Identification of microorganisms using maldi-tof-ms on-plate extraction

Abstract

Rapid methods that identify sepsis-causing bacteria or yeast aid the physician in critical therapeutic decision-making, thus decreasing patient mortality rates. The methods described herein employ plating microorganisms directly on to a MALDI-MS plate, adding concentrated formic acid, and identifying the microorganism by mass spectrometry. Optionally, an organic solvent may be combined with the formic acid, or added to the sample before or after the concentrated formic acid is added thereto. The methods enable direct extraction of proteins from microorganisms without the need for liquid protein extraction methods and yields positive identification results for gram-positive bacteria, gram-negative bacteria and yeast in minutes.

1 . A kit for preparing a positive blood culture sample for identification of microorganisms therein, the kit comprising:
 a lysis buffer, wherein the lysis buffer is selected to lyse blood cells in the sample while microorganisms, if any, remain intact;   a volatile acid solution, wherein a volume percent of the volatile acid is at least 70% of the volatile acid solution; and   a matrix.   
     
     
         2 . The kit of  claim 1 , wherein the volatile acid is selected from the group consisting of formic acid, acetic acid, trifluoracetic acid and hydrochloric acid. 
     
     
         3 . The kit of  claim 1 , further comprising an organic solvent, wherein the organic solvent is selected from the group consisting of ethanol, methanol, isopropanol, or acetone. 
     
     
         4 . The kit of  claim 1 , wherein the kit comprises a volatile acid solution that is 70% volatile acid. 
     
     
         5 . The kit of  claim 3 , wherein the organic solvent is a solution is 100% organic solvent. 
     
     
         6 . The kit of  claim 3 , wherein the organic solvent is a solution is 30% organic solvent to about 70% organic solvent. 
     
     
         7 . The kit of  claim 1 , where the kit further comprises a fixative, wherein the fixative is selected from the group consisting of an organic solvent and a fixative. 
     
     
         8 . The kit of  claim 7 , wherein the organic solvent is ethanol. 
     
     
         9 . The kit of  claim 7 , wherein the fixative is formaldehyde. 
     
     
         10 . A method for characterizing at least one microorganism in a sample for identification of microorganisms therein, the method comprising:
 (a) obtaining a sample containing at least one microorganism;   (b) suspending the sample in a solution consisting of water to form a microbial suspension;   (c) combining the microbial suspension with an organic solvent;   (d) depositing at least a portion of the microbial suspension on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by mass spectrometry;   (e) drying the sample;   (f) treating the sample with a solution selected from the group consisting of formic acid in water and formic acid in an organic solvent, wherein the formic acid is at a volume percent of at least 70% when combined with the sample;   (g) drying the sample;   (h) placing a matrix over the treated sample;   (i) drying the sample; and   (j) identifying the at least one microorganism by a mass spectrometry.   
     
     
         11 . The method of  claim 10 , wherein a final concentration of organic solvent in the microbial suspension is in a range of about 30% by volume to about 70% by volume. 
     
     
         12 . The method of  claim 10 , wherein a concentration of the sample in the water is greater than about 0.5 McFarland. 
     
     
         13 . The method of  claim 10 , wherein the organic solvent is selected from the group consisting of ethanol, methanol, isopropanol, acetonitrile, acetone, and ethyl acetate. 
     
     
         14 . The method of  claim 10 , wherein the method further comprises adding a non-ionic detergent to the sample either when the sample is obtained or when suspended in water or both. 
     
     
         15 . The method of  claim 10 , wherein, after the sample is obtained in step a):
 i) adding a lysis buffer to the sample;   ii) concentrating the at least one microorganism in the sample; and   iii) separating a concentrated portion of the sample from a remainder; and then suspending the separated sample in water.