EP2694672A1

EP2694672A1 - Method, loc and analysis device for the lysis of cells and pcr amplification

Info

Publication number: EP2694672A1
Application number: EP12704033.5A
Authority: EP
Inventors: Peter Rothacher
Original assignee: Robert Bosch GmbH
Current assignee: Robert Bosch GmbH
Priority date: 2011-04-08
Filing date: 2012-02-08
Publication date: 2014-02-12
Anticipated expiration: 2032-02-08
Also published as: WO2012136400A1; DE102011007035A1; EP2694672B1

Abstract

The invention relates to a LOC (lab-on-a-chip) (10) for the lysis of cells and PCR amplification, having a substrate (11), a fluidic network (12) in the substrate (11), an externally temperature-controlled filter (16) on the substrate (11), an externally temperature-controlled PCR chamber (17) on the substrate (11), and optionally an array (18) on the substrate (11), wherein the filter (16), the PCR chamber (17) and the array (43) are connected to the fluidic network (12). A method according to the invention for the lysis of cells and PCR amplification comprises the method steps of: a) positioning the cells on a filter (16); b) dyeing the cells; c) adding a PCR treatment solution; d) photolysis of the cells; and e) amplifying DNA of the cells by means of PCR; wherein method steps a) and b) as well as c) and d) can occur in any sequence.

Description

Description title

Method, LOC and analyzer for cell lysis and PCR amplification

State of the art

The invention relates to a method, a LOC (Lab-on-a-Chip) and an analysis device for lysing cells and PCR amplification of the DNA of the cells.

In the diagnosis of infectious diseases, the detection of antibiotic resistance is gaining in importance 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. The polymerase chain reaction (PCR, polymerase chain reaction) became mid-80's

Discovered and used extensively in diagnostics, research and forensics for DNA amplification. In this case, a temperature-stable DNA polymerase is added to the DNA to be amplified and a temperature cycle consisting of denaturation, i. Splitting DNA into single strands, at about 95 ° C;

- Primeranlagerung at about 55 ° C; and

Elongation. i.e. 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 is a particular

possess 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 is described in 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.

Disclosure of the invention

Advantages of the invention

With the method according to the invention, the LOC and the analysis device can be compared with the prior art little effort cells, in particular

Lysing bacteria, fungi, viruses, blood cells and cell aggregates in a filter without enzymes. This is possibly also possible while avoiding purification after lysis.

The invention is easily transferred to the current biochemical assay and provides a fully integrated LOC. 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 an LOC as μΤΑβ (Micro Total Analysis System), since the sequence of the processing and amplification steps before detection is simplified compared to the prior art and a transferable into a biochip,

automated simplification of the protocol has been created.

Brief description of the drawings

Fig. 1 shows a schematic representation of an LOC in an analysis apparatus for lysis of cells and PCR amplification, both according to one embodiment of the present invention.

2 shows a schematic representation of an LOC in an analysis device for lysis of cells and PCR amplification, both in each case according to another

Embodiment of the present invention.

3 shows a flowchart of the method for lysis of cells and PCR amplification according to an embodiment of the present invention without

Purification step.

4 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 shows an LOC 10 in a cell lysis and PCR amplification analyzer 30, both in accordance with an embodiment of the present invention for DNA evaluation with an array 18. The LOC 10 has a rigid, flat substrate 1 1 fluidic network 12 in the substrate 1 1 on. 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 a first external temperature control 43 under the substrate 1 1 tempered. The filter 16 fills a flow-through cross section of the

Multifunction chamber 19 fully off. The array chamber 17 is via a second

Temperature control element 44 under the substrate 11 tempered. 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 over 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 can focus the filter 16 and scan in a raster shape according to the beam diameter. 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

Micro-mirror 35, the camera 39, the array illumination lightwave 40, the micromirror 35 and the tempering elements 43 and 44 connected.

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.

5

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 by means of the fluorescence exciting an array illumination light wave 40 and the fluorescent light

observed camera 39, alternatively another (not shown) photometer checked. The evaluation of a DNA analysis is carried out by observing at which positions on an array 18 a fluorescence takes place, with the camera 39. 5 The LOC 10 for lysis of cells and PCR amplification has a common, a

Filter 16 having multi-function chamber 19 for cell accumulation, staining of the cells, cell lysis and PCR.

2 shows an LOC 46 in an analysis device 47 for lysing cells and PCR amplification, both in each case according to a further embodiment of the present invention for DNA evaluation. The elements known from Fig. 1 again have the same reference numerals. In this embodiment, the detection takes place 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 real-time PCR by means of a

5 light source 41 is excited and with a detector 42, the resulting

Fluorescence radiation, possibly at different wavelengths observed. In this embodiment, an array chamber is not required.

FIG. 3 shows a flowchart 50 of the method for lysis of cells and PCR amplification of the DNA of the cells according to an embodiment of the present invention

Invention. The method comprises the method steps:

a) positioning the cells on a filter;

b) staining of the cells;

c) adding a PCR treatment solution;

5 d) photolysis of the cells; and

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 relates to the analysis by means of an array, corresponding to a device such as shown in Fig. 1. In an alternative embodiment without an array, corresponding to a device such as shown for example in FIG. 2, method steps f) and g) and method step e), in this case real-time PCR, are omitted directly in the multifunctional chamber 19 in FIG. 2 instead of. The individual process steps are followed by further explanations, optionally with reference to the analysis device 30 and the LOC 10 from FIG. 1 or 2. In method step a), a cell-containing liquid, usually body fluid such as urine, sputum, is placed on the filter for positioning the cells. Serum, plasma, smears, BAL (bronchoalveolar lavage), etc. via a filter medium, in Figs. 1 and 2 over the silica fiber pad of the filter 16, pumped. The cells are separated and washed with a few 100 μΙ buffer. The silica fiber pad contains glass fibers with diameter d = 0.5 μηι -10 μηι.

With regard to the sequence of process steps a) and b) are two alternative

Embodiments possible, namely the cells before or after application to the

To dye filter 16.

To stain the cells in process step b), a dye solution, about 50 μΙ, is added to the filter and incubated for about 30 s. It is then washed, e.g. first with 200 μΙ ethanol and then with 200 μΙ water. As dyeing agents can be used:

1) Gram stain - crystal violet solution, wash with 200 μΙ water, fix for 2 min with 50 μΙ Lugol's solution (Lugol's Solution, Kl ₃ solution);

2) methylene blue solution;

3) other dyes such as malachite green, safranin, fuchsin (with PAS reaction,

Periodic acid-Schiff reaction), Fuchsinschwefelige acid for mushrooms, brilliant green, methyl green, ethyl green, brilliant blue, Coomassie purple, etc. These dyes have an ammonium group, typically in a quinoid 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 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 leads to

Inactivation of the polymerase, can lead to 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 can be

Buffer system and a salt to increase a salt concentration include. The method according to the invention can be easily transferred both to the filter PCR and to a fully integrated LOC.

Fig. 4 shows a flow chart 60 of the method for lysis of cells and PCR amplification of the DNA of the cells according to an embodiment of the present invention with a purification step. The method comprises the steps of: 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.

Advantageously, the further process step follows

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 the method steps known from the description of FIG. 3, 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

claims

1. A method of lysis of cells and PCR amplification with the method steps a) positioning of the cells in a chamber (19);

b) staining of the cells;

c) adding a PCR treatment solution;

d) photolysis of the cells; and

e) amplification of DNA of the cells by PCR;

wherein process steps a) and b) and c) and d) can each take place in any desired order.

2. The method according to claim 1, characterized by the further process step d1) purification of the DNA of the cells after process step d).

3. The method according to claim 1 or 2, characterized by the other

step

f) Delivery of a hybridization buffer to the amplified DNA.

4. The method according to claim 3, characterized by the further process step g) analysis of the amplified DNA.

5. The method according to any 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. The method according to any one of claims 1 to 5, characterized in that cells, bacteria, especially Gram-positive bacteria, fungi, spores or viruses are used.

7. The method according to any one of claims 1 to 6, characterized in that in step b) the staining of the cells with at least one dye from the amount comprising crystal violet solution, methylene blue solution, and dyes containing at least one ammonium group and / or a Have amino group.

8. The method according to any one of claims 1 to 7, characterized in that in process step c) the treatment solution is a mixture of DNTPs

(deoxynucleotide triphosphate), primers, polymerase and buffer, as well as a substance for blocking the filter surface has.

9. The method according to any one of claims 1 to 8, characterized in that in step d) the photolysis of the cells by means of laser beam, high-energy LEDs, flash lamps or light sources with high energy density.

10. The method according to any one of claims 1 to 9, characterized in that the light used for photolysis is substantially in a wavelength range which is strongly absorbed by the colored cells, but is weakly absorbed by water.

1. LOC (10, 46) for lysis of cells and PCR amplification with a common chamber (19) having a filter for accumulation of cells, staining of cells,

Cell lysis and PCR.

12. LOC according to claim 10, characterized in that the LOC (10) has a further chamber (17) with an array (18).

13. A cell lysis and PCR amplification analyzer for a LOC having a filter (16), the analyzer (30, 47) comprising a photolysis light source (31) for irradiating the filter (16) and a controller (38) Performing the lysis by irradiation of the filter (16) and the PCR amplification.

14. Analysis device according to claim 13, characterized in that the

Analysis device (30, 47) has a receptacle for the LOC (10, 46), so that the filter (16) is arranged at a defined position on the analysis device (30, 47).

15. Analysis device according to claim 13 or 14, characterized in that the analysis device (47) comprises an analysis light source (41) for irradiating the filter (16) and a detector (42) for observing the filter for a real-time PCR ,

16. The method according to any one of claims 1 to 10, characterized in that a wavelength range of a light used for photolysis on the color of the cells is agreed that an absorption coefficient of the colored cells is many times greater than an absorption coefficient of a buffer medium present around the cells.