Methods for detecting and quantifying viable bacterial endo-spores

Abstract

Methods and systems for detecting viable bacterial endospores in a sample and related methods to quantify viable bacterial endospores in a sample.

What is claimed is: 
     
         1 . A method for detecting a viable bacterial endospore in a sample, the method comprising
 contacting the sample with an agent capable of labeling a first form of nucleic acid to produce a labeled first form of nucleic acid, the agent further capable to interfere with amplification of the labeled first form of nucleic acid, the first form of nucleic acid consisting of a nucleic acid not comprised in the viable endospore;   treating the sample following the contacting to produce an extract, the treating performed for a time and under conditions to allow extraction from the sample of a nucleic acid comprising the first form of nucleic acid and a second form of nucleic acid, the second form of nucleic acid consisting of a nucleic acid comprised in the viable bacterial endospore;   performing nucleic acid amplification of the extract;   detecting an amplification product in the extract following nucleic acid amplification to detect viable endospores in the sample.   
     
     
         2 . The method of  claim 1 , wherein the agent is selected from the group consisting of propidium monozide (PMA), ethidium monoazide bromide (EMA), ethidium bromide, berberine, proflavine, daunomycin, doxorubicin, and thalidomide. 
     
     
         3 . The method of  claim 1 , further comprising pretreating the sample for a time and under conditions suitable to kill viable cells or a portion thereof before contacting the sample with the agent. 
     
     
         4 . The method of  claim 3 , wherein the pretreating comprises heating the sample up to about 85° C. for about 15 minutes. 
     
     
         5 . The method of  claim 1 , wherein contacting the sample with the agent and/or contacting the treated sample with the agent is performed by mixing the sample and/or treated sample with the agent in a solution. 
     
     
         6 . The method of  claim 1 , wherein the agent is a nucleic acid intercalating agent. 
     
     
         7 . The method of  claim 1 , wherein the treating comprises heating the sample to a temperature comprised within the range from about 90 to about 100° C. 
     
     
         8 . The method of  claim 7 , wherein the treating is performed for a duration from about 5 minutes to about 30 minutes. 
     
     
         9 . The method of  claim 1 , wherein the treating comprises exposing the sample to UV radiation. 
     
     
         10 . The method of  claim 9 , wherein the UV radiation has a wavelength of about 245 nm. 
     
     
         11 . The method of  claim 9 , wherein the UV radiation is provided in a dose of from about 0.2 to about 3 KJ/m 2 . 
     
     
         12 . The method of  claim 1 , wherein the treating comprises contacting the sample to urea. 
     
     
         13 . The method of  claim 12 , wherein the urea is at a buffer concentration of about 8M. 
     
     
         14 . The method of  claim 13 , wherein buffer comprises about 5 mM cyclohexylaminoethane sulfonic acid, about 50 mM dithioerythritol, about 0.8% sodium dodecyl sulfate, and about 5 mg lysozyme. 
     
     
         15 . The method of  claim 1 , wherein the amplification is real-time quantitative PCR amplification. 
     
     
         16 . The method of  claim 15 , further comprising comparing the detected amplification product to a look-up table, the look-up table comprising pre-determined amplification product associated to quantities of viable bacterial endospores to quantitatively detect the viable bacterial endospore in the sample.