Microorganism nucleic acid purification from host samples

Abstract

The present disclosure provides systems, devices, and methods for purifying microorganism nucleic acid from a host sample, such as a whole blood sample from a human. In certain embodiments, devices and systems with multiple filters are employed and provide for the selective removal of blood cells and host nucleic acids from a sample in order to enrich for microorganism nucleic acid.

We claim: 
     
         1 . A method for purifying microorganism nucleic acid from a sample comprising:
 a) moving a biological sample through a system or device, wherein said biological sample comprises human cells and is suspected of containing microorganisms, and wherein said system or device comprises:
 i) a first size-exclusion filter that: excludes passage therethrough of lymphocytes, monocytes, neutrophils, eosinophils, and basophils, and allows passage therethrough of erythrocytes, lymphocytes, and platelets; 
 ii) either:
 A) a second size-exclusion filter that: excludes passage therethrough of erythrocytes, lymphocytes, and platelets, and allows passage therethrough of microorganisms smaller than platelets; or 
 B) both of the following:
 I) a second size-exclusion filter that excludes passage therethrough of erythrocytes and lymphocytes, but allows passage therethrough of platelets and microorganisms smaller than platelets, and 
 II) a third size-exclusion filter that excludes passage therethrough of platelets, but allows passage therethrough of microorganisms smaller than platelets; and 
 
 
 iii) a shearing filter that mechanically shears said microorganisms such that microorganism nucleic acid is released; and 
   b) collecting a purified sample in or from said system or device, wherein said purified sample is free or at least 99% free of said human cells and comprises at least a portion of said microorganism nucleic acid if microorganisms are originally present in said sample.   
     
     
         2 . The method of  claim 1 , wherein said biological sample comprises whole blood. 
     
     
         3 . The method of  claim 1 , wherein said system or device further comprises an affinity capture filter comprising affinity binding molecules that bind human or animal nucleic acid. 
     
     
         4 . The method of  claim 3 , wherein said affinity binding molecules bind human repetitive nucleic acid sequences. 
     
     
         5 . The method of  claim 1 , wherein said system or device further comprises a sample containment vessel, wherein said sample containment vessel comprises a proximal end and a distal end, and wherein said first and second size exclusion filters, and said third size exclusion filter if present, and said shearing filter are located in said sample containment vessel. 
     
     
         6 . The method of  claim 5 , wherein said shearing filter is located distal to said second size exclusion filter, wherein said second size exclusion filter is located distal to said first size exclusion filter, and wherein said third size exclusion filter, if present, is located between said shearing filter and said second size exclusion filter. 
     
     
         7 . The method of  claim 1 , wherein said system or device is part of a component selected from the group consisting of: a spin column, a plate filter, a filter manifold, and a microfluidic device. 
     
     
         8 . The method of  claim 1 , wherein said biological sample is moved through said system or device by: centrifugal force, positive pressure, or negative pressure. 
     
     
         9 . A method of purifying a sample comprising:
 a) providing:
 i) a sample comprising human cells and suspected of containing pathogens comprising pathogen nucleic acid; and 
 ii) a multi-stage purification device comprising:
 A) a sample containment vessel, wherein said sample containment vessel comprises a proximal end and a distal end, 
 B) a first filter comprising pores approximately 10-20 μm in size, positioned inside said vessel; 
 C) a second filter comprising pores approximately 1-2 μm in size, positioned inside said vessel and distal to said first filter; and 
 D) a third filter comprising pores approximately 0.1 μm in size, positioned inside said vessel distal to said first and second filters; 
 wherein said first, second, and third filters are further positioned inside said sample containment vessel such that fluid passing from said proximal end to said distal end of said sample containment vessel passes through said first, second, and third filters; and 
 
   b) pouring said sample into said proximal end of said sample containment vessel and treating said multi-stage purification device such that a purified sample is collected in, or eluted from, said distal end of said sample containment vessel, wherein said purified sample is free or at least 99% free of said human cells and comprises at least a portion of said pathogen nucleic acid if originally present in said sample.   
     
     
         10 . The method of  claim 9 , wherein said treating said multi-stage purification device comprises centrifuging or agitating said multi-stage purification device. 
     
     
         11 . The method of  claim 9 , wherein said sample comprises whole blood.