Detection method using eukaryotic cells

Abstract

Provided herein are materials and methods for isolation of eukaryotic nucleic acid from a human or non-human animal stool sample. Also provided are methods of analysis of eukaryotic biomarkers present in a human or non-human animal stool sample.

1 - 37 . (canceled) 
     
     
         38 . A method of identifying one or more eukaryotic biomarkers in a stool sample, the method comprising:
 a) mixing the sample with a buffer, a surfactant and a ribonuclease inhibitor to form a suspension;   b) separating the suspension into a portion enriched for eukaryotic cells and a portion enriched for bacterial cells and retaining the portion enriched for eukaryotic cells;   c) adding a chaotropic agent and optionally a surfactant to the portion enriched for eukaryotic cells to form a lysate;   d) fractioning the lysate into a cell debris layer, a layer comprising eukaryotic nucleic acids and a lipid layer; and   e) collecting the layer comprising eukaryotic nucleic acids and optionally the lipid layer, wherein the layer comprising eukaryotic nucleic acids comprises a plurality of extracted nucleic acids, and   f) analyzing the extracted nucleic acids by microarray sequencing, molecular barcoding, probe capture, polymerase chain reaction (PCR), ddPCR, RT-PCR, RT -qPCR, or nucleic acid sequencing to identify the eukaryotic biomarkers.   
     
     
         39 . The method of  claim 38 , wherein the stool sample is a human stool sample. 
     
     
         40 . The method of  claim 38 , wherein the separating step comprises centrifugation or a column-based method. 
     
     
         41 . The method of  claim 38 , wherein the separating step utilizes targeted probes that bind eukaryotic cells. 
     
     
         42 . The method of  claim 38 , wherein the separating step utilizes differential filtration. 
     
     
         43 . The method of  claim 38 , wherein i) step a) is repeated one, two, three, four or more times, ii) step b) is repeated one, two, three, four or more times, or iii) both step a) and step b) are repeated one, two, three, four or more times. 
     
     
         44 . The method of  claim 38 , wherein the fractionating step comprises centrifugation or differential filtration. 
     
     
         45 . The method of  claim 38 , wherein the fractionating step utilizes targeted probes that specifically bind eukaryotic nucleic acid. 
     
     
         46 . The method of  claim 38 , wherein i) step d) is repeated two or more times, ii) step e) is repeated two or more times, or iii) both step d) and step e) are repeated two or more times. 
     
     
         47 . The method of  claim 38 , further comprising extracting the eukaryotic nucleic acids from the collected layer comprising eukaryotic nucleic acids. 
     
     
         48 . The method of  claim 38 , wherein the extraction method is a magnetic particle-based, column-based, filter-based, bead-based, or organic solvent-based method. 
     
     
         49 . The method of  claim 38 , wherein the nucleic acid comprises DNA, RNA, total RNA, mRNA, tRNA, rRNA, ncRNA, smRNA, or snoRNA, or a combination of any of DNA, RNA, total RNA, mRNA, tRNA, rRNA, ncRNA, smRNA, or snoRNA. 
     
     
         50 . The method of  claim 38 , wherein the separating step comprises centrifugation. 
     
     
         51 . The method of  claim 38 , wherein the extraction method comprises a magnetic particle-based method. 
     
     
         52 . The method of  claim 38 , wherein the nucleic acid comprises RNA. 
     
     
         53 . The method of  claim 38 , wherein the nucleic acid comprises mRNA. 
     
     
         54 . The method of  claim 38 , further comprising a step of analyzing the extracted nucleic acid via ddPCR. 
     
     
         55 . The method of  claim 38 , further comprising a step of analyzing the extracted nucleic acid via PCR. 
     
     
         56 . The method of  claim 38 , wherein the eukaryotic biomarkers comprise at least one of ACY1, TNFRSF10B, EGLN2, KRAS, AREG, and CDH1. 
     
     
         57 . The method of  claim 38 , wherein the eukaryotic biomarkers comprise at least one of ACY1, TNFRSF10B, EGLN2, KRAS, AREG, CDH1, and GAPDH.