EP2694672B1 - Method for the lysis of cells and pcr amplification - Google Patents

Description

State of the art

The invention relates to a method for the lysis of cells and PCR amplification of the DNA of the cells.

In the diagnosis of infectious diseases, the detection of antibiotic resistance is becoming increasingly important due to the increasing prevalence of these resistances. Methicillin-resistant Staphylococcus aureus (MRSA), pneumonia, sepsis, tuberculosis, and fluoroquinolone resistance in Escherichia coli are some examples. In conventional diagnostics, these resistances are usually determined based on cell culture. The cell culture-based determination of resistance, however, is a relatively lengthy process, since the culture and evaluation 2-3 days must be scheduled. By means of DNA analysis of the bacterial DNA, resistances can be determined significantly faster. However, the DNA of the cells must be amplified, i. be increased.

• Denaturierung, d.h. Aufspaltung DNA in Einzelstränge, bei ca. 95 °C;
• Primeranlagerung bei ca. 55 °C; und
• Elongation. d.h. Synthese der neuen DNA, bei ca. 72 °C;

mehrfach - bis zu 40 x - wiederholt, wobei sich bei jedem Zyklus die DNA-Menge verdoppelt. Für die Verzehnfachung der DNA-Menge sind also im Schnitt 3,3 Zyklen notwendig. Generell wird dazu das DNA-haltige Material vor der PCR aufgereinigt, wobei nach einem Assay-Ablauf-Protokoll vorgegangen wird.The polymerase chain reaction (PCR, polymerase chain reaction) was discovered in the mid-80's and is widely used in DNA amplification in diagnostics, research and forensics. In this case, a temperature-stable DNA polymerase is added to the DNA to be amplified and a temperature cycle consisting of

• Denaturation, ie splitting DNA into single strands, at about 95 ° C;
• Primer attachment at about 55 ° C; and
• Elongation. ie synthesis of the new DNA, at about 72 ° C;

repeatedly - up to 40 x - repeated, with each cycle doubling the amount of DNA. For the tenfold increase in the DNA amount, 3.3 cycles are necessary on average. In general, the DNA-containing material is purified before the PCR, following an assay procedure protocol.

In Gram-negative cells, the PCR works even if cell material is used directly after a prolonged first high-temperature step without purification, as at temperatures near the boiling point the cells are destroyed and DNA is released. However, this does not work with Gram-positive cells, which have a particularly resistant cell membrane. Although Gram-positive bacteria die during this heating, the DNA contained in them can not leave the bacterial envelope and can not be duplicated accordingly.

Various methods are currently used for the lysis of Gram-positive cells, including enzymatic lysis, ultrasound lysis and electroporation. A photolysis with a laser describes the EP 4342914 A1 , There, a cell-containing solution is first placed on a metal oxide carrier, wherein the metal oxide binds cells. Subsequently, the metal oxide is irradiated with the laser, destroying the cell walls and releasing DNA. Subsequently, unbound cell components are washed out in a purification step. These procedures require a lot of effort and handling before the PCR can begin.

EP 1797956 A1 shows a microfluidic device for the concentration and lysis of cells or viruses and an associated method, the microfluidic device comprising: a reaction chamber in which magnetic beads can be accommodated, comprising a plurality of electrodes 20, 20 'for generating a nonuniform electric field, a vibrating member for moving the magnetic beads in the chamber, and a laser source for emitting a laser beam onto the magnetic beads in the reaction chamber.

Dhawan MD et al. "Development of a laser-induced cell lysis system", Analytical and Bioanalytical Chemistry, Vol. 374, No. 3, pp. 421-426 discloses cell lysis based on laser-induced destruction of bacterial and yeast cells. The cells are broken up by the targeted heating of the water in the cell by the use of a 100 mW laser. The wavelengths used ranged from 500 nm to 1550 nm, with optimal wavelengths between 1250 nm and 1550 nm.

US 2003/096429 A1 shows a method for determining laser parameters for cell lysis, exposing the cells from a sub-sample of a sample to the laser light. At least one parameter of the laser is varied and damage to intracellular molecules of the subsamples of the sample becomes at the parameters so different measured. At least one of the parameters is determined based on the measured damage.

Rau Kaustubh et. Al. "Pulsed laser microbeam-induced cell lysis: Time resolved imaging and analysis of hydrodynamic effects"), BIOPHYSICAL JOURNAL, BIOPHYSICAL SOCIETY, US, Vol. 91, No. 1, 1 July 2006 (2006-07-01), pages 317 -329 discloses lysis of cells by disrupting the cell wall by irradiation of the cells by laser to create cavitation bubbles.

T. Baier et al. "Hands-free sample preparation platform for nucleic acid analysis", LAB ON A CHIP, Vol. 9, No. 23, January 1, 2009 (2009-01-01), page 3399 shows a lab-on-chip system for automatic extraction of nucleic acid from human cell samples in a methanol-based solution.

US 2008/171366 A1 discloses a method and apparatus for disrupting cells and amplifying nucleic acids using gold nanorods which are laser irradiated to disrupt the cells.

Disclosure of the invention Advantages of the invention

With the method according to the invention, it is possible to lyse cells, in particular bacteria, fungi, viruses, blood cells and cell aggregates in a filter without enzymes, in comparison to the prior art. This is possibly also possible while avoiding purification after lysis.

The invention can be easily transferred to the current biochemical assay.

The invention also makes it possible to amplify Gram-positive bacteria or other cells which are difficult to lyse, such as fungi or spores, which makes possible a platform with applications for urinary tract infections, MRSA, pneumonia, sepsis, mycoses, etc.

A laser scanner for scanning a sample can be conveniently realized with a micromirror in a very small construction and allows a compact analyzer. The laser lysis can be realized compactly with standardized components for optics, scanners and lasers with a considerably lower outlay than lysis devices according to the prior art.

Compared to enzymatic / chemical lyses, the clear simplification of the assay is crucial.

Opposite Ultraschalllysen is in addition to the effort for the generator simpler design and tuning of the chip crucial.

The invention makes possible a Lab-on-a-Chip (LOC for short) as μTAS (Micro Total Analysis System), since the sequence of the processing and amplification steps before the detection is simplified compared to the prior art and an automatable to be transferred into a biochip Simplification of the Protocol.

Brief description of the drawings

• Fig. 1 shows a schematic representation of an LOC in an analysis device for lysis of cells and PCR amplification.
• Fig. 2 shows a schematic representation of an LOC in an analysis device for lysis of cells and PCR amplification.
• Fig. 3 Figure 12 shows a flow chart of the method for lysis of cells and PCR amplification according to an embodiment of the present invention without purification step.
• Fig. 4 Fig. 10 shows a flow chart of the method for lysis of cells and PCR amplification according to another embodiment of the present invention with purification step.

Embodiments of the invention

Fig. 1 1 shows an LOC 10 in an analysis device 30 for lysis of cells and PCR amplification, both for DNA evaluation with an array 18. The LOC 10 has a rigid, flat substrate 11 with a fluidic network 12 in the substrate 11. To the fluidic network 12
include microchannels 13 shown by way of example, valves 14 and chamber 15, and a multi-function chamber 19 with a filter 16, in which inter alia, a PCR takes place, and an array chamber 17 with the array 18. The filter 16 contains here as a filter medium a silica Pad and is temperature controlled by a first external temperature control element 43 under the substrate 11. The filter 16 fills a flow-through cross-section of the multi-function chamber 19 fully. The array chamber 17 can be tempered via a second tempering element 44 under the substrate 11. Elements of the fluidic network 12 can also run in the interior of the LOC 10 and are liquid-tight at the top with a membrane or film, not shown. The actuators for the operation of the fluidic network 12 and a fluidic interface to the analyzer 30 are not shown.

The analyzer 30 includes a photolysis light source 31 for irradiating the filter 16 and a controller 32 for performing lysis by irradiating the filter. The photolysis light source 31 in this embodiment has a laser 33, a beam expander 34, a micromirror 35 and a lens 36. A light beam 37 leaving the laser 33 passes via the beam expander 34 to the micromirror 35 and is deflected there and continues to pass through the lens 36 to the filter 16. In this embodiment, the micromirror 35 is a controllable micromirror, with which the laser light beam 37 forms the filter 16 focused and raster-shaped according to the beam diameter can sweep. The analyzer 30 further includes a camera 39 and an array illumination lightwave 40, each directed to the array 18.

The controller 32 is connected via electrical control lines 38 to the laser 33, the micromirror 35, the camera 39, the array illumination lightwave 40, the micromirror 35 and the tempering elements 43 and 44.

The analysis device 30 further has a mechanical interface (not shown) with the LOC 10 with which the LOC 10 is positioned in the analysis device 30 in a defined manner. Due to the defined positioning of the filter 16 and the array 18 in the LOC, LOCs are interchangeable in the analyzer 30 without re-optical adjustment. When the LOC 10 is inserted, the photolysis light source 31 is directed to the position of the filter 16 and the camera 39 is directed to the position of the array 18, here in the array chamber 17. When LOC 10 is inserted, the filter 16 is arranged above the first external tempering element 43, and the array chamber 17 is arranged above the second tempering 44. As tempering Peltier elements, micro-hotplates or convective heating / cooling elements, optionally electrical resistors, can also be used in combination. The mechanical interface allows a suitable heat transfer.

In operation, the controller 32 controls the flow of the assay protocol on the LOC 10. The fluidic network 12 is controlled via the fluidic interface. Temperatures and temperature changes are controlled by the temperature control elements 43 and 44. The detection is carried out with an array / biochip and is checked by means of the one fluorescence exciting an array illumination light wave 40 and the fluorescent light observing camera 39, alternatively another (not shown) photometer. The evaluation of a DNA analysis is carried out by the observation at which positions fluorescence takes place on an array 18, with the camera 39.

The LOC 10 for lysis of cells and PCR amplification has a common, filter 16 having a multi-function chamber 19 for cell accumulation, cell staining, cell lysis and PCR.

Fig. 2 Figure 4 shows an LOC 46 in an analysis device 47 for cell lysis and PCR amplification, both for DNA evaluation. From Fig. 1 known elements again have the same reference numerals. In this embodiment, the detection by means of real-time PCR in the same chamber as the lysis, in the multi-function chamber 19. For this purpose, the fluorescent dyes used in the real-time PCR are excited by a light source 41 and a detector 42, the resulting fluorescence radiation , possibly at different wavelengths, observed. In this embodiment, an array chamber is not required.

1. a) Positionierung der Zellen auf einem Filter;
2. b) Färbung der Zellen;
3. c) Zugeben einer PCR-Behandlungs-Lösung;
4. d) Photolyse der Zellen; und
5. e) Amplifikation von DNA der Zellen mittels PCR.

Fig. 3 Figure 12 shows a flow chart 50 of the method for lysing cells and PCR amplifying the DNA of the cells according to an embodiment of the present invention. The method comprises the method steps:

1. a) positioning the cells on a filter;
2. b) staining of the cells;
3. c) adding a PCR treatment solution;
4. d) photolysis of the cells; and
5. e) Amplification of DNA of the cells by PCR.

Thus, the DNA of the cells is present in sufficient quantity for DNA examinations. The method is suitable for Gram-negative and Gram-positive cells.

Advantageously, the further process step follows

• f) Mix the amplified DNA with a hybridization buffer. This serves as preparation for the further process step
• g) Analysis of the amplified DNA. This concerns the analysis by means of an array, corresponding to a device such as in Fig. 1 shown.

In an alternative embodiment without array, corresponding to a device such as in Fig. 2 shown, omitted process steps f) and g) and process step e), in this case real-time PCR, takes place directly in the multi-function chamber 19 in Fig. 2 instead of.

For the individual method steps, further explanations, optionally with reference to the analysis device 30 and the LOC 10, follow Fig. 1 or 2 , In method step a), a cell-containing liquid, usually body fluid such as urine, sputum, serum, plasma, smears, BAL (bronchoalveolar lavage), etc. via a filter medium, in order to position the cells on the filter Fig. 1 and 2 over the filter 16 silica fiber pad, pumped. The cells are separated and washed with a few 100 ul buffer. The silica fiber pad contains glass fibers with diameter d = 0.5 μm -10 μm.

With regard to the sequence of process steps a) and b), two alternative embodiments are possible, namely to dye the cells before or after application to the filter 16.

1. 1) Gram-Färbung - Kristallviolett-Lösung, waschen mit 200 µl Wasser, 2 min fixieren mit 50 µl Lugolscher Lösung (Lugol's solution, Kl₃-Lösung);
2. 2) Methylenblau-Lösung;
3. 3) weitere Farbstoffe wie Malachitgrün, Safranin, Fuchsin (mit PAS-Reaktion, Periodic acid-Schiff reaction), Fuchsinschwefelige Säure für Pilze, Brillant-Grün, Methylgrün, Ethyl-Grün, Brillant-blau, Coomassie violett, etc.. Diese Farbstoffe weisen eine Ammoniumgruppe auf, typischerweise in einem Chinoiden System mit weiteren Alkyl-/Aryl-aminogruppen. Viele gehören zur Klasse der Triphenylmethan-Fabstoffe. Die Farbstoffe können auch als Feststoffe in Depots auf dem LOC 10, z.B. lyophilisiert, gelagert werden und mit Wasser in den Filter 16 geschwemmt werden. Die gefärbten Bakterien lassen sich mit Licht mit vergleichsweise geringen Intensitäten lysieren.

For staining the cells in method step b), a dye solution, about 50 μl, is added to the filter and incubated for about 30 s. Then it is washed, eg first with 200 .mu.l of ethanol and then with 200 .mu.l of water. As dyeing agents can be used:

1. 1) Gram stain - crystal violet solution, wash with 200 μl of water, fix for 2 minutes with 50 μl of Lugol's solution (Lugol's solution, Kl ₃ solution);
2. 2) methylene blue solution;
3. 3) other dyes such as malachite green, saffron, fuchsin (with PAS reaction, periodic acid-Schiff reaction), fuchsinschwefelige acid for mushrooms, brilliant green, methyl green, Ethyl Green, Brilliant Blue, Coomassie Violet, etc. These dyes have an ammonium group, typically in a quinonoid system with other alkyl / arylamino groups. Many belong to the class of triphenylmethane dyes. The dyes can also be stored as solids in depots on the LOC 10, for example lyophilized, and washed with water into the filter 16. The stained bacteria can be lysed with light of comparatively low intensities.

In method step c), the filter is filled with the PCR treatment solution, the so-called master mix. This PCR treatment solution consists of a mixture of DNTPs (deoxynucleotide triphosphates), primers, polymerase and buffer. Further, in the mixture, a substance for blocking the filter surface is contained, e.g. BSA (Bovine serum albumin), PEG (Polyethylene glycol), PPG (Polypropylene glycol). It is also possible to add the substances required only in process step e) only after process step d).

The photolysis of the cells in process step d) is preferably carried out by means of laser lysis. During photolysis, the DNA of the cells is released. The filter on which the stained cells are located is irradiated with a commercially available laser. In this case, the laser beam is preferably focused and guided with a micromirror on the filter, wherein as far as possible the entire filter surface is swept over completely with the laser beam. Alternatively, the filter can also be irradiated with high-energy LEDs, flash lamps or focused light sources with high energy density. The decisive factor is that the bacteria absorb as much light as possible, whereas the matrix and the surrounding liquid should absorb as little energy as possible. This is achieved by having the light used for photolysis substantially in a wavelength range which is strongly absorbed by the colored cells but weakly absorbed by water. The color of the stained cells is determined by the dye, but may be different from the color of the dye. The wavelength or wavelengths of the photolysis light are matched to the color of the stained cells, so that the absorption coefficient of the stained cell is a multiple of the absorption coefficient of a fluid or a stationary phase of a buffer medium present around the cells, usually water. A multiple may be, for example, at least the factor 2.5, for example 10. A high energy intake of the fluid or the stationary phase would result in a strong, poorly controlled increase in temperature, which may lead to inactivation of the polymerase, evaporation of the liquid medium and bursting of the LOCs. The photolysis light source advantageously has a Wavelength in the visible region which is complementary to the color of the stained cells is, for example, 532 nm in the case of red staining of the cells with methylene blue, so that the absorption by the stained bacteria is as high as possible.

Between the process steps d) and e) no purification takes place in this embodiment. The process steps a) to d) all take place in the filter 16. Subsequently, the content of the filter 16 is pumped into the array chamber 17, in which then the hybridization and detection takes place.

During the PCR amplification of the DNA in method step e), the filter is subjected to the usual thermal cycles for the PCR. At these temperature cycles, the DNA is amplified as described above. Now the DNA of the cells is in sufficient quantity for DNA examinations. The method is suitable for Gram-negative and Gram-positive cells. Subsequently, the DNA can be eluted from the filter element. Thus, for example, from 10 ml of urine with 10 ⁵ cells after 20 cycles, 10 ¹¹ DNA molecules are isolated in 50 μl. Alternatively, other amplifications, including isotherms, are also possible, for example NASBA (Nucleic Acid Sequence Based Amplification).

After adding a so-called hybridization buffer in method step f) and applying it to the DNA array 18 by means of the fluidic network 12, the DNA for the detection or analysis of the amplified DNA in method step g), by means of a DNA array, for example array 18 in Fig. 1 , prepared. The hybridization buffer may comprise a buffer system and a salt to increase a salt concentration.

The method according to the invention can be easily transferred both to the filter PCR and to a fully integrated LOC.

• a) Positionierung der Zellen auf einem Filter;
• b) Färbung der Zellen;
• d) Photolyse der Zellen;
• d1) Reinigung von DNA der Zellen;
• c) Zugeben einer PCR-Behandlungs-Lösung; und
• e) Amplifikation der DNA der Zellen mittels PCR.

Fig. 4 Figure 12 shows a flow diagram 60 of the method for lysing cells and PCR amplifying the DNA of the cells according to an embodiment of the present invention with a purification step. The method comprises the method steps:

• a) positioning the cells on a filter;
• b) staining of the cells;
• d) photolysis of the cells;
• d1) purification of DNA of the cells;
• c) adding a PCR treatment solution; and
• e) Amplification of the DNA of the cells by means of PCR.

Thus, the DNA of the cells is present in sufficient quantity for DNA examinations. The method is suitable for Gram-negative and Gram-positive cells.

• f) Mischung der amplifizierten DNA mit einem Hybridisation Buffer. Dies dient der Vorbereitung für den weiteren Verfahrenschritt
• g) Analyse der amplifizierten DNA. Die Analyse umfasst die Hybridisierung und die Detektion.

Advantageously, the further process step follows

• f) Mix the amplified DNA with a hybridization buffer. This serves as preparation for the further process step
• g) Analysis of the amplified DNA. The analysis includes hybridization and detection.

In this embodiment of the present invention, the method steps of the same name correspond to those of the description of FIG Fig. 3 known method steps, taking into account the following differences. After the photolysis of the cells in process step d), the DNA of the cells is then purified in process step d1). This purification can be accomplished by one or more washes in which cell components other than DNA are removed. Because of the liquid exchange associated with the cleaning, the PCR treatment solution is added only after the purification - process step c) thus takes place after process step d1).

Claims (9)

1. Method for the lysis of cells and PCR amplification, having the following method steps

   a) positioning the cells in a chamber (19);

   b) staining the cells;

   c) adding a PCR treatment solution;

   d) photolysis of the cells; and

   e) amplification of DNA from the cells by means of PCR;

   wherein method steps a) and b) and also c) and d) can in each case take place in any desired order and the light used for the photolysis is substantially in a wavelength range which is strongly absorbed by the stained cells, but is poorly absorbed by water, characterized in that in method step b) the cells are stained with at least one stain from the group consisting of crystal violet solution and methylene blue solution.
2. Method according to Claim 1, characterized by the further method step d1) purifying the DNA from the cells after method step d).
3. Method according to Claim 1 or 2, characterized by the further method step

   f) supplying a hybridization buffer to the amplified DNA.
4. Method according to Claim 3, characterized by the further method step

   g) analysing the amplified DNA.
5. Method according to one of Claims 1 to 4, characterized in that the method steps a) to d) take place in the same volume element of an analysis chip.
6. Method according to one of Claims 1 to 5, characterized in that cells, bacteria, especially Gram-positive bacteria, fungi, spores or viruses are used.
7. Method according to one of Claims 1 to 6, characterized in that in method step c) the treatment solution has a mixture of dNTPs (deoxynucleotide triphosphates), primers, polymerases and buffer, and also a substance for blocking the filter surface.
8. Method according to one of Claims 1 to 7, characterized in that in method step d) the photolysis of the cells is carried out by means of laser beam, high-energy LEDs, flash lamps or light sources with a high energy density.
9. Method according to one of Claims 1 to 8, characterized in that a wavelength range of a light used for the photolysis is adapted to the staining of the cells such that an absorption coefficient of the stained cells is greater by a multiple than an absorption coefficient of a buffer medium present around the cells.

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