JP2004159648A - Method for preserving cell and nucleic acid target - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for preserving a cell and a nucleic acid in a sample by exposing the sample to a composition producing an inhibitory effect on a proteolytic substance and/or a nucleic acid-degrading substance in the sample. <P>SOLUTION: The composition useful for the method contains a chelating agent which is bound to trace metals necessary for proteolytic activity of the proteolytic substance and/or nucleic acid-degrading activity of the nucleic acid-degrading substance in the sample, wherein sodium citrate, sodium borate, sodium fluoride, and EDTA are each exemplified as the chelating agent. <P>COPYRIGHT: (C)2004,JPO

Description

  The field of the invention relates generally to maintaining the integrity of such nucleic acids, in order to make them suitable for certain diagnostic methods such as hybridization and amplification. More particularly, the present invention relates to the use of compositions that cause an inhibitory effect on proteolytic and / or nucleolytic substances.

  Diagnostic methods utilizing nucleic acid molecules include nucleic acid probe hybridization for measuring the presence and / or amount of target nucleic acid, nucleic acid primer hybridization for nucleic acid amplification and nucleic acid primer hybridization for enzymatic activity including nucleic acid extension, nicking and / or cleavage. included. Present in a sample using nucleic acid amplification methods such as strand displacement amplification (SDA), polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), and transcription-mediated amplification (TMA) Increase the copy number of the particular nucleic acid sequence of interest (target sequence) with a lower copy number.

  A known method for inactivating DNAse and protease is to heat a sample at 100 ° C. for 15 minutes (for example, see Non-Patent Document 1). Other methods of inactivating proteolytic enzymes use a chaotropic agent such as guanidinium thiocyanate or sodium thiocyanate to denature these enzymatic proteins (e.g., incorporated herein by reference). Patent Document 1). In addition to inactivating nucleases and proteolytic enzymes, chaotropic agents are also known to promote the lysis of cell walls of a wide range of biological materials (see patents incorporated herein by reference). Reference 2). Therefore, the use of chaotropic agents that promote inactivation of proteases or nucleases should be avoided when cell wall preservation is also desired.

  A more general description of methods for storing nucleic acids is provided by U.S. Patent No. 6,037,097 to Exact Laboratories, which uses EDTA and EGTA as nuclease inhibitors in a method for extracting DNA from exfoliated human epithelial cells in stool samples. (See Patent Document 3). In addition, Kyoto Medical Science Institute, KK owns two Japanese patent disclosures on methods for preserving cells in urine. The use of citric acid as a buffer in addition to EDTA as an antibacterial agent is disclosed (see Patent Document 4), and the use of sodium fluoride as a fluorine compound in addition to citric acid and EDTA is disclosed. (See Patent Document 5). Sierra Diagnostics includes ethylenediaminetetraacetic acid (EDTA), ethylenebis (oxyethylenenitrile) tetraacetic acid (EGTA), 1,2-bis (2-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid (BAPTA) Also disclosed is the use of at least one "chelating agent-enhancing component" (lithium chloride, guanidine, sodium salicylate, sodium perchlorate or sodium thiocyanate) together with their salts (Patent Document 6). Lithium chloride, guanidine and sodium thiocyanate are chaotropic agents known to those skilled in the art. The effective concentration range of the chelator enhancing component is from about 0.1M to about 2.0M.

U.S. Pat. No. 4,843,155 U.S. Pat. No. 5,234,809 WO 00/50640 pamphlet JP-A-04-118557 JP 05-249104 A WO 99/29904 pamphlet European Patent Application No. 0,915,171 U.S. Pat. No. 5,973,138 Sambrook, J., Fritsh, E.F., and Maniatis, T..Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

  Many of these methods are time consuming, labor intensive, and / or have cumbersome safety requirements associated with them. Another problem with methods that use relatively stringent processing steps or conditions is the loss of certain target nucleic acid sequences. Despite the ability of nucleic acid amplification methods to make multiple copies of a target sequence (amplicon) from very few original targets, amplification efficiency and detection capacity when the original target is present in large amounts in a sample Is improved. Higher detectability can be very important when processing samples that are particularly difficult to detect, such as acid-fast Bacillus (AFB), smear-negative Mycobacterium tuberculosis samples.

  Another common problem with samples performing molecular diagnostics is the stability of the sample with respect to time. When a sample is taken at one location and then shipped to another location, such as an intensive laboratory for molecular diagnostics, the stability of the sample becomes more important.

  For example, many clinically significant organisms do not maintain their integrity in urine samples and vaginal and cervical swabs for more than about 24 hours at room temperature. Therefore, such samples need to be refrigerated during transportation and / or storage to the centralized laboratory. One subject commonly tested from urine samples and swabs and known to be unstable in samples stored at room temperature is Nisseria gonorrhoeae.

  SUMMARY OF THE INVENTION To solve the problems associated with maintaining the integrity of target nucleic acids and maintaining sample stability, and thus realizing the benefits of improved detection of target nucleic acid sequences, the present invention provides for proteolytic substances and / or nucleic acids in a sample. Methods are provided for preserving cells and nucleic acids in a sample by exposing the sample to a composition that causes an inhibitory effect on degradants. Such compositions useful in the methods of the present invention include chelating agents that bind to trace metals required for proteolytic activity of proteolytic substances and / or nuclease activity of nucleolytic substances in a sample, such as sodium citrate, Includes sodium borate, sodium fluoride and EDTA. The compositions of the present invention do not contain an effective concentration of a "chelator enhancing component" or a chaotropic agent.

  As described above, the present invention relates to a method for preparing a composition that does not contain an effective concentration of a "chelate enhancer" or chaotropic agent, but which still causes an inhibitory effect on proteolytic and / or nucleolytic agents in the sample. To preserve cells and nucleic acids in a sample by exposing the cells. In this method, the sample is exposed to such a composition prior to cell lysis, so that the nucleic acid-containing cells remain stable in the sample.

  Sample types in which the methods of the present invention can be performed include substantially all types of samples, such as sputum samples, blood samples, urine samples, cerebrospinal fluid ("CSF") samples, vaginal and cervical swabs, and the like. Includes human and livestock clinical samples, environmental samples such as water, air and soil samples, and food samples. Samples in which the method of the invention can be performed include cells having target nucleic acid sequences for performing a diagnostic method that can include hybridization, such as direct probe hybridization or primer hybridization for initiation of an amplification method. Is assumed.

  Proteolytic substances commonly found in such samples include proteolytic enzymes, such as endopeptidases such as renin, exopeptidases such as trypsin and chymotrypsin, and proteases including cathepsins. When present in a sample, proteolytics cause the degradation of cell walls, thus releasing nucleic acids, including the target nucleic acids to be diagnosed. The nucleic acid thus released is then subjected to other agents which have a negative effect on the diagnostic method. The agents include degrading enzymes (such as DNase and RNase) and substances that inhibit nucleic acid hybridization and / or amplification methods (such as porphyrin compounds obtained from heme and hematin), Serum enzymes (such as aprotinin, leupeptin PMSF and pepstatin), and urea. Exposure of a nucleic acid, particularly a target nucleic acid, to such agents adversely affects any of the diagnostic methods performed on the nucleic acid. Therefore, by preventing early lysis of the cell wall and thereby preventing contact between the nucleic acid and such an agent, it is possible to maintain a stable sample for a long period before the diagnostic method can be performed on the sample. Become.

  The method of the invention involves exposing the sample to a composition that does not contain an effective concentration of a chelating enhancer or chaotropic agent, but causes an inhibitory effect on proteolytic and / or nucleolytic agents in the sample. This exposure can occur at any time before the cells are lysed and the target nucleic acid is released. This exposure results in a reaction mixture containing the cell sample and a composition that binds to at least one cofactor required for proteolytic and / or nucleolytic activity.

  Many such compositions are useful in the methods of the present invention. Examples of such useful compositions include chelating agents such as sodium citrate, sodium borate, sodium fluoride and EDTA. Such chelating agents bind to trace metals required for proteolytic activity of proteolytic substances in the sample. Thus, proteolytic substances cannot act effectively or at all to degrade cell walls. This keeps the cell wall intact and maintains the integrity of the nucleic acid molecule within the cell. This is because the nucleic acid molecule is not exposed to agents that cause degradation.

  Compositions that cause inhibition of proteolytic substances can be used in any form or condition. For example, such compositions may be prepared as a solid or liquid in a dry granule form, a compressed tablet, a dissolvable capsule containing the composition, or a permeable excipient such as a bag containing the composition. Can be exposed to The composition (in any form) can be added to the container holding the sample, or incorporated into the container to which the sample is added.

  The concentration of such compositions useful in the methods of the present invention will vary from about 2.5 mM to about 350 mM, with a preferred range from about 5 mM to about 100 mM. The temperature range at which the method of the present invention is effective varies from about 5 ° C to about 60 ° C.

  Other compositions useful in the methods of the present invention may provide a reasonable predictor of success by performing routine screening tests directed at optimal properties of such compositions (eg, causing inhibition of proteolytics). Can be confirmed by those skilled in the art.

  The examples provided herein provide a simple and straightforward routine screening test that allows one of ordinary skill in the art to quickly determine whether a particular composition is likely to be useful in the methods of the present invention. .

  When the composition and sample are exposed to each other, magnesium, calcium, zinc and manganese, which are co-factors required for proteolytic substances such as proteases and nucleases such as DNases and RNases Trace metals bind to the composition. In such a bound state, the trace elements cannot utilize the functions of those cofactors. Therefore, the decomposing action of the proteolytic substance on the cell wall and the nuclease on the nucleic acid is inhibited.

  The length of time for effective exposure of the composition and sample to each other for the methods of the present invention is generally minimal and no maximum. Trace metal binding is initiated almost immediately upon exposure of the composition and sample to each other, and such binding does not disappear measurably over time. In general, for the method of the present invention to be effective, the exposure of the composition and the sample to each other should be at least 30 minutes.

  Optionally, after such exposure, the composition and the sample may be separated. Such separation can be performed by various means such as centrifugation, filtration, and the like, if a permeable excipient was used, the permeable excipient was physically removed from the sample or processed. The sample can be removed simply by pipetting.

  Currently, various methods are used to prepare target nucleic acids in a sample for hybridization or amplification. For example, sputum samples that are processed to amplify mycobacterial nucleic acid sequences are commonly subjected to the NALC / NaOH method. Similarly, other types of clinical samples are subjected to other well-known standard methods (eg, centrifugation for large samples such as blood and urine). The method of the present invention can be used before, as part of, or after such standard methods.

  In addition to the utility of the method of the invention for maintaining cell wall integrity and nucleic acid integrity, the method also stabilizes the sample for transport at room temperature. The inhibition of proteolytics and / or nucleolytics produced by the method of the present invention allows for storage and transport of the sample at room temperature for longer periods of time than currently available methods using conventional sample storage methods. For example, the use of a mixed bed ion exchange resin as disclosed in US Pat. No. 6,074,097 allows the storage of urine samples at room temperature for as long as four days, while the invention of the present invention shown in the following examples. The method allows for the storage of a urine sample at room temperature for at least 14 days.

  The following examples illustrate certain embodiments of the invention described herein. As will be apparent to those skilled in the art, various changes and modifications are possible and are considered to be within the scope of the described invention.

Prevention of N. gonorrhoeae DNA degradation in urine The purpose of this example was to determine whether the method of the present invention eliminates the degradation of gonococcal plasmid DNA in human urine.

material:
BDProbeTec ™ ET Neisseria gonorrhoeae priming microwell (BD)
BDProbeTec ™ ET Neisseria gonorrhoeae amplified microwell (BD)
BDProbeTec ™ ET Neisseria gonorrhoeae plasmid DNA (BD)
BDProbeTec ™ ET instruments and instrument plates
BDProbeTec ™ ET melting heater
BDProbeTec ™ ET priming and heating heater
BDProbeTec ™ ET Pipettor and Pipette Tips Sodium Citrate, Trisodium Salt (Sigma)
Ethylenediaminetetraacetic acid, disodium salt "EDTA" (Sigma)
Negative pooled urine glycine HCl (Sigma)
Pea Ridge Iron Ore
Dimethyl sulfoxide “DMSO” (Sigma)
Bicine (Sigma)
Glycerol (Sigma)
Potassium hydroxide "KOH" (Sigma)

Method:
A volume of 20 ml of negative pooled urine was prepared using 10, 40, and 100 mM (final concentration) sodium citrate, 10, 20, and 40 mM (final concentration) EDTA, and without any chemical additives. . In addition, a volume of 20 ml of negative pooled urine was heated for 30 minutes (heat treated urine). The sample was incubated at room temperature for 60 minutes. After the incubation step, the samples were supplemented with Neisseria gonorrhoeae plasmid DNA (2500 DNA copies / ml). The samples were incubated at room temperature for 60 minutes. After the incubation step, a 2 ml volume from each sample (eight replicates for each sample) was heated to 114 ° C. for 30 minutes to denature the DNA.

  Heat denatured DNA was isolated from the sample using paramagnetic particles in an acidic environment as described in US Pat. A 1 ml volume of 8 repetitions for each of the above samples was added to ferric oxide. A volume of 100 μl of 6 M glycine HCl was added to each sample. The DNA-iron tetroxide complex was separated with a magnet and washed twice with 900 μl of 1 mM glycine HCl to reduce ionic strength and remove non-specifically bound material. Using a volume of 480 μl elution buffer (88.3 mM KOH, 177.8 mM bicine, 12.1% (v / v) glycerol and 11.0% (v / v) DMSO (final concentration), Was released from ferric oxide.

  Strand displacement amplification (SDA) was performed according to the manufacturer's instructions (BDProbeTec ™ ET Chlamydia trachomatis and Neisseria gonorrhoeae amplified DNA Assay Package Insert L-000203 (November 1999)). A volume of 150 μl of each sample was added to the priming microwell. The priming microwell plate was incubated at room temperature for 20 minutes, after which the plate was incubated at 72.5 ° C. The amplification microwell plate was incubated at 54 ° C. After 10 minutes, a 100 μl volume of each priming microwell was added to the corresponding amplification microwell. The amplification microwell plate was placed in a BDProbeTec ET instrument. Data were recorded as MOTA values (metric other than acceleration).

Results The results are shown in the table below as the average amplified MOTA value for each treated sample (repeated eight times for each sample).

Conclusion The data in this example show that 10, 20, and 40 mM sodium citrate and 10, 40, and 100 mM EDTA protected gonorrhoeae plasmid DNA in human urine from degradation by endogenous nucleases. The data also indicate that heat-treated urine protects against gonococcal plasmid DNA degradation.

Stability of Neisseria gonorrhoeae cells by sodium citrate The purpose of this example was to determine the stability of gonococci intact cells in human urine for 14 days at room temperature.

Materials Same amplification reagents and equipment as described in Example 1 Sodium citrate (Sigma)
BSA / PBS (0.2% bovine serum albumin, 10 mM potassium phosphate buffer (pH 7.6) and 150 mM sodium chloride)
Negative pooled urine CT / GC diluent (BD)
Neisseria gonorrhoeae ATCC 19424
Dynac II centrifuge (BD)

Methods A volume of 80 ml of negative pooled urine was prepared with and without 100 mM (final concentration) sodium citrate. Neisseria gonorrhoeae cells were added to the samples at a final concentration of 2500 cells / ml. Each sample was incubated at room temperature for 14 days. At specific time points (days 0, 6, and 14), 2.0 ml of each sample (8 replicates for each sample) was centrifuged at 2000 xg for 30 minutes at room temperature. The supernatant was decanted and the cell pellet was resuspended in 2.0 ml of BSA / PBS and then centrifuged at room temperature for 30 minutes at 2000 × g. The supernatant was again decanted and the cell pellet was resuspended in 2.0 ml of CT / GC diluent. Each sample was heated at 114 ° C. for 30 minutes to lyse the cells. SDA was performed on cell lysates as described in Example 1.

Results The results are shown in the table below as the average amplified MOTA value for each sample treated for 14 days (repeated eight times for each sample).

Conclusions The data in this example show that the addition of 100 mM sodium citrate to human urine inhibited lysis of gonococcal intact cells and maintained its stability at room temperature for a minimum of 14 days. In contrast, in urine without sodium citrate, cells were not stable and tended to undergo lysis before centrifugation as indicated by a decrease in MOTA values compared to day 0.

Stability of Neisseria gonorrhoeae cells by other chemical additives The purpose of this example was to determine if other chemical additives could maintain the stability of Neisseria gonorrhoeae intact cells in human urine .

Materials Same amplification reagents and equipment as described in Example 1 BSA / PBS (0.2% bovine serum albumin, 10 mM potassium phosphate buffer (pH 7.6) and 150 mM sodium chloride)
Negative pooled urine CT / GC diluent (BD)
Neisseria gonorrhoeae ATCC 19424
Sodium citrate (Sigma)
Sucrose (Sigma)
Polyvinylpyrrolidone “PVP” (average molecular weight 40000) (Sigma)
Magnesium chloride “MgCl ₂ ”
Sodium fluoride (Sigma)
Sodium formate (Sigma)
Sodium borate (Sigma)
Ethylenediaminetetraacetic acid, disodium salt "EDTA" (Sigma)
Ethylene bis (oxyethylene nitrilo) tetraacetic acid "EGTA" (Sigma)
Dynac II centrifuge (BD)

Methods 100 mM sodium citrate (final concentration), 5, 10 and 15% (v / v) sucrose (final concentration), 5, 10 and 15% (v / v) PVP (final concentration), 5, 10 and 15 mM chloride Magnesium (final concentration), 10 and 100 mM sodium fluoride (final concentration), 10 and 100 mM sodium formate (final concentration), 10 and 100 mM sodium borate (final concentration), 0.5 and 5 mM EDTA (final concentration), A volume of 40 ml negative urine was prepared with 0.5 and 5 mM EGTA (final concentration) and without any chemical additives. Neisseria gonorrhoeae cells were added to the samples at a final concentration of 2500 cells / ml. Each sample was incubated at room temperature for 6 days. At specific time points (days 0 and 6), samples were processed by centrifugation at 2000 × g as described in Example 2. Each sample was heated at 114 ° C. for 30 minutes to lyse the cells. SDA was performed on cell lysates as described in Example 1.

Results The results are shown in the table below as the average amplified MOTA values for each sample treated for 6 days (repeated eight times for each sample).

Conclusions The data in this example show that for 100 mM sodium citrate, 100 mM sodium fluoride, 100 mM sodium borate, and 5 mM EDTA, there was a significant difference in the mean amplified MOTA value of gonococcal cells on day 0 and day 6. Indicates that there is no These results indicate that the stability of gonococcal intact cells is maintained in human urine by these chemical stabilizers. Furthermore, these results indicate that these chemical stabilizers functioned in a manner similar to sodium citrate to prevent lysis of intact cells of Neisseria gonorrhoeae.

Stability of Neisseria gonorrhoeae cells in urine at different temperatures The purpose of this example was to examine the urinary concentration of sodium citrate and other chemical stabilizers described in Example 3, 5, 25, 35 and 45 in urine. It was to determine if the stability of the gonococcal intact cells could be maintained at 6 ° C for 6 days.

Materials Same amplification reagents and equipment as described in Example 1 BSA / PBS (0.2% bovine serum albumin, 10 mM potassium phosphate buffer (pH 7.6) and 150 mM sodium chloride)
Negative pooled urine CT / GC diluent (BD)
Neisseria gonorrhoeae ATCC 19424
Sodium citrate (Sigma)
Sodium fluoride (Sigma)
Sodium borate (Sigma)
Ethylenediaminetetraacetic acid "EDTA" (Sigma)
Dynac II centrifuge

Methods A volume of 70 ml with 100 mM sodium citrate (final concentration), 100 mM sodium fluoride (final concentration), 100 mM sodium borate (final concentration), 5 mM EDTA (final concentration), and without any chemical additives. A negative pooled urine was prepared. Neisseria gonorrhoeae cells were added to this sample at a final concentration of 2500 cells / ml. Each sample was incubated at 5, 25, 35 and 45 ° C. for 6 days. At specific time points (days 0 and 6), samples were processed by centrifugation at 2000 × g as described in Example 2. Each sample was heated at 114 ° C. for 30 minutes to lyse the cells. SDA was performed on the cell lysate as described in Example 1.

Results The data are shown in the table below as the average MOTA value of each sample for 6 days at 5, 25, 35 and 45 ° C (8 replicates for each sample).

Conclusions The data of this example show that for days 0 and 6 at 5, 25, 35 and 45 ° C., in human urine with 100 mM sodium citrate, 100 mM sodium fluoride, 100 mM sodium borate and 5 mM EDTA. Shows that there is no significant difference in mean MOTA values of intact cells of Neisseria gonorrhoeae. These results indicate that these chemical stabilizers confer good stability on intact gonococcal cells in urine and prevent cell lysis for a minimum of 6 days. In contrast, cells are not stable in urine without chemical compounds and within 6 days at temperatures 5, 25, 35 and 45 ° C., as indicated by a decrease in MOTA value compared to day 0. Showed a tendency to undergo cell lysis before centrifugation.

Stability of Neisseria gonorrhoeae DNA in urine The purpose of this example was to determine whether gonococcal plasmid DNA could be maintained in human urine with the chemical additives described in Example 3 at room temperature for three days. Was.

Materials The same amplification reagents and equipment as described in Example 1
BDProbeTec ET Neisseria gonorrhoeae plasmid DNA (BD)
Ethylenediaminetetraacetic acid, disodium salt "EDTA" (Sigma)
Negative pooled urine trioxide (Pea Ridge Iron Ore)
Dimethyl sulfoxide “DMSO” (Sigma)
Bicine (Sigma)
Glycerol (Sigma)
Potassium hydroxide "KOH" (Sigma)

Method Using 100 mM sodium citrate (final concentration), 100 mM sodium fluoride (final concentration), 100 mM sodium borate (final concentration), 10 and 20 mM EDTA (final concentration), and without chemical additives, volume 60 ml of negative pooled urine was prepared. The sample was incubated at room temperature for 60 minutes. After the incubation step, the sample was supplemented with Neisseria gonorrhoeae plasmid DNA at a final concentration of 5000 DNA copies / ml. The sample was incubated at room temperature for 3 days. At specific time points (days 0 and 3), a 2 ml volume of each sample (8 replicates for each sample) was heated to 114 ° C. for 30 minutes to denature the DNA. As described in Example 1, heat-denatured DNA was isolated from samples using paramagnetic particles in an acidic environment. SDA was performed on the isolated DNA as described in Example 1.

Results The results are shown in the table below as the average MOTA value for each sample after 3 days incubation at room temperature (8 replicates for each sample).

Conclusions The data in this example shows that 100 mM sodium citrate, 100 mM sodium fluoride, 100 mM sodium borate and 10 mM EDTA and 20 mM EDTA were expressed in urine at room temperature for a minimum of 3 days at room temperature with endogenous nuclease-induced gonococcal plasmid DNA. To prevent the decomposition of Without chemical additives in urine, gonococcal plasmid DNA was rapidly degraded.

Long-term stability of Neisseria gonorrhoeae cells in urine with EDTA at different temperatures and comparison of commercial urine preservatives with EDTA The purpose of this example was to demonstrate that in urine with EDTA, 5, 27, 33, 45 and Determine whether the stability of intact cells of Neisseria gonorrhoeae can be maintained at 60 ° C. for 6 and 14 days and evaluate the efficacy of EDTA against a commercial urine preservative, DNA / RNA Protect ™, from Sierra Diagnostics, LLC Is to compare.

Materials The same amplification reagents and equipment as described in Example 1 Ethylenediaminetetraacetic acid, dipotassium salt "EDTA" (Sigma)
DNA / RNA Protect ™ (Sierra Diagnostics)
BSA / PBS (0.2% bovine serum albumin, 10 mM potassium phosphate buffer (pH 7.6) and 150 mM sodium chloride)
Negative pooled urine CT / GC diluent (BD)
Neisseria gonorrhoeae ATCC 19424
Dynac II centrifuge

Method i) Potassium EDTA: A 70 ml volume of pooled negative urine was prepared using 10 mM potassium EDTA (final concentration). Neisseria gonorrhoeae cells were added to this sample at a final concentration of 2000 cells / ml. Each sample was incubated at 5, 25, 33, 45 and 60 ° C. for 14 days. At specific time points (days 0, 6, and 14), the samples were processed by centrifugation at 2000 xg for 30 minutes as described in Example 3. Each sample was heated at 114 ° C. for 30 minutes to lyse the cells. SDA was performed on the cell lysate as described in Example 1.

ii) A 70 ml volume of pooled negative urine was prepared using 10% (v / v) DNA / RNA Protect ™ (final concentration) in 70 ml of collected negative urine. Neisseria gonorrhoeae cells were added to this sample at a final concentration of 2000 cells / ml. Each sample was incubated at 5, 25, 33, 45 and 60 ° C. for 14 days. At specific time points (days 0, 6, and 14), the samples were processed by centrifugation at 2000 xg for 30 minutes as described in Example 3. Each sample was heated to 114 ° C. for 30 minutes to lyse the cells. SDA was performed on the cell lysate as described in Example 1.

Results The data are shown in the table below as the average MOTA values for each sample (8 replicates) after 6, and 14 days at 5, 27, 33, 45 and 60 ° C.

Conclusions The data in this example show that the addition of 10 mM EDTA to human urine inhibits lysis of gonococcal intact cells and maintains their stability at 5, 27, 33, 45 and 60 ° C. for a minimum of 14 days. Indicates that In contrast, the cells were not stable in urine without EDTA at 27 ° C. and tended to undergo lysis before centrifugation, as indicated by reduced MOTA values compared to day 0. Was. Also, in DNA / RNA Protect ™ treated human urine, cells became unstable after 6 days at 27 and 33 ° C and 14 days at 5 and 60 ° C.

Although the present invention has been described with reference to certain specific examples, modifications obvious to those skilled in the art may be made without departing from the scope of the invention. Various features of the invention are set forth in the following claims.

Claims (10)

  A method of preserving the nucleic acid integrity of cells in a sample, comprising exposing the sample to a composition that causes inhibition of proteolytic substances in the sample, wherein the composition comprises an effective concentration of a chelating agent. A method characterized by not containing an agent-enhancing component.   The method of claim 1, wherein the composition that causes inhibition of a proteolytic substance is a composition that binds at least one cofactor required for the activity of the proteolytic substance.   A method of preserving the nucleic acid integrity of cells in a sample, comprising exposing the sample to a composition that causes inhibition of nucleolytics in the sample.   3. The method of claim 2, wherein the composition that causes inhibition of a nucleolytic agent is a composition that binds at least one cofactor required for the activity of the nucleolytic agent.   5. The method according to claim 2, wherein the cofactor is a trace metal.   The method according to claim 2 or 4, wherein the composition that binds to at least one cofactor required for the activity of the nucleolytic substance is a chelating compound.   27. The method of claim 26, wherein said chelating compound is selected from the group consisting of sodium citrate, sodium borate, sodium fluoride and EDTA.   Claims: 1. A reaction mixture for preserving nucleic acid integrity, comprising a sample containing cells and a composition that binds to at least one cofactor required for the activity of a proteolytic or nucleolytic substance. Is a reaction mixture containing no effective concentration of a chelating agent enhancing component.   9. The reaction mixture of claim 8, wherein the composition that binds to at least one cofactor required for the activity of a proteolytic or nucleolytic substance is a chelating compound. The method of claim 9, wherein the chelating compound is selected from the group consisting of sodium citrate, sodium borate, sodium fluoride, and EDTA.