Saliva collection, processing, stabilization, and storage method

Abstract

Provided herein is an all-in-one saliva collection apparatus that collects saliva to allow for the filtration of saliva in order to separate saliva components, such as extracellular proteins and nucleic acids that are not present in intact cells, from the intact cells and debris remaining in the extracted sample. The filtered saliva samples can be aliquoted into two fractions for protein and/or nucleic acid analysis. The present invention further describes long term storage at ambient temperatures of filtered salivary nucleic acids, and long term storage at ambient temperatures of filtered salivary proteins added to an ethanol solution. The filtered cell-free saliva samples have diagnostic usefulness.

1 . A method for stabilizing nucleic acid and protein samples isolated from a saliva sample, the method comprising:
 a) collecting a saliva sample from a subject;   b) filtering the saliva sample to produce a filtered sample that is free of cells;   c) collecting the filtered sample in at least a first and a second receiving device;   d) adding an alcohol solution to the first receiving device to produce an alcohol-containing filtered sample comprising a protein sample, with the proviso that alcohol is not added to the second receiving device to produce an alcohol-free filtered sample comprising a nucleic acid sample; wherein the protein sample and the nucleic acid sample are stabilized for at least 3 days when stored at 25 degrees Celsius; and   e) performing an analysis on the filtered sample collected in the first and second receiving devices comprising one or more of: a protein analysis on the alcohol-containing filtered sample or a nucleic acid analysis on the alcohol-free filtered sample.   
     
     
         2 . The method of  claim 1 , wherein the nucleic acid is DNA. 
     
     
         3 . The method of  claim 1 , wherein the nucleic acid analysis is polymerase chain reaction (PCR). 
     
     
         4 . The method of  claim 1 , wherein the nucleic acid is RNA. 
     
     
         5 . The method of  claim 4 , wherein the nucleic acid analysis is RT-PCR. 
     
     
         6 . The method of  claim 5 , wherein the RT-PCR is reverse transcription quantitative real-time PCR (RT-qPCR). 
     
     
         7 . The method of  claim 1 , wherein the alcohol solution comprises 20% ethanol. 
     
     
         8 . The method of  claim 1 , wherein the protein analysis comprises western blot, mass spectrometry protein identification, or ELISA. 
     
     
         9 . The method of  claim 1 , wherein the filtered sample is stored at ambient temperature. 
     
     
         10 . The method of  claim 1 , wherein the filtered sample is stored at ambient temperature for at least two weeks without more than 50% degradation of proteins or nucleic acids present in the filtered sample. 
     
     
         11 . The method of  claim 1 , wherein the filtered sample is stored at ambient temperature for at least two weeks without more than 25% degradation of proteins or nucleic acids present in the filtered sample 
     
     
         12 . The method of  claim 1 , wherein the filtered sample is stored at ambient temperature for at least ten weeks without more than 50% degradation of proteins or nucleic acids present in the filtered sample. 
     
     
         13 . The method of  claim 1 , wherein the filtered sample is stored at ambient temperature for at least ten weeks without more than 25% degradation of proteins or nucleic acids present in the filtered sample. 
     
     
         14 . The method of  claim 1 , wherein the alcohol solution comprises 15-25% ethanol. 
     
     
         15 . The method of  claim 1 , wherein the alcohol solution comprises 5-35% ethanol. 
     
     
         16 . The method of  claim 1 , wherein the filter is selected from the group consisting of a 0.22 μm, 0.45 μm and 5.0 μm hydrophilic membrane. 
     
     
         17 . The method of  claim 16 , wherein the filter is a 0.22 μm hydrophilic membrane. 
     
     
         18 . An apparatus for the collection of saliva comprising a sample collection pad, a filter, two or more receiving devices, wherein the receiving devices are selected from an mRNA collection tube, a polypeptide collection tube, and a DNA collection tube, wherein the polypeptide collection tube comprises an ethanol solution, and the DNA collection tube comprises a DNA stabilizer, wherein the filter is operably connected to the receiving devices. 
     
     
         19 . The apparatus of  claim 18 , wherein the filter is selected from the group consisting of a 0.22 μm, 0.45 μm and 5.0 μm hydrophilic membrane. 
     
     
         20 . The apparatus of  claim 19 , wherein the filter is a 0.22 μm hydrophilic membrane. 
     
     
         21 . A method of using the apparatus of  claim 18 , the method comprising inserting the sample collection pad into the oral cavity for sufficient time to moisten the sample collection pad, inserting the collection pad into the receiving tube, applying sufficient force to cause the materials collected in the collection pad to pass through the filter thereby forming a filtered sample, and collecting the filtered sample into one or more receiving devices. 
     
     
         22 . A method for stabilizing nucleic acid and protein samples isolated from a saliva sample, the method comprising:
 a) collecting a saliva sample from a human subject;   b) filtering the saliva sample using a 0.22 μm to 5.0 μm hydrophilic membrane to produce a filtered sample that is free of cells;   c) collecting the filtered sample in at first and a second receiving device;   d) adding an ethanol solution to the first receiving device to produce a 20% ethanol-containing filtered sample comprising a protein sample, with the proviso that alcohol is not added to the second receiving device to produce an alcohol-free filtered sample comprising a nucleic acid sample; wherein the protein sample and the nucleic acid sample are stabilized for at least 3 days when stored at 25 degrees Celsius.   
     
     
         23 . The method of  claim 22 , wherein the protein sample is stabilized for at least 2 weeks when stored at 25 degrees Celsius. 
     
     
         24 . The method of  claim 22 , wherein the nucleic acid sample is stabilized for at least 10 weeks when stored at 25 degrees Celsius. 
     
     
         25 . The method of  claim 22 , further comprising the step of: (e) performing an analysis on the filtered samples collected in the first and second receiving devices comprising one or more of: a protein analysis on the ethanol-containing filtered sample or a nucleic acid analysis on the alcohol-free filtered sample. 
     
     
         26 . The method of  claim 22 , wherein the ethanol solution comprises 15-25% ethanol. 
     
     
         27 . The method of  claim 22 , wherein the ethanol solution comprises 5-35% ethanol. 
     
     
         28 . The method of  claim 22 , wherein the hydrophilic membrane in 0.22 μm.