Detection of cells in a liquid sample
The invention relates to a method for counting cells, such as bacteria and/or somatic cells in liquid samples, such as in dairy products, preferably raw milk. Disclosed is a method comprising a combination of steps that apply dimeric nucleic acid dyes that normally do not penetrate cells (=cell-impermeant dyes), which are rendered cell-permeant by using the right combination of pH, buffer and temperature.
1. A method of counting cells in a liquid sample, said method comprising the steps of:
a) mixing a staining composition comprising a dimeric nucleic acid dye and a buffering agent with said sample;
b) optionally sonicating the mixture of step a);
c) incubating the mixture at a temperature from about 50° C. to about 95° C. for less than 10 minutes, wherein the mixture is incubated without an ion-chelating agent or detergent;
d) optionally sonicating the incubated mixture of step c); and
e) counting the cells that are stained with said dye within said mixture, or a part thereof, wherein
the dimeric nucleic acid dye is cell-impermeant to HeLa cells after 30 minutes of incubation at 37° C. and neutral pH and has the formula Q1-BRIDGE-Q2, wherein Q1 and Q2 are nucleic acid dye moieties and BRIDGE is connecting Q1 and Q2 and has the formula:
—(CH 2 ) x —C(═O)NH—(CH 2 ) α —[O—(CH 2 ) β ] b —[O—(CH2) γ ] c -NH(O═C)—(CH 2 ) x —,
wherein:
each x, independently, is an integer selected from 1 to 11,
α may be an integer selected from 2 to about 20,
β and γ, are independently zero, 2 or 3,
b is zero or an integer selected from 1 to about 20, and
c is zero, 1 or 2, and wherein
the buffering agent is an organic buffering agent comprising an amine which is effective in rendering the dimeric nucleic acid dye cell-permeant under the incubating conditions of step c) to achieve a reliable counting of the cells.
2. The method according to claim 1 , wherein the dimeric nucleic acid dye is capable of binding to DNA via a release-on-demand mechanism.
3. The method according to claim 2 , wherein the BRIDGE is a substantially aliphatic, substantially neutral linker comprising from about 8 to about 150 non-hydrogen atoms.
4. A method according to claim 1 , wherein
each x is 5,
α is 2 or 3,
β is 2,
b is 1 or 2
c is zero, 1 or 2, and
γ is 3, when c is 1 or 2.
5. The method according to claim 4 , wherein BRIDGE has a formula selected from the group consisting of:
—(CH 2 ) 5 —C(═O)NH—(CH 2 ) 3 —[O—(CH2) 2 ] 2 -O—(CH 2 ) 3 —NH(O═C)—(CH 2 ) 5 —, and a)
—(CH 2 ) 5 —C(═O)NH—(CH 2 ) 2 —O—(CH 2 ) 2 —NH(O═C)—(CH 2 ) 5 —. b)
6. The method according to claim 1 , wherein Q1 and/or Q2 is a fluorescent nucleic acid dye moiety.
7. The method according to claim 6 , wherein the fluorescent nucleic acid dye moiety is derived from a nucleic acid dye selected from the group consisting of an acridine-based nucleic acid dye, an asymmetric cyanine-based nucleic acid dye, a phenanthridinium-based nucleic acid dye, a symmetric cyanine-based nucleic acid dye, a pyronin nucleic acid dye, a styryl nucleic acid dye, a derivative of DAPI, and a derivative of a Hoechst dye.
8. The method according to claim 7 , wherein the fluorescent nucleic acid dye moiety is derived from a nucleic acid dye selected from the group consisting of an acridine-based nucleic acid dye and a phenanthridinium-based nucleic acid dye.
9. The method according to claim 1 , wherein Q1 and Q2 are the same.
10. The method according to claim 1 , wherein the staining composition further comprises a protease.
11. The method according to claim 1 , wherein said cells are somatic and/or bacterial cells.
12. The method according to claim 1 , wherein the liquid sample is milk, said method comprising the steps of:
a) mixing a staining composition comprising a dimeric nucleic acid dye, a protease and a buffering agent with said milk;
b) optionally sonicating the mixture of step a) for about 1 to about 10 seconds at from about 15 to about 50 kHz;
c) incubating the mixture at a temperature from about 62° C. to about 68° C. for about 1 minute to about 5 minutes;
d) optionally sonicating the incubated mixture of step c) for about 1 to about 10 seconds at from about 15 to about 50 kHz, and
e) counting the cells that are stained with said dye within said mixture, or a part thereof, using a flow cytometer;
wherein,
the dimeric nucleic acid dye has the formula Q1-BRIDGE-Q2, wherein Q1 and Q2 are nucleic acid dye moieties connected by BRIDGE, and
Q1 and Q2 are the same and are selected from the group consisting of nucleic acid dye moieties having structure I and structure III,
wherein:
each R1 is independently selected from the group consisting of H, a C1-C2 group, and an alkyl,
BRIDGE is attached to R2 or R3,
when BRIDGE is attached to R2, R3 is selected from the group consisting of H or —CH3,
when BRIDGE is attached to R3, R2 is selected from the group consisting of H, —CH3, —NH2, —NHCH3, —CN, and —C(═O)NH2,
each of R6 and R7 is independently selected from the group consisting of H, a C1-C2 group and an alkyl, and
ψ is an anion that balances positive charge(s) associated with the dye
wherein,
BRIDGE is attached to R1, and
ψ is an anion that balances positive charge(s) associated with the dye;
wherein
BRIDGE has a formula selected from the group consisting of
—(CH 2 ) 5 —C(═O)NH—(CH 2 ) 3 —[O—(CH2) 2 ] 2 -O—(CH 2 ) 3 —NH(O═C)—(CH 2 ) 5 —, and
—(CH 2 ) 5 —C(═O)NH—(CH 2 ) 2 —O—(CH 2 ) 2 —NH(O═C)—(CH 2 ) 5 —, and wherein
the buffering agent is Tris.
13. The method according to claim 12 , wherein the milk is cow milk.
14. The method according to claim 10 , wherein the protease is a serine endopeptidase.
15. The method according to claim 1 , wherein said staining composition has a pH from about 8 to about 11.5.
16. The method according to claim 1 , wherein step c) is performed at a temperature from about 60 to about 70° C.
17. The method according to claim 1 , further comprising preheating the staining composition to a temperature of from about 45 to about 95° C., prior to mixing with the sample.
18. The method according to claim 1 , wherein the dimeric nucleic acid dye is 10, 10′-(6, 22-dioxo-11, 14, 17-trioxa-7, 21-diazaheptacosane-1, 27-diyl) bis (3, 6-bis (dimethylamino) acridin-10-ium) iodide) and the buffering agent is Tris.
19. A method of counting cells in a liquid sample, said method comprising the steps of:
a) mixing a staining composition comprising a dimeric nucleic acid dye and an amine buffering agent with said sample;
b) optionally sonicating the mixture of step a);
c) incubating the mixture at a temperature from about 50° C. to about 95° C. for less than 10 minutes, wherein the mixture is incubated without an ion-chelating agent or detergent;
d) optionally sonicating the incubated mixture of step c); and
e) counting the cells that are stained with said dye within said mixture, or a part thereof, wherein
the dimeric nucleic acid dye is cell-impermeant to HeLa cells after 30 minutes of incubation at 37° C. and neutral pH and has the formula Q1-BRIDGE-Q2, wherein Q1 and Q2 are nucleic acid dye moieties and BRIDGE is connecting Q1 and Q2 and has the formula:
—(CH 2 ) x —C(═O)NH—(CH 2 ) α —[O—(CH 2 ) β ] b —[O—(CH2) γ ] c -NH(O═C)—(CH 2 ) x —,
wherein:
each x, independently, is an integer selected from 1 to 11,
α may be an integer selected from 2 to about 20,
β and γ, are independently zero, 2 or 3,
b is zero or an integer selected from 1 to about 20, and
c is zero, 1 or 2.
20. The method of claim 1 , wherein step c) is performed at a temperature from about 62° C. to about 68° C.