CA2708846A1 - Method and kit for the collection, preservation and transport of nucleic acids from saliva samples - Google Patents

Abstract

The present invention provides a method and kit for the collection, preservation and transport of nucleic acids from saliva samples.

Description

TITLE: Method and Kit for the Collection, Preservation and Transport of Nucleic Acids from Saliva Samples FIELD OF INVENTION

100011 The present invention relates to methods and kits useful for the collection, preservation and transport of nucleic acids from saliva samples.

BACKGROUND
100021 The collection, storage and transport of biological samples containing analytes of interest are required for many applications in molecular biology and diagnostic analysis. There are currently a number of problems and disadvantages associated with biological sample collection, storage and transport. First of all, the stability of the nucleic acids and analytes within the sample must be maintained. Indeed, the ability to maintain the stability of nucleic acids within biological samples determines whether the nucleic acids can be successfully analyzed, be it for research or diagnostic purposes. Nucleic acids in a biological sample quickly degrade and/or denature at ambient temperature and must generally be stored and shipped under cold to freezing temperatures to remain stable. This requirement for rcfrigeration/freezing is a significant problem when samples are collected at remote field sites, or at significant distances from central testing facilities. Furthermore, the requirement to ship samples on ice (typically dry ice) is associated with a large cost.

[00031 Another major concern with collection and transport of biological samples for analyte analysis is that these samples are often infectious, as the biological sample may contain live virus, fungi or bacteria. The presence of live infectious pathogens in these biological samples poses a health and safety risk to the individuals involved in the collection, transfer and testing of the samples if the samples are kept viable and/or biologically intact. Due to the potential dangers of shipping biologically intact samples the expense and effort required in shipping these samples is increased.

2 [0004J Recently there has been a shift towards non-invasive sample collection for research and diagnostics, including the use of saliva. Non-invasive specimen collection has a number of advantages including the fact that it is preferred by patients, requires less specialized personnel and equipment, and can be performed in various different settings, not just doctor's offices (Cook et al., 2005). Over the last decade it has been established that saliva is an excellent source of DNA for many types of genetic studies. Salivary DNA is equivalent in both quantity and purity to DNA obtained from blood (Rylander-Rudqvist et al., 2006; Quinque et al., 2006;
Rogers et al., 2007). Whole saliva is a mixture of the secretions from the salivary glands located in the mouth and it may also contain nasal and bronchial secretions, tears, blood from injuries in the mouth, serum exudates from the gums as well as food and cellular debris.
The DNA in the saliva originates from cells that are shed from the inner linings of the mouth and from white blood cells. These DNA containing cells can therefore be collected and the DNA
extracted for use in diagnostic assays.

100051 A number of different groups have investigated methods to overcome the problems associated with biological sample collection, storage and transport, particularly for non-invasive samples. Different reagents for the collection, storage and transport of biological samples have been formulated and marketed with varying degrees of success. One problem being encountered is that the reagents themselves frequently require cold temperatures or other special care to maintain stability. Therefore, when using these reagents there is still stability issues regarding transport of the reagents to a field site, storage at the field site before use and transport of the biological specimens-containing reagents back to a testing site is a primary concern. Furthermore, these reagents and systems are often not user-friendly, and are packaged in such a way that skill or special equipment is required in order to collect and preserve the sample. This could make the use of the reagent and collection system difficult, particularly in resource-limited areas. Furthermore, particularly for saliva, there is the problem of user safety. It is not safe to have an individual spit into a tube that contains a liquid preservative, as the chemical preservative may slash up onto the user. Therefore this is another area of concern that must be taken into account for saliva collection and preservation.

3 100061 Thus in order to fully realize the use of saliva as a biological source of analytes a method for the sate and simple collection, preservation and shipping of the saliva is required. In U.S. Patent No. 7,482,116, compositions and methods for preserving and extracting nucleic acids from saliva are disclosed. The compositions include a chelating agent, a denaturing agent, and buffers to maintain the pH of the composition within ranges desirable for DNA
and/or RNA. The compositions may also include a reducing agent and/or antimicrobial agent.
U.S. Patent No.
7,482,116 further discloses methods of using the compositions of the invention to preserve and isolate nucleic acids from saliva as well as to containers for the compositions of the invention.
While this patent discloses a method for preserving nucleic acids in saliva at room temperature, it does not address the presence of viable bacteria or virus which may be present in the sample.
[00071 Therefore, there still exists a need for safe, easy, user-friendly collection, storage and transport systems for saliva that maintain the integrity and quality of the nucleic acids contained within the sample. Moreover, there is a need for collecting, preserving and transporting the samples at ambient environmental conditions for extended periods of time without refrigeration, freezing or special handling of the collection reagents or the sample itself, such that samples can be collected in resource-] imited areas. Furthermore, there is a need for a convenient collection/preservation/storage system for saliva that minimizes the risk of pathogen exposure thereby resulting in a safe sample for subsequent biological and diagnostic analysis.
SUMMARY OF INVENTION

100081 In a first aspect, the present invention provides a kit for collecting, preserving and transporting nucleic acids in saliva, said kit comprising: saliva collection container having a sealable opening; and a pre-aliquoted, sealed ampule containing a preservation solution.

100091 In one embodiment, the kit further comprises a shipping container sized to contain the saliva collection container.

1000101 In another embodiment, the kit further comprises a label sized for attachment to the saliva collection container.

4 1000111 In another embodiment, the pre-aliquoted sealed ampule is a dropper, a syringe, a micro-doser, a pipette, a vial, or a pouch.

1000121 In another embodiment, the preservation solution comprises a lysis agent.
1000131 In another embodiment, the preservation solution is Norgen Saliva Preservation Buffer T9 [000141 In another embodiment, the kit further comprises instructions for use of the kit.
1000151 In another aspect, the present invention provides a method for the collection, preservation and transport of nucleic acids from a saliva sample using the kit of the present invention, comprising the steps of:

a) transferring saliva into the collection container;

b) opening the sealed ampule containing the preservation solution adding the preservation solution to the saliva in the container;

c) sealing the collection container; and d) mixing the saliva with the preservation solution.

[000161 In one embodiment, in step a), the saliva is transferred into the collection container by spitting into the collection container.

BRIEF DESCRIPTION OF THE FIGURES

1000171 Preferred embodiments of the invention will now he described, by way of example, with reference to the accompanying drawings, in which:

1000181 Figure 1 is side view of one embodiment of a saliva collection kit;

1000191 Figure 2 illustrates the use of the kit of Figure 1 for the collection, preservation and transport of nucleic acids from saliva samples;

1000201 Figure 3 is a line graph showing the successful amplification and detection of saliva RNA after 0, 1 and 2 weeks of storage at room temperature in the preservation solution.
1000211 Figure 4 is a line graph showing the successful amplification and detection of saliva DNA after 1, 2, 3, 4, 8, 16, 32 and 40 weeks of storage at -20 C, 4 C, 22 C and 55 C in preservation solution; and 1000221 Figure 5 is a line graph showing the successful amplification and detection of saliva DNA using real-time PCR after 40 weeks of storage at -20 C, 4 C, 22 C
and 55 C in preservation solution.

DETAILED DESCRIPTION

1000231 Conventional methods of the collection and preservation of saliva samples require the use of collection devices such as those disclosed in US Design Patent Nos.
D574,507 and D507,351 which can be expensive to manufacture and may not be user friendly.
In contrast, the collection and preservation kit and method disclosed herein provide a safe, easy and user-friendly collection, storage and transport system for saliva that maintains the integrity and quality of the nucleic acids contained within the sample. By providing the preservation solution is a pre-aliquoted, separate sealed ampule, there is reduced risk of the user being exposed to the preservation solution (for example, splash back from spitting into a collection container already containing a preservation solution) or the saliva sample not being correctly treated with the preservation solution. Furthermore, the disclosed collection and preservation kit and method allows for the collection, preservation and transport of the saliva samples at ambient environmental conditions for up to 1 year without refrigeration, freezing or special handling of the reagent or the sample. The disclosed collection and preservation kit and method also minimize the risk of pathogen exposure thereby resulting in a safe sample for subsequent analysis.
1000241 As shown in Figure 1, in one preferred embodiment of the invention, the kit 10 comprises a larger shipping container 16 sized to contain a smaller saliva collection container 12, a sealed ampule 20 containing a preservation solution and a post collection label 22 sized for attachment on the saliva collection container 12.

1000251 The saliva collection container 12 is provided with a sealable opening for receiving the saliva sample. The saliva collection container 12 is further provided with a lid or cap 14 to provide a leak-proof seal such that the saliva sample and the preservation solution are contained when the collection container 12 is closed. In one embodiment, saliva collection container 12 is provided with a threaded screw cap. In other embodiments the lid 14 may remain attached to the collection container, such as a flip-top lid. In other embodiments the lid 14 may also fit tightly on the container like a cork.

1000261 The shipping container 16 is sized for containing the saliva collection container 12 and is provided with a removable lid or cap 18. In one embodiment, the shipping container 16 is provided with a threaded screw cap.

1000271 The shape of shipping container 16 and saliva collection container 12 may be cylindrical, rectangular, tubular or any other shape which accommodates a removable lid. The shipping container 16 and saliva collection container 12 may be made from any type of material that creates the interior space, including but not limited to plastic, metal or glass. Typically, the shipping container 16 and saliva collection container 12 will be cylindrical in shape and have a diameter of 4.5cm and 3 cm, respectively.

1000281 The sealed ampule 20 may be a syringe, a vial, a pipette, a dropper, or a pouch containing variable, predetermined, volumes of a preservation solution. The sealed ampule 20 can be easily opened to release the preservation solution when needed. In one preferred embodiment, the sealed ampule 20 is a micro-doser sized to contain 2 mL of preservation solution.

1000291 In one preferred embodiment, the preservation solution contained within the sealed ampule 20 is Norgen's Saliva Preservation Buffer (2X) (Cat# 21451). In further embodiments, the preservation solution may comprise cell lysis agents such as SDS, Sarkosyl, and NaCl. In these embodiments, the preservation solution allows for cell lysis and preservation of the nucleic acids in saliva.

1000301 The disclosed kit can be used for the collection, preservation and transport of nucleic acids from saliva samples. As used herein, the term "nucleic acids"
refers to RNA or DNA that is linear or branched, single or double stranded or a fragment thereof, of any origin found in saliva. As used herein, the term "preserved" refers to nucleic acids which are protected from degradation and can be successfully isolated and analyzed using molecular biology techniques.

1000311 As shown in Figure 2, saliva is first collected into the saliva collection container 12 by a user spitting into the saliva collection container 12. Next, the smaller, sealed end of ampule 20, which is shown as a micro-doser, is removed by scissors or by breaking the tip off, and the preservation solution contained within is transferred into the collection container 12 with the saliva by squeezing the micro-doser 20. Once the preservation solution has been added to the container the lid 14 is screwed onto the saliva collection container 12 to seal the saliva collection container 12, and the saliva collection container 12 is then shaken by hand in order to mix the saliva and the preservation solution. In embodiments, wherein the preservation solution further functions as a lysis solution, any cells present in the saliva, including bacterial, yeast and viral cells, will be lysed. The saliva collection container 12 can be labeled using the post collection label 22 and placed in the shipping container 16 for storage and/or shipping.

100032 Using the disclosed collection and preservation kit and method, the nucleic acids present in a saliva sample can be stored at room temperature for up to 1 year in the container with the nucleic acids being preserved and stabilized. The required time period for nucleic acid preservation will vary depending on the particular collection circumstances and may be as short of a time as necessary to transfer a sample from the point of collection to the point of analysis, or it may be for extended periods of time. Using the disclosed collection kit and method, the preservation of saliva nucleic acids may be for a period of several minutes, hours, days, months or greater as required.

1000331 Collected biological specimens may be stored in the container once the preservation solution has been added under a wide temperature range. In preferred embodiments, the storage and shipping temperature is room temperature, which is generally from 15 C to 40 C.
In other embodiments, the samples may be stored in cool environments. Samples collected using the disclosed collection kit and method can be stored at 4 C for 10 months with subsequent isolation and analysis of the intact saliva DNA or at -20 C for 10 months with subsequent isolation and analysis of the intact saliva DNA. In other embodiments, collected samples can be stored in a warm environment, for example at 55 C, for about 10 months with subsequent isolation and analysis of the intact saliva DNA.

1000341 After storage, the saliva DNA and/or RNA are isolated using methods know in the art, including spin column chromatography. Those skilled in the art will recognize that there are many different methods which can be used to isolate the DNA and/or RNA for downstream analysis. Once isolated, the nucleic acids are then analyzed by any technique known in the art which can be used for analyzing nucleic acids, preferentially by gel electrophoresis or PCR.
amplification techniques.

1000351 While only specific embodiments of the invention have been described, it is apparent that variations can be made thereto without departing from the scope of the invention.
The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. It is the intention in the appended claims to cover all variations that may fall within the true scope of the invention.

1000361 Example 1 - Storage and Preservation of Saliva RNA for 2 Weeks at Room Temperature 1000371 For saliva collection a container with a screw-on lid with a diameter of 3 cm was obtained. The lid was removed and 2 mL of saliva was collected by spitting into the container.
A micro-doser containing 2 mL of Norgen's Saliva Preservation Buffer (2X) (Cat# 21451) was then obtained. The end of the sealed micro-doser containing pre-aliquoted saliva preservation solution was then opened using scissors, and the preservation solution was added to the saliva sample in the container. The container was then sealed by screwing the lid back on, and the saliva and the preservative were mixed by hand. The saliva sample was then stored at room temperature (22 C), and 500 tL aliquots of the preserved sample were removed at time 0, 1 week and 2 weeks. RNA was isolated from the saliva samples using a modified version of Norgen's Total RNA Purification Kit (Cat # 17200). Briefly, 250 L of RNA Lysis Solution was added to 500 L of preserved saliva and mixed. Next, 25 pL of Triton X 100 was added to the solution and mixed. Next, the solution was mixed and centrifuged for 2 minutes at 14000 rpm. The supernatant was then removed and mixed with 500 pL of isopropanol then loaded onto a silicon carbide column by spinning for I minute at 14,000 rpm. The bound RNA was then washed twice with 500 t, of RNA Wash Solution by spinning at 14,000 rpm. A dry spin at 14,000 rpm was then performed, followed by elution of the bound RNA using 50 tL of nuclease-free water and spinning the column for 2 minutes at 2,000 rpm followed by 2 minutes at 14,000 rpm.

[000381 After saliva RNA isolation, the purified RNA was then analyzed by RT-qPCR
amplification. For the RT reaction, 5 tL of RNA was used in a 20 L total reaction using Invitrogen's Superscript III system with Oligo dT. The conditions of the PCR
are as follows:
qPCR mix:
0.1 pL each primer (50 M stock S 15 primer) 3 L RT product L of SYBR Green qPCR Mastermix Complete to 20 L, with water qPCR Program:
3 min at 95 C

sec at 95 C
30 sec at 59 C
45 sec at 72 C
Repeat 40 cycles Run melt curve End program 1000391 Figure 3 shows the successful amplification and detection of saliva RNA after 0 weeks (circle), I week (triangle) and 2 weeks (diamond) of storage at room temperature in the preservation solution.

Table 1- Ct values associated with the successful amplification and detection of saliva RNA
after 0. 1 and 2 weeks of storage at room temperature in the preservation solution Week Ct 0 22.1 1 20.0 2 20.6 NTC 34.7 1000401 The results set out in Figure 3 and Table 1 indicate that the RNA is still detectable and therefore in tact after 2 weeks of storage at room temperature. This indicates that the saliva RNA was preserved following collection.

[000411 Example 2 - Storage and Preservation of Saliva DNA for up to 40 Weeks at -C, 4 C, Room Temperature (22 C) and 55 C

[000421 In order to test the preservation of saliva DNA at different temperatures in the saliva preservation buffer, forty 250 L samples of saliva were obtained, and each sample was mixed with 250 pL of Norgen's Saliva Preservation Buffer (2X) (Cat# 21451).
Ten of the samples were placed at -20 C, ten of the sample at 4 C, ten of the samples at room temperature (22 C) and ten of the samples at 55 C. DNA was then isolated from I sample at each temperature at the following time points: I week, 2 weeks, 3 weeks, 4 weeks, 8 weeks, 16 weeks, 24 weeks, 32 weeks and 40 weeks. DNA was isolated using Norgen's Saliva DNA
Isolation Kit (Cat# 21410). Briefly, Add 100 tL of Binding Solution was added and mixed by vortexing.
Next, 6 pL of Purification Additive was added and mixed by vortexing. Next, 60 ltL of isopropanol was added and mixed by vortexing. The solution was then centrifuged for -7 minutes at 14,000 x g ('-14,000 RPM) to precipitate cells. Clarified supernatant was added to spin columns containing silicon carbide and centrifuged for 2 minutes at 14,000 x g (-14,000 RPM).
The bound DNA was then washed with the provided wash solutions by spinning for 1 minute at 14,000 xg. Columns were then spun for 2 minutes to dry the resin. To elute the DNA 100 tL of Elution Buffer was spun through the column for 2 minutes at 200 xg followed by 1 minute at 14,000 xg.

[000431 After saliva DNA isolation, the DNA was analyzed by qPCR amplification using 5S primers. The conditions of the PCR are:

qPCR Mix:

2x PCR master mix: 10 L
5S primer (5 uM mix): 2 L
Tempate (DNA): 2 L
Water: 6 L
Total : 20 L
qPCR program:
95 C for 3min.
95 C for 16 sec.
60 C for 30 sec.
For 40 cycles For disassociation curve analysis (part of the real-time PCR) 95 C for 1 min 0.5 C was reduced in I min. from 95 C to 60 C

1000441 Figure 4 shows the successful amplification and detection of saliva DNA after 1, 2. 3, 4, 8, 16, 32 and 40 weeks of storage at -20 C, 4 C, 22 C and 55 C in preservation solution.
Figure 5 shows the successful amplification and detection of saliva DNA using real-time PCR
after 40 weeks of storage at -20 C, 4 C, 22 C and 55 C in preservation solution. From the results shown in Figures 4 and 5, it can be seen that all the DNA templates were successfully amplified without any loss to the quality of the template, indicating that saliva DNA was stable at different temperatures up to 40 weeks.

1000451 Example 3 - Lysis of Bacterial and Fun&al Cells in Saliva with Preservation Solution 1000461 Samples of I mL of saliva were collected, and were spiked with either I x 106 E.
coli cells, or I x 106 Saccharomvices cerevisiae. Immediately I mL of Norgen's Saliva Preservation Buffer (2X) (Cat# 21451) was added to each spiked saliva sample and mixed well.
After the preservation solution was added, 100 L samples of the preserved saliva were removed at 0 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes and 60 minutes and were plated immediately onto agar plates. The plates were then incubated overnight at 37 C
for the bacteria and 30 C for the yeast. The plates were incubated overnight (16 hours), and the number of colonies were then counted and observed. For the saliva samples spike with bacteria, no bacterial growth was observed on the plates that had been preserved with the perseveration solution for 5 minutes or greater, indicating that the bacterial cells were lysed. For the saliva samples spiked with yeast, no yeast growth was observed on the plates that had been preserved with the perseveration solution for 5 minutes or greater, indicating that the yeast cells were lysed. The addition of preservation solution resulted in cell lysis of both bacteria and yeast found in saliva after 5 minutes (see Table 2), resulting in a saliva sample that is free of infectious and harmful materials and providing a sample which is safer to ship than a preserved saliva sample with intact pathogens.

Table 2 Summary of the number of viable yeast and bacteria present in saliva after increasing amounts of time in the preservation solution Time of Exposure to Preservation Solution minutes minutes minutes minutes minutes minutes # of colonies <50,000 0 0 0 0 0 (bacteria) colonies # of colonies <50,000 0 0 0 0 0 (yeast) colonies

Claims (9)

What is claimed is:

1. A kit for collecting, preserving and transporting nucleic acids in saliva, said kit comprising:

saliva collection container having a sealable opening; and a pre-aliquoted, sealed ampule containing a preservation solution.

2. The kit according to claim 1 further comprising a shipping container sized to contain the saliva collection container.

3. The kit according to claim 1 or 2, wlierein the kit further comprises a label sized for attachment to the saliva collection container.

4. The kit according to any one of claims 1 to 3, wherein the pre-aliquoted sealed ampule is a dropper, a syringe, a micro-doser, a pipette, a vial, or a pouch.

5. The kit according to any one of claims 1 to 4, wherein the preservation solution comprises a lysis agent.

6. The kit according to any one of claims 1 to 5, wherein the preservation solution is Norgen Saliva Preservation Buffer .TM..

7. The kit according to any one of claims 1 to 6, wherein the kit further comprises instructions for use of the kit.

8. A method for the collection, preservation and transport of nucleic acids from a saliva sample using the kit of any one of claims 1 to 7, comprising the steps of.

a) transferring saliva into the collection container;

b) opening the sealed ampule containing the preservation solution adding the preservation solution to the saliva in the container;

c) sealing the collection container; and d) mixing the saliva with the preservation solution.

9. The method of claim 8 wherein in step a), the saliva is transferred into the collection container by spitting into the collection container.