Detection of micro-organisms

Abstract

The present invention concerns methods for detecting the presence of a micro-organism in a fluid, gaseous and solid samples, together with apparatus for use in same. The invention comprises the use of a plurality of hollow fibres to filter the fluid and the cpture of the micro-organisms on the membrane by means of an specific binding pair.

1 . A method for detecting the presence of a micro-organism in a fluid sample, comprising the steps of: 
 i) passing said sample through the sample inlet of a filter device comprising a plurality of hollow fibre filter membranes which have attached to them a first member of a specific binding pair, said micro-organism displaying the second member of said specific binding pair, said membranes having first and second ends, an outer surface and an inner surface defining a lumen, said first end of each of said membranes being open and communicating with said sample inlet and flow through said second end of each of said membranes being restricted such that said flow occurs only through said first end and the pores of said membranes, the sample mixture being filtered through the pores of said membranes, leaving a filtrand in said lumen of said membranes;    ii) washing the unbound part of said filtrand from said lumen of said membranes;    iii) detecting the presence of any of said specific binding pairs attached to said membranes; and    iv) correlating the results of detection step (iii) with the presence of said micro-organism in said fluid sample.    
     
     
         2 . A method according to  claim 1 , said sample having a volume of at least 25, 50, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900 or 1000 ml.  
     
     
         3 . A method according to either one of claims  1  or  2 , said first member of a specific binding pair being an antibody or an antigen binding fragment thereof.  
     
     
         4 . A method according to any one of the preceding claims, said membranes being selected from the group consisting of polypropylene, polysulfone, cellulose acetate and nylon membranes.  
     
     
         5 . A method according to any one of the previous claims, additionally comprising prior to step (i) the steps of: 
 a) passing a gaseous sample through a gas filtration device comprising a gas filter; and    b) contacting said filter of said gas filtration device with a sample fluid to suspend or solubilise any of said micro-organisms entrapped in said filter to produce a fluid sample.    
     
     
         6 . A method for testing the contents of a package for the presence of a given micro-organism, comprising the steps of: 
 a) placing said package in a first chamber which communicates by way of a piercing implement with a second chamber containing a sample fluid;    b) contacting said package with said piercing implement such that any solid material in said package is able to pass via said piercing implement to said sample fluid in said second chamber, to give a fluid sample; and    c) performing the method of any one of claims  1 - 4  upon said fluid sample.    
     
     
         7 . A method according to  claim 6 , additionally comprising the step of effecting a relative gaseous pressure change in said first and second chambers such that the pressure in said first chamber is greater than that in said second chamber.  
     
     
         8 . A method according to either one of claims  6  or  7 , said package being provided with a self-sealing pad, said self-sealing pad being contacted by said piercing implement.  
     
     
         9 . A method for detecting the presence of a micro-organism in a solid sample, comprising the steps of: 
 a) contacting said solid sample with a sample fluid such that said micro-organism is placed in fluid-phase, to give a fluid sample; and    b) performing the method of any one of claims  1 - 4  upon said fluid sample.    
     
     
         10 . A method according to any one of the preceding claims, detection step (iii) being selected from the group consisting: enzyme immunosorbent assay, detection of a radio label, detection of fluorescence, an ATP assay, plating out, and PCR.  
     
     
         11 . A method according to any one of the preceding claims, said membranes being coated with the first members of a plurality of specific binding pairs.  
     
     
         12 . A method according to any one of the preceding claims, said membranes being treated with at least one of the group consisting a wetting agent and a detergent prior to said first member of said specific binding pair being attached to them.  
     
     
         13 . A method according to  claim 12 , said membranes being treated with isopropanol prior to said first member of said specific binding pair being attached to them.  
     
     
         14 . A method according to either one of claims  12  or  13 , said membranes being treated with Tween-20 prior to said first member of said specific binding pair being attached to them.  
     
     
         15 . A device having a sample inlet and a plurality of hollow fibre filter membranes which have attached to them a first member of a specific binding pair, the second member of said specific binding pair being displayed by a given micro-organism, said membranes having first and second ends, an outer surface and an inner surface defining a lumen, said first end of each of said membranes being open and communicating with said sample inlet and flow through said second end of each of said membranes being restricted such that said flow occurs only through said first end and the pores of said membranes, such that a sample mixture passed into said device through said sample inlet is filtered through the pores of said membranes, leaving a filtrand in said lumen of said membranes. sample inlet and flow through said second end of each of said membranes being restricted such that said flow occurs only through said first end and the pores of said membranes, such that a sample mixture passed into said device through said sample inlet is filtered through the pores of said membranes, leaving a filtrand in said lumen of said membranes.