, including chemical, enzymatic, mechanical, sonication, thermal, electrical, and focused radiation methods. As a next step to lysis, amplification inhibition is minimized by further purifying and/or extracting the nucleic acid from the lysed sample and its inhibitory substances. Nucleic acid of interest can be isolated using a variety of different methods, including those based on solution-based chemistry, such as alcohol precipitation; silica-binding chemistry; cellulose-binding chemistry; or ion exchange chemistry (Ali N el al. Current Nucleic Acid Extraction