Method for differentiating between living and dead cells

Abstract

The present invention relates to a method for quantitatively determining living and dead cells in a biological sample. The method according to the present invention is based on the determination of the amount of DNA in the sample with the aid of a DNA amplification reaction which does not impair the membrane integrity of living cells.

Claims

1. A method for quantitatively determining the number of living and dead cells in a biological sample, comprising the steps of: (a) providing an untreated aliquot of the sample, (b) providing an aliquot of the sample, wherein all cells have been inactivated, resulting in a complete loss of integrity of the cell membranes, (c) carrying out separate DNA amplification reactions with the aliquots provided in steps (a) and (b), respectively, wherein the DNA amplification reactions do not impair the membrane integrity of living cells, (d) determining the respective DNA amount in the aliquots by the DNA amplification reactions carried out in step (c), and (e) determining the number of living and dead cells in the biological sample from the amounts of DNA determined in step (d).

2. The method according to claim 1, wherein the DNA amplification reactions are isothermal amplification reactions.

3. The method according to claim 1, wherein the DNA amplification reactions are isothermal or non-isothermal amplification reactions, which run at temperatures below 50° C.

4. The method according to claim 3, wherein the DNA amplification reactions run at a temperature between 36 and 42° C.

5. The method according to claim 1, wherein the DNA amplification reactions are recombinase polymerase amplification reactions.

6. The method according to claim 1, wherein in the inactivation of the cells of the aliquot provided in step (b) the DNA of these cells is extracted, and wherein in step (c) said DNA is used in the corresponding DNA amplification reaction.

7. The method according to claim 1, wherein the determination of the respective amount of DNA in step (d) is carried out using fluorescence-labeled probes or DNA-binding fluorescent dyes.

8. The method according to claim 7, wherein the determination of the respective amount of DNA in step (d) is carried out using a fluorometer.

9. The method according to claim 8, wherein the fluorometer is a real-time fluorometer.

10. The method according to claim 1, wherein the determination of the respective amount of DNA in step (d) is carried out using gel electrophoresis and a subsequent comparison of the band intensities.

11. The method according to claim 1, wherein the determination of the number of living and dead cells in the biological sample in step (e) is carried out with the aid of a standard curve, which is obtained by DNA amplification reactions with parallel reaction batches containing known DNA concentrations.

12. The method according to claim 1, wherein the cells are prokaryotic cells.

Description

(1) The figures show:

(2) FIG. 1: Target amplicon at 430 bp on agarose gel after completed RPA reaction.

(3) FIG. 2: Standard curve using quantified E. coli DNA.

(4) FIG. 3: Fluorescence curves of samples 1 and 2 as well as negative controls.

(5) FIG. 4: Fluorescence curves of samples 3 and 4 as well as negative controls.

(6) FIG. 5: Standard curve using quantified E. coli DNA.

(7) FIG. 6: Fluorescence curves of vital and inactivated E. coli cells as well as negative controls.

(8) The present invention is illustrated in further detail by the following non-limiting examples.

EXAMPLE 1

(9) A reaction batch according to the present invention comprises, for example, the following components:

(10) 200 μl reaction vessel with at least one recombinase, an SSB (single-stranded DNA-binding protein), a DNA polymerase, a “crowding agent” (e.g. polyethylene glycol), a buffer, a reductant, ATP or an ATP analogon, optionally a “recombinase loading protein”, one or more primer(s), a target DNA, as well as magnesium ions.

(11) The RPA kit “TwistAmp fpg” of the company TwistDx has been used, wherein a part of said components is already provided as a freeze-dried pellet in 200 μl reaction vessels. The user merely has to add buffer solution, the desired primer, the sample and magnesium acetate and optionally a detection probe, in order to complete the reaction batch.

(12) In example 1, a 50 μl reaction batch has the following composition:

(13) Lyophilized pellet of the RPA kit “TwistAmp fpg”

(14) 29.5 μl buffer of the RPA kit “TwistAmp fpg”

(15) 420 nM primer having the sequence:

(16) TABLE-US-00001 (SEQ ID NO: 1) 5′-CAGGATTAGATACCCTGGTAGTCCACGCCGTAAAC-3′
420 nM primer having the sequence:

(17) TABLE-US-00002 (SEQ ID NO: 2) 5′-TAAGGGGCATGATGATTTGACGTCATCCCCACCTT-3′
14.2 μl sample
12 mM magnesium acetate

(18) An RPA reaction begins only after adding magnesium acetate, since magnesium ions are essential for the activity of the DNA polymerase.

(19) In order to guarantee exactly the same start time for samples running in parallel, magnesium acetate is pipetted in the lid of the reaction vessels, such that the magnesium acetate can be simultaneously introduced in all samples by a brief centrifugation and thus all reactions start in parallel. A so-called “magnesium start” is necessary, since RPA reactions already run at room temperature, although with low efficiency.

(20) In example 1 a Bacillus subtilis cultivation (in exponential phase) and an Escherichia coli cultivation (in exponential phase) were diluted 1:100 and each was directly used in an RPA reaction with the above-described reaction batch composition at 37° C. After a reaction time of 30 min the complete reaction batches (50 μl each) were plated on nutrient agar and incubated at 37° C. Both E. coli and B. subtilis formed a bacterial lawn within 24 hours.

(21) Thus, according to example 1 it could be shown that also after the isothermal amplification both B. subtilis cells (gram positive) and E. coli cells (gram negative) are still vital and no inactivating substances or reactions are present during the amplification.

EXAMPLE 2

(22) According to an embodiment of the present invention the band thicknesses after RPA and gel electrophoresis are evaluated in a qualitative manner.

(23) As a sample material, the gram-negative bacterium E. coli both in the exponential phase and also after complete inactivation by ultrasound was used. As an example of a gram-positive species, B. subtilis was used in the stationary phase and after complete inactivation.

(24) The experimental conditions were selected as follows:

(25) 30 min amplification at 37° C. with the following components per 50 μl reaction batch:

(26) Lyophilized pellet of the RPA kit “TwistAmp fpg”

(27) 29.5 μl buffer of the RPA kit “TwistAmp fpg”

(28) 420 nM primer having the sequence:

(29) TABLE-US-00003 (SEQ ID NO: 1) 5′-CAGGATTAGATACCCTGGTAGTCCACGCCGTAAAC-3′
420 nM primer having the sequence:

(30) TABLE-US-00004 (SEQ ID NO: 2) 5′-TAAGGGGCATGATGATTTGACGTCATCCCCACCTT-3′
14.2 μl sample
12 mM magnesium acetate

(31) After the finished amplification reaction the complete reaction batches were purified with the aid of a silica-based membrane for DNA isolation in the presence of chaotropic compounds for the subsequent agarose gel electrophoresis (FIG. 1).

(32) In example 2 it could be shown that an E. coli cultivation in the exponential phase does not result in a DNA amplification (no bands on the agarose gel), since it is a 100% vital cultivation in the ideal case. Furthermore, it could demonstrated that a B. subtilis cultivation in the stationary phase results in thin DNA bands (amplicon at 430 bp), since a die off of cells already occurs in the stationary phase. As expected, previously inactivated B. subtilis and E. coli samples (10 min ultrasound), however, resulted in clearly thicker DNA bands at 430 bp.

EXAMPLE 3

(33) Each of the following samples has been analyzed three times by RPA in a comparative manner, in order to demonstrate the quantification potential of the method according to the present invention. 1. E. coli (exponential phase) was completely inactivated by ultrasound for 10 min and then directly used in the RPA reaction. 2. E. coli (exponential phase) was completely inactivated by ultrasound for 10 min, the DNA was isolated with an extraction kit based on a silica membrane and then used in the RPA reaction. 3. A DNA spike of 6.5×10.sup.6 genome units (GE) per RPA reaction of a quantified genomic E. coli DNA was added to E. coli (exponential phase) and then isothermally amplified. 4. A DNA spike of 6.5×10.sup.6 genome units (GE) per RPA reaction of a quantified genomic E. coli DNA was added to water (PCR grade) and then isothermally amplified.

(34) A standard curve with quantified genomic DNA and negative controls (controls without template; adding “PCR grade” water instead of the sample) were run in parallel in the RPA. The reaction ran at 37° C. within a time period of 30 min. Fluorescence data were recorded every 30 seconds.

(35) A 50 μl reaction batch contained the following components:

(36) Lyophilized pellet of the RPA kit “TwistAmp fpg”

(37) 29.5 μl buffer of the RPA kit “TwistAmp fpg”

(38) 420 nM primer having the sequence:

(39) TABLE-US-00005 (SEQ ID NO: 1) 5′-CAGGATTAGATACCCTGGTAGTCCACGCCGTAAAC-3′
420 nM primer having the sequence:

(40) TABLE-US-00006 (SEQ ID NO: 2) 5′-TAAGGGGCATGATGATTTGACGTCATCCCCACCTT-3′
1 μl “SYBR Green I” (diluted 1:50,000)
13.2 μl sample
12 mM magnesium acetate

(41) The standard curve as well as the corresponding values are shown in FIG. 2 and table 1.

(42) TABLE-US-00007 TABLE 1 Values of the standard curve using quantified E. coli DNA Time until increase of fluorescence Concentration [seconds] [Copies/ Quantified DNA standard (average, n = 2) reaction] E. coli standard diluted 1:10 337 8.58 × 10.sup.7 E. coli standard diluted 1:100 398 8.58 × 10.sup.6 E. coli standard diluted 1:1000 419 8.58 × 10.sup.5

(43) The values obtained for samples 1 to 4 and fluorescence curves are shown in FIGS. 3 and 4 as well as in table 2.

(44) TABLE-US-00008 TABLE 2 Determined concentrations of the genome units per reaction Time until increase of fluorescence Concentration Sample [seconds] [Copies/reaction] E. coli 10 min ultrasound 378 1.62 × 10.sup.7 (1) 368 375 E. coli 10 min ultrasound + 420 2.35 × 10.sup.6 DNA isolation (2) 403 404 E. coli exponential phase + 408 1.62 × 10.sup.6 6.5 × 10.sup.6 GE (3) 416 422 H.sub.2O + 6.5 × 10.sup.6 GE (4) 398 2.77 × 10.sup.6 412 408 Control without template 509 2.06 × 10.sup.4 491 486

(45) The results in table 2 as well as in FIGS. 3 and 4 demonstrate that the present invention allows a quantitative differentiation between living and dead microorganisms.

(46) Regarding the curve progression and the determined concentration of genome units, only a small difference is apparent between samples 1 and 2, which can be explained by the DNA losses in the silica membrane-based DNA isolation carried out in sample 2. However, this approach has shown that DNA of bacteria having a disturbed membrane integrity may be completely reproduced by RPA.

(47) The curve progressions of samples 3 and 4 (FIG. 4) as well as the determined concentrations of genome units (copies/reaction; table 2) are almost identical, which illustrates that vital cells remain intact during an RPA reaction and that their DNA is not amplified.

(48) The negative controls (“controls without template” in FIGS. 3 and 4 as well as in table 2) also show signals in the framework of the fluorescence detection. However, the same could be clearly differentiated from the samples and thus were not considered. These signals in the negative controls are the result of non-specific amplifications, for example, primer artifacts, since the non-specific dye “SYBR Green I” has been used for the detection in these experiments. A primer screening in further experiments, which is adapted to the respective application, allows to select optimal primer sequences for the respective target application. Furthermore, specific fluorescence-labeled probes may be used instead of “SYBR Green I” in order to avoid signals in the negative controls.

EXAMPLE 4

(49) Both completely vital as well as inactivated E. coli samples each have been analyzed four times by RPA and the number of living cells has been determined in parallel by plating on nutrient agar in different dilution stages (determination of colony numbers after an incubation time of 24 hours at 37° C.).

(50) A standard curve with quantified genomic DNA and a negative control (control without template; adding “PCR grade” water instead of the sample) were run in parallel in the RPA. Fluorescence data were recorded during 30 min at a constant reaction temperature of 37° C. every 30 seconds.

(51) A 50 μl reaction batch contained the following components

(52) Lyophilized pellet of the RPA kit “TwistAmp fpg”

(53) 29.5 μl buffer of the RPA kit “TwistAmp fpg”

(54) 420 nM primer having the sequence:

(55) TABLE-US-00009 (SEQ ID NO: 1) 5′-CAGGATTAGATACCCTGGTAGTCCACGCCGTAAAC-3′
420 nM primer having the sequence:

(56) TABLE-US-00010 (SEQ ID NO: 2) 5′-TAAGGGGCATGATGATTTGACGTCATCCCCACCTT-3′
1 μl “SYBR Green I” (diluted 1:50,000)
13.2 μl sample
12 mM magnesium acetate

(57) The standard curve as well as the corresponding values are shown in FIG. 5 and table 3.

(58) TABLE-US-00011 TABLE 3 Values of the standard curve using quantified E. coli DNA Time until increase of fluorescence Concentration [seconds] [Copies/ Quantified DNA standard (average, n = 2) reaction] E. coli standard diluted 1:10 517 8.58 × 10.sup.7 E. coli standard diluted 1:100 586 8.58 × 10.sup.6 E. coli standard diluted 1:1000 615 8.58 × 10.sup.5

(59) The values obtained for the samples and fluorescence curves are shown in FIG. 6 as well as in table 4.

(60) TABLE-US-00012 TABLE 4 Determined concentrations of the genome units (number of cells) per reaction and per ml Time until Concen- Average Average increase of tration concentration concen- Fl [Copies/ [Copies/ tration Sample [seconds] reaction] reaction] [Copies/ml] E. coli 10 min 526 7.87 × 10.sup.7 4.60 × 10.sup.7 3.48 × 10.sup.9 ultrasound = 527 7.34 × 10.sup.7 GE.sub.total 549 2.66 × 10.sup.7 583 5.25 × 10.sup.6 E. coli 665 1.15 × 10.sup.5 2.12 × 10.sup.5 1.60 × 10.sup.7 exponential 636 4.32 × 10.sup.5 phase = 656 1.73 × 10.sup.5 GE.sub.dead 662 1.27 × 10.sup.5 Control without 677 6.53 × 10.sup.4 template

(61) The following results with regard to the number of living cells in the sample (R.sub.living) and for the corresponding number of genome units (GE.sub.living):
GE.sub.living=GE.sub.total−GE.sub.dead=3.48×10.sup.9−1.60×10.sup.7=3.47×10.sup.9 GE/ml

(62) The data from table 4 show that the E. coli culture in the exponential phase contained 3.47×10.sup.9 living cells per milliliter and only 1.60×10.sup.7 dead bacteria, corresponding to 0.46%. The associated fluorescence curves are shown in FIG. 6.

(63) The parallel plating on nutrient agar resulted in lower, however, feasible results, since it has to be assumed that there are at no time 100% of the living bacteria growing and forming colonies, since, for example, VBNC may be present. Many bacteria change to a VBNC phase, for example, in case of unfavorable environmental conditions. The metabolism is reduced, such that the growth on standard nutrient agar is not possible anymore. Furthermore, it has to be considered that a colony frequently has not grown from a single germ of the original sample, but has to be ascribed to several not completely separated microorganisms. A number of living cells of E. coli in the exponential phase of about 5.0×10.sup.8 bacteria per milliliter has been determined by plating.