EP2299985A1 - Réactif de diagnostic contenant des bioparticules, procédé pour sa préparation, et son utilisation en tant qu'étalon interne dans des procédés de préparation et de détection d'acides nucléiques - Google Patents

Réactif de diagnostic contenant des bioparticules, procédé pour sa préparation, et son utilisation en tant qu'étalon interne dans des procédés de préparation et de détection d'acides nucléiques

Info

Publication number
EP2299985A1
EP2299985A1 EP09753787A EP09753787A EP2299985A1 EP 2299985 A1 EP2299985 A1 EP 2299985A1 EP 09753787 A EP09753787 A EP 09753787A EP 09753787 A EP09753787 A EP 09753787A EP 2299985 A1 EP2299985 A1 EP 2299985A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
diagnostic reagent
preparation
pcr
bioparticles
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP09753787A
Other languages
German (de)
English (en)
Inventor
Ralf Himmelreich
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qiagen GmbH
Original Assignee
Qiagen GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qiagen GmbH filed Critical Qiagen GmbH
Priority to EP09753787A priority Critical patent/EP2299985A1/fr
Publication of EP2299985A1 publication Critical patent/EP2299985A1/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2059—Starch, including chemically or physically modified derivatives; Amylose; Amylopectin; Dextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2009—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/2027—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/2031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyethylene oxide, poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2068—Compounds of unknown constitution, e.g. material from plants or animals

Definitions

  • An example of simply feline cells are white blood cells These can be comparatively gently lysed, for example by the enzyme proteinase K in the presence of a detergent (eg sodium dodecyl sulfate or Triton X-1 00) At each Zell ⁇ uf gleich nucleic acids are sheared and fragmented by Doppelstr ⁇ ngbruche in Erbm ⁇ te ⁇ l, However, the fragmentation of genetic information is not so extensive that thereby the desired analyzes were at risk.
  • a detergent eg sodium dodecyl sulfate or Triton X-1 00
  • cells that are difficult to break up are to be lysed, this is done, for example, by adding heat. Then, cells that are difficult to break up are treated for 1 0 minutes at 95 ° C. Typically, many bacteria are lysed with the aid of heat.
  • LoC lab-on-a-ch ⁇ p
  • LoC ie disposable consumables as a laboratory on a chip in the size of a credit card
  • molecular biological determinations For example, the determination of pathogens of an infection, food control, veterinary diagnostics, analysis of biological warfare agents, environmental analysis
  • LoC Only a small amount of personnel and no special training or training is required to operate LoC become a large-scale operator
  • Em LoC combines microfluidic functions for sample extraction and enrichment of the analyte, for signal amplification (amphfication) and detection in one chip.
  • the operation of the LoC and the control unit is very simple and the analysis is extremely fast compared to conventional
  • Fungi, parasites, eukaryotic cells or plant cells or also called spores Fungi, parasites, eukaryotic cells or plant cells or also called spores. It is described as particularly advantageous that both the nucleic acid preparation and the amplification can be checked by direct addition of the cells to the test sample. Genetically modified cells (i.e., those into which the control DNA sequence has been engineered by genetic engineering) or natural cells, such as those of the invention, can be used. For example, spores of B. globigii can be used as control cells. Solid preparations are not disclosed, but suspensions containing the genetically modified spores by introducing the control DNA.
  • the object of the invention is to provide a suitable internal standard for the problems described To provide nuclear acidification and nuclear acid detection methods
  • the dyes are added to the mixture for the preparation of the reagent according to the invention either as solids or in the form
  • gentian violet solution in water
  • fuchsine solution in ethanol
  • eosin solution in water
  • methylene blue solution in 95% ethanol
  • malachite green solution may be added
  • solutions are commercially available, for example from Fluka or Sigma-Ald ⁇ ch
  • the reagent may also contain anti-foaming agents. These are suitably selected from the known anti-foaming agents
  • the reagent according to the invention may also contain particles of a hard material such as glass, silicon carbide or zirconium carbide. Magnetic silica particles or anion exchange resin particles are also suitable. The diameter of these particles is preferably from 1 00 to 800 nm Reagent very difficult to lyse microorganisms, especially certain bacteria such as Corynebacterium glutamicum, Mycobacterium phlei, yeast fungi; or spores of Bacillus anthracls.
  • glass particles with different diameters are used:
  • the preparation of the reagent can be carried out by the ingredients, some of which are also in liquid form, for. B, mixed with a spatula or any mixers and optionally with distilled water until a viscous to viscous homogeneous mass is formed.
  • the microorganisms to be incorporated are added to the other constituents in the form of a bacterial culture fluid or a suspension of microorganisms such as, for example, phage supernatant.
  • the order of addition only matters if the reagent is to additionally contain hard particles too long stirring In this case, the bacteria can already be lysed by grinding effects, so the suspension of microorganisms in this case is added as the very last ingredient and also very carefully mixed with the other ingredients. Otherwise, the order of addition and mixing of the individual components is irrelevant
  • the resulting viscous mass is z B in a syringe überschreibt and with a blunt cannula (z B as small balls (diameter z B 1 -2 mm)) on a suitable film such as parafilm or polyethylene film or Teflonfohe applied in portions and then at
  • microfluidic preparation and detection methods where they can be added to the sample to be prepared by placing them on the microfluidic device.
  • the reagent according to the invention in the microfluidic device such that it can be used the task of the sample to be prepared is suspended in this and then further prepared together with it, ie as part of a LoC or a detection Cart ⁇ dge
  • buffer G GITC (guanidinium thiocyanate) / Nonidet® (nonylphenyl ethylene glycol)
  • GITC guanidinium thiocyanate
  • Nonidet® nonylphenyl ethylene glycol
  • the temperature and time for incubation is suitably selected Since some lysis procedures using enzymes, the selected temperature is also Depending on the activity of the enzymes zymolase, lyticase and lysozyme are usually used in the temperature range of 20 0 C - 37 0 C Incubation with proteases, such as proteinase K or subtilisin, is usually at 50 - 60 0 C The duration The incubation is generally 5 to 20 minutes, preferably about 10 minutes. The incubation may be carried out in one
  • Thermomixer done by Schuttein, z B at about 1 400 rpm, or on a Vortex® device (maximum power level)), optionally with the addition of glass beads, the inventive preparation itself already particles of a hard material and / or enzymes to support may contain the lysis
  • nucleic acids liberated by the lysis are isolated by any purification method known in the art.
  • a suitable solvent can be added to the resulting mixture, for example ethanol.
  • the nucleic acids contained in the lysate are generally purified by adsorption
  • filter elements which are formed, for example, from silica gel and which on the one hand are porous or mat-like in order to allow a liquid to pass through the filter element. and, on the other hand, have a surface to which the biomolecules bind in a specific or nonspecific process.
  • biomolecules on filter elements are simply retained by the effect of large exclusion.
  • a vessel which is open and closed at the top, At the bottom of the vessel is a membrane as a filter element, Below the membrane is an opening in the form of a nozzle, which with, for example, a hose connected is. Liquid can be sucked out of the vessel via this hose
  • wash buffers are added from above into the vessel and aspirated down out of the vessel or centrifuged.
  • the wash buffer maintains this binding while simultaneously washing unwanted cell constituents out of the membrane
  • Membrane acting as a filter A membrane serving as a filter has a certain dead volume of, for example, 40 ⁇ l. It can therefore not be prevented that a corresponding amount of ethanol always remains in the filter. On the one hand, ethanol contributes undesirably to binding. On the other hand, ethanol can also falsify the result of a later analysis.
  • ethanol is first removed, for example, by centrifuging the vessel. The ethanol escapes from the membrane and can be removed downwards. Thereafter, the membrane is sufficiently free of ethanol. By means of vacuum and heat supply, ethanol can alternatively be removed from the membrane. To remove the nucleic acid from the membrane is eluted. This is done regularly with water or a weak saline wass ⁇ gen pH-stabilized solution Following the elution, the nucleic acid can be removed through the membrane from the vessel downwards.
  • Ethanol usually centrifuging again after each washing step to remove the washing buffers.
  • the buffer TE 10 mM Tris / Cl pH 8.0; 1 mM EDTA
  • the primers can be prepared synthetically by methods known to those skilled in the art or are commercially available from the companies Operon or MWG Biotech
  • the tablet was prepared in exactly the same way as tablet C with the exception that 1 ml of page supernatant solution was replaced by 1 ml bidistilled water. After drying, 4 .mu.l of phage supernatant solution (obtained as described above) were pipetted onto each dried tablet and dried again overnight in a fume hood.
  • the results are shown in FIG. 2.
  • the histogram shows the amount of C glutamicum DNA (in pg) detected by the PCR per reaction at a sample quantity of 4.
  • the Ct values in each case were in the RT-PCR Determined by fluorescence spectroscopy.
  • the respective initial concentration of DNA in the respective eluent was determined.
  • the values of the 4 different approaches and representations are within one standard deviation.
  • the amount of C, glutamicum is approximately the same in all samples and approaches. This shows that the inventive
  • Preparations of C, glutamicum were prepared according to the tablet formulation B (Example I b). In three independent determinations, in each case 1 preparation was mixed with 200 ⁇ l whole blood. Following the protocol of Example 2, the nucleic acids were prepared, and in each case with 2 ul eluate a quantitative duplex RT-PCR with the specific for C.
  • the cell numbers used were a) 5 ⁇ 10 5 b) 1 ⁇ 10 6 c) 5 ⁇ 10 4 d) 1 ⁇ 10 4 e) 5 ⁇ 10 3 f) 1 ⁇ 10 3
  • 1 tablet C was mixed with 1 40 .mu.l of plasma and 560 .mu.l of buffer AVL for 2 minutes at 1400 rpm in the Thermomixer until the tablet was dissolved. Then lysed for 10 minutes under normal conditions with the addition of 560 .mu.l of ethanol on a Vortex® device Das Mixture was placed on a QIAamp column twice for binding and centrifuged at 6000 xg for 1 minute.
  • FIG. The figure shows the development of the fluorescence signal as a function of the number of the PCR cycle, 24 individual samples are shown simultaneously, the mean value of the 24 quantitative PCRs is 1.7.8 and the coefficient of variation CV.sub.2, 1%.
  • the example shows that the preparations according to the invention are outstandingly suitable in a reproducible manner for use as internal standard in RNA preparation and subsequent RNA amplification methods.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Inorganic Chemistry (AREA)
  • Botany (AREA)
  • Zoology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention porte sur un réactif de diagnostic se présentant sous la forme d'une composition ayant une stabilité de forme dans les conditions normales, contenant au moins des bioparticules choisies dans le groupe constitué par les bactéries, les virus, les champignons, les protozoaires, les bactériophages, les levures, les spores, les parasites, les cellules végétales, les cellules animales ou humaines, les gamètes, les plasmides et les viroïdes, ainsi que des adjuvants pharmaceutiques usuels.  L'invention porte également sur un procédé pour la préparation de ce réactif de diagnostic, et sur son utilisation en tant qu'étalon interne dans des procédés de préparation et de détection d'acides nucléiques, sur un procédé de détection des bioparticules, ainsi que sur une trousse contenant ce réactif de diagnostic.
EP09753787A 2008-05-27 2009-05-11 Réactif de diagnostic contenant des bioparticules, procédé pour sa préparation, et son utilisation en tant qu'étalon interne dans des procédés de préparation et de détection d'acides nucléiques Pending EP2299985A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP09753787A EP2299985A1 (fr) 2008-05-27 2009-05-11 Réactif de diagnostic contenant des bioparticules, procédé pour sa préparation, et son utilisation en tant qu'étalon interne dans des procédés de préparation et de détection d'acides nucléiques

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP08156939A EP2163239A1 (fr) 2008-05-27 2008-05-27 Produits comprenant des bioparticules, leur procédé de fabrication
EP09753787A EP2299985A1 (fr) 2008-05-27 2009-05-11 Réactif de diagnostic contenant des bioparticules, procédé pour sa préparation, et son utilisation en tant qu'étalon interne dans des procédés de préparation et de détection d'acides nucléiques
PCT/EP2009/055646 WO2009144132A1 (fr) 2008-05-27 2009-05-11 Réactif de diagnostic contenant des bioparticules, procédé pour sa préparation, et son utilisation en tant qu'étalon interne dans des procédés de préparation et de détection d'acides nucléiques

Publications (1)

Publication Number Publication Date
EP2299985A1 true EP2299985A1 (fr) 2011-03-30

Family

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Family Applications (2)

Application Number Title Priority Date Filing Date
EP08156939A Withdrawn EP2163239A1 (fr) 2008-05-27 2008-05-27 Produits comprenant des bioparticules, leur procédé de fabrication
EP09753787A Pending EP2299985A1 (fr) 2008-05-27 2009-05-11 Réactif de diagnostic contenant des bioparticules, procédé pour sa préparation, et son utilisation en tant qu'étalon interne dans des procédés de préparation et de détection d'acides nucléiques

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP08156939A Withdrawn EP2163239A1 (fr) 2008-05-27 2008-05-27 Produits comprenant des bioparticules, leur procédé de fabrication

Country Status (3)

Country Link
US (1) US20110189654A1 (fr)
EP (2) EP2163239A1 (fr)
WO (1) WO2009144132A1 (fr)

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US10260111B1 (en) * 2014-01-20 2019-04-16 Brett Eric Etchebarne Method of detecting sepsis-related microorganisms and detecting antibiotic-resistant sepsis-related microorganisms in a fluid sample
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Also Published As

Publication number Publication date
EP2163239A1 (fr) 2010-03-17
US20110189654A1 (en) 2011-08-04
WO2009144132A1 (fr) 2009-12-03

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